THE DEVELOPMENT OF AN ASSAY AND MHHOD @F PUMFECATIQN FOR HUMAN BLOOD CLOTTING FACTOR XI (PLASMA THRQMBQP‘LASHN ANTECEDERT) Thesis {for ”10 Degree of D“. D. MECHEGAN STEFE UNWERSETY Harold Gallick 1965 MICHIGAN Sum; .‘AWtRSITY o - . . .0 N“.- .- a- q A 4s-.\ ‘ “1 LANSlNG. MICHW ABSTRACT THE DEVELOPMENT OF AN ASSAY AND METHOD OF PURIFICATION FOR HUMAN BLOOD CLOTTING FACTOR XI (PLASMA THROMBOPLASTIN ANTECEDENT) by Harold Gallick The purpose of this study was to devise a method of preparing human blood clotting factor XI (plasma thromboplastin antecedent) of sufficient purity to be useful for elucidating its biochemical role in the blood clotting sequence of re- actions, and to be available for possible clinical application. In order to undertake the purification of the factor XI protein, it was first necessary to develop a means of determin- ing its potency that was not dependent upon a patient's factor XI-deficient plasma. Such an assay was achieved through modification of the existing Thromboplastin Generation Test (TGT). Replacement of the crude TGT reagents with puri- fied factor XI—free reagents such as calf-brain platelet sub- stitute, factor V, factor VIII and factors IX + X reagents was accomplished in this study. The addition of factor XI was required for the generation of intrinsic thromboplastin (intrinsic prothrombin activator). The assay was capable of quantitatively determining factor XI potency in plasma, serum or a plasma derivative, and in this respect, could determine the extent of a factor XI defect in a patient's serum. 1 Harold Gallick Employing the newly devisedassay for following factor XI activity, a method for purifying this clotting component was developed. Cohn's fraction IV-l was used as the starting material and was subjected to the following treatments: Albumins and other inert proteins were removed from the paste by two extractions with water. Ceruloplasmin, as well as factor XI were then extracted from the washed paste with an acetate buffer of pH 5.2 and ionic strength 0.15. Ceruloplasmin was separated out of the acetate extract by selective adsorption on DEAE cellulose. The solution of pro- teins not adsorbed by DEAE cellulose was fractionated by addition of (NH4)2804. The proteins precipitating between 5 40-45% saturation yielded the greatest factor XI activity. This precipitate dissolved in an isotonic citrate buffer and dialyzed free of (NH4)250g gave a product suitable for intra- venous administration. An 18‘fold enrichment in activity over that in fraction IV-l was achieved. This represented about a ZOO-fold enrich- ment over that in plasma. The composition of the purified preparation, as determined by free boundary electrophoretic analysis in veronal buffer at pH 8.6 and 0.05 ionic strength, was 24% orglobulin, 22% B-globulins and 54% y-globulins. It was found that commercial BaSO4 did not adsorb factor XI from solution whereas considerable quantities were adsorbed by freshly precipitated BaSO4. Heating a 1% solution Harold Gallick of the preparation at 600 for 5 minutes destroyed one-third of the activity and the activity was totally destroyed at 800 under the same conditions. The factor XI preparation was very stable on storage. None of its activity was lost when a solution was stored at 2-40 for two months and similarly a lyophillized product retained full activity for one year. THE DEVELOPMENT OF AN ASSAY AND METHOD OF PURIFICATION FOR HUMAN BLOOD CLOTTING FACTOR XI (PLASMA THROMBOPLASTIN ANTECEDENT) BY Harold Gallick A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1965 ACKNOWLEDGEMENTS The author wishes to express sincere appreciation to Dr. Hans A. Lillevik in the Department of Biochemistry at Michigan State University for his patience, encouragement and guidance during the course of this study. Acknowledgement is due the Michigan Department of Health for providing facilities, materials and technical analysis and to Dr. L. A. Hyndman and other associates in the Division of Laboratories for their helpful advice and suggestions. Portions of this investigation were financially sup‘ ported by funds from the Michigan Department of Health, The National Institutes of Health of the U. S. Public Health Service (Grant H—l465) and the Difco Laboratories of Detroit, Michigan. ii TABLE OF CONTENTS I. INTRODUCTION . . . . . . . . II. HISTORICAL . . . . . . . . . III. EXPERIMENTAL . . . . . . . . A. Apparatus. . . . . . . B. Materials and Reagents 0 1. Chemical Reagents. . . . . . . . . . 2. Biologic Reagents. . . . . . . . . C. Methods Employed for Determining Clotting Abnormalities . . . . . . . . . . . . . 1. General Considerations . . . . . . . . The Calcium Clotting Time of Plasma. . The One-Stage Prothrombin Time . . . . The Partial Thromboplastin Time Test . The Thromboplastin Generation Test-- Reagents and Procedure. . . . . . (fir-POI“) D. Development of the Procedure for Assaying Factor XI (PTA) . . . . . . . . . . . . 1. Considerations Concerning the Thromboplastin Generation Test. . 2. Directions for a Modified TGT Pro- cedure. . Replacement of the TGT Platelet Reagent . . . . . . . o . . . . . . Replacement of the TGT Adsorbed Plasma Reagent. . . . . . . . . . . Replacement of the TGT Serum Reagent . Identification of Factor XI (PTA). . . Applications of the Factor XI (PTA) Assay . . . . . . . . \umm tP (N E. The Procedure for Isolating Factor XI (PTA) 1. Selection of Some Possible Starting Materials . . . . . . . . . . 2. Effect of (NH4)2SO4 Fractionation of Fraction IV-1 on Factor XI Activity. . . . . . . . . . . . . 5. Extraction of Fraction IV-1 with 0.05 M NaCl . . . . . . . . . . . iii Page 25 25 26 26 27 51 51 52 55 54 55 59 41 42 45 48 49 52 54 57 57 62 65 TABLE OF CONTENTS - Continued IV. DISCUSSION. V. SUMMARY BIBLIOGRAPHY. APPENDIX I. Page Extraction of Fraction IV-1 with Distilled Water . . . . . . . . . 65 Extraction of Precipitate 2 with Acetate Buffer. . . . . . . . . . 67 Removal of Ceruloplasmin by Adsorp- tion on DEAE Cellulose. . . . . . 68 (NH4)2SO4 Fractionation of Effluent 4 . . . . . . . . . . . . . . . . 70 Physical and Chemical Properties of Purified Factor XI. . . . . . . . 77 . . . . . . . . . . . . . . . . . . 99 . . . . . . . . . . . . . . . . . 102 . . . . . . . . . . . . . . . . . . 115 iv TABLE II. III. IV. VI. VII. VIII. IX. XI. LIST OF TABLES Page The Most Common Synonyms for the Blood Clot- ting Factors. . . . . . . . . . . . . . . . 8 Properties of the Intrinsic Thromboplastic Clotting Factors. . . . . . . . . . . . . . 19 Some Physical and Chemical Properties of the Blood Clotting Factors. . . . . . . . . 24 Clotting Factors Present in Normal Plasma and in Thromboplastin Generation Test Re- agenUSPrepared from Normal Blood. . . . . . 58 A Summary of the Effect of Thromboplastin Generation Test Reagents on Clotting Time and the Deficiency Indicated. . . . . . . . 40 Distribution of Proteins in Cohn's Plasma Fraction IV—1 Analyzed from the Tiselius Free Moving Boundary Pattern of Figure 8 . 61 Subfractionation of a pH 4.9, 0.05 M NaCl Extract of Fraction IV-1 by Dilution With water 0 O O O O O O O O O O O O O I O O O O 64 Protein Redistribution and Factor XI Activ— ity Resulting from the Extraction of Fraction IV-1 With Water. . . . . . . . . . 67 Redistribution of Factor IX by the 2 Tech- niques of (NH4)2SO4 Addition for the Frac- tionation of Effluent 4 . . . . . . . . . . 75 Recovery of Protein and Factor XI Activity Resulting from the Purification Procedure Developed in this Study . . . . . . . . . . 75 A Comparison of the Protein Distribution in Normal Human Plasma, Fraction IV-1 and a Purified Factor XI Preparation. . . . . . . 76 LIST OF TABLES - Continued TABLE Page XII. The Capacity of Different BaSO4 Preparations to Adsorb Factor XI . . . . . . . . . . . . 78 XIII. Thermostability of Purified Factor XI in pH 7.2 Citrate Buffer, Ionic Strength 0.15 . . 79 vi LIST OF FIGURES FIGURE 10. A current and tentative mechanism for the conversion of prothrombin to thrombin in human plasma via the intrinsic clotting system. . . . . . . . . . . . . . . . . . . . Thromboplastin generation curves resulting from TGT reagents prepared from a patient' with a hemorrhagic tendency and the corrective effect of normal reagents . . . . . . . . . . Thromboplastin generation curves resulting from the replacement of the TGT platelet re- agent with calf brain platelet substitute (P-S) and factor V (AcG). . . . . . . . . . . Thromboplastin generation curves demonstrat- ing the effect of replacing the TGT normal plasma and serum reagents with purified clot- ting reagents . . . . . . . . . . . . . . . . Thromboplastin generation curves with TGT re- agents from a factor XI (PTA)-deficient patient and the corrective ability of normal TGT reagents and purified factor XI reagent . Thromboplastin generation curves obtained with purified clotting reagents and varying quanti- ties of purified factor XI reagent. . . . . . Diagramatic representation of the preparation of fraction IV-1 from normal human plasma by Cohn's method 6 . . . . . . . . . . . . . . . Free moving boundary electrOphoretic pattern of a Cohn plasma fraction IV-1 obtained in a Klett-Tiselius apparatus. . . . . . . . . . . The scheme for the purification of factor XI from Cohn's fraction IV-1 . . . . . . . . . . Free moving boundary electrOphoretic pattern of a preparation of purified factor XI obtain- ed in a Klett-Tiselius apparatus. . . . . . . vii Page 18 44 47 50 55 56 59 61 74 76 I. INTRODUCTION The reactions involved in blood coagulation are not as yet completely understood for they involve many proteins, called clotting factors, where one serves as the substrate for another. Few of these clotting factors have been iso- lated in purified form; most have been studied in crude reagents. It is in this latter category that factor XI (plasma thromboplastin antecedent, PTA) must be classified. This component has only recently been accepted as a partici- pant in the coagulation scheme by most investigators, but some still doubt its existence (105). The advantages of a purified protein over a crude one for eliciting its mole— cular structure and biological function are many. Thus there exists a great need for a means of preparing purified (factor XI. Because of heredity, certain individuals are deficient in factor XI. These persons face extensive blood loss and even exsanguination following trauma or surgical procedures. At present, the deficiency is corrected by the intravenous administration of whole blood or plasma, but often such therapy is more harmful than beneficial. The administration oflarge quantities aggravate internal hemorrhaging. Furthermore, by using whole blood or plasma, which is in great demand for other clinical applications, the already short supply is diminished. What is needed to serve as a substitute is a concentrate of factor XI, depleted of other plasma proteins. It was the purpose of this study to devise a method for isolating factor XI to be used for studying its bio- chemical role in coagulation and in quantities sufficient for clinical application. A readily available "waste" product from fractionated human plasma (Cohn's fraction IV-1), in which factor XI is already concentrated, was chosen as the starting material. In order to follow the progress of purification of factor XI, a means of determining its potency at all stages of purification had to be devised. Heretofore, available assays have involved specific factor XI-deficient patients' plasma which is difficult to locate and unstable on storage. An improved assay had to be formulated with easily obtain— able materials from normal sources. With this objective in mind, as well as the ultimate isolation of factor XI, this investigation was carried out. II. HISTORICAL The maintenance of normal hemostasis in the human blood circulatory system requires the delicate integration of three distinct mechanisms: the vascular mechanism, the platelets, and the clotting mechanism.4‘ A degree of im- portance cannot be assigned to any of these mechanisms for their participation is dependent upon the extent of an injury, the size of the traumatic vessel, and the pressure of the blood flowing through the vessel. For capillaries and small venules, the vascular mechanisms may be sufficient to arrest a hemorrhage. For somewhat larger vessels, plate- lets assist the vascular mechanism. But in cases of extensive trauma or injury to the large blood vessels, the formation of a fibrin clot, the result of the clotting mechanism, may be the deciding factor in returning the circulatory system to normal hemostasis. The malfunctioning of any one mechanism does not usually result in a bleeding tendency. Conversely, spon- taneous bleeding is often the result of the abnormal func- tioning of more than one of the mechanisms. 1Throughout the course of this dissertation a consider- able terminology of coagulation will be used and to aid in familiarization a glossary of terms has been provided in the Appendix. A. The Vascular Mechanism The response of the vascular mechanism to trauma is not well defined, but its action appears to be as necessary for the control of bleeding as the other mechanisms. Vaso- constriction of short duration at the site of injury is brought about through an axonic reflex, resulting in the reduction or complete stopping of blood flowage through the vessel (117). Intimal endothelial cells, pressed together by the contraction, develop a stickiness and the resulting adhesion is sufficient to seal capillaries and often larger vessels. B. The Platelets Platelets are anuclear cell fragments of megakaryocytic cytoplasm which vary considerably in size and shape. The majority are 2 to 4 micra in width. Normal blood contains between 250,000 and 500,000 platelets/mm3 and 60,000/mm3 or more are required for normal hemostasis (16,78). IndiSpensi- ble for coagulation, platelets must be present not only in sufficient number, but must possess normal activity. Instances of thrombocytoasthenia,in which the platelet count is normal but in which the platelets are qualitatively deficient, have been observed (114). The agglutination of platelets at the point of dis- continuity in a vascular wall mechanically assists endothelial adhesion by forming a plug (115), but the constituents re- leased by disrupted platelets are perhaps of greater conse- quence. Among these-are serotonin (5-hydroxytryptamine) (124) which effects persistent vasoconstriction, retracto— enzyme which mediates the shrinking of the fibrin clot, and the factors that participate in the clotting mechanism (58). Clumped platelets also act as the nuclei upon which the fibrin clot is built. C. The Blood Clotting Mechanism 1. The Classical Theory The classical theory or mechanism for the coagulation of blood was postulated by Morawitz (68) and by Fuld and Spiro (52) in 1904. A two step process, enzymic in nature, was visualized. It was hypothesized that the cells of an injured blood vessel released the enzyme thrombokinase (thromboplastin) which, in the presence of calcium, brought about the conversion of prothrombin to thrombin. In turn, the enzyme thrombin converted fibrinogen to fibrin. This may be represented by the following equations: Tissue‘ Step 1. PROTHROMBIN ThromgggiaStIHP~ THROMBIN Thrombin Step 2. FIBRINOGEN %‘ FIBRIN 2. Revisions of the Classical Theory Even when the classical theory was proposed it met with objections because platelets were not taken into con- sideration for Bizzozero (15) had previously demonstrated that platelets were involved in blood coagulation. A revision of the theory was necessitated when Collingwood and MacMahon (22) showed that blood drawn in a manner which avoided the inclusion of tissue juices was capable of clotting at a rate comparable to that brought about by tissue thromboplastin. This observation led to a distinction between the thromboplastin released by injured tissue cells (extrinsic thromboplastin) and the thrombo- plastin formed by plasma constituents (intrinsic thrombo- plastin) (65,66). Brinkhous (17) deduced that platelets contained a thromboplastin comparable to that found in tissue cells and that its liberation required a lytic factor present in plasma which he termed thrombocytolysin. Quick (85), however, found that platelets yielded little material capable of converting prothrombin to thrombin. He concluded that plasma thromboplastin evolved from the union of a platelet component with a plasma factor. Quick named the plasma constituent thromboplastinogen. Thrombocytolysin and thromboplastinogen have become synpnyms for the same clotting factor. Today, this component is most frequently referred to as factor VIII or antihemophilic globulin (AHG). A "bleeder"-was called a hemophiliac and the syndrome hemophilia was believed to be due to the absence of factor VIII in the plasma. Much of the confusion now existing in coagulation literature has originated from the enormous diversity of terminology. In an effort to alleviate this problem, the International Committee for the Nomenclature of Blood Clot— ting Factors has assigned Roman numerals to the accepted factors (125) with the hope that they will be adopted universally. This nomenclature, with the most common synonyms, is presented in Table 1. 5. Hemophilia an Ambiguous Term The term "hemophilia," which described a bleeding syndrome and carried the connotation of a factor VIII (AHG) deficiency, became inadequate by 1952 when Aggeler gt_al. (5) and Biggs g£_al. (14), demonstrated that for intrinsic thromboplastin to be elicited, a second clotting component hadto be supplied by plasma. The newly discovered factor was called plasma thromboplastin component (PTC, factor IX) and Christmas factor by the respective investigators. The clinical symptoms of individuals lacking this component, and the hereditary mode of transmission were found indis- tinguishable from factor VIII deficiency. In 1955, Rosenthal, Dreskin and Rosenthal (95) re- ported the discovery of a previously unrecognized clotting 8 Table I. The Most Common Synonyms for the Blood Clotting Factors Most Common Synonym Symbol Other Synonyms s Fibrinogen Factor I Prothrombin Factor II Serozyme Thromboplastin Factor III Tissue Thromboplastin, Extrin- sic Thromboplastin, Extrinsic Prothrombin Activator, Thrombo- . kinase, Thromboplastic Cell Component Calcium Factor IV Accelerator Factor V Accelerin, Labile Factor, Pro- Globulin (AcG) thrombin Accelerator, Serum Accelerator, Thrombogen. Serum Prothrombin Factor VII Proconvertin, Stable Factor, Conversion Accelerator (SPCA) Antihemophilic Globulin (AHG) Plasma Thromboplastin Component (PTC) Stuart Factor Plasma Thromboplastin Antecedent (PTA) Hageman Factor Factor VIII Factor IX Factor X Factor XI Factor XII Cothromboplastin, Autopro- thrombin I, Prothrombinogen Antihemophilic Factor (AHF), Platelet Cofactor I, Plasma Thromboplastic Factor A, Thromboplastinogen, Thrombo- cytolysin, Thromboplastic Plasma Component Christmas Factor, Autopro- thrombin II, Platelet Cofactor II, Antihemophilic Globulin B, Plasma Thromboplastic Factor B Stuart—Prower Factor Plasma Thromboplastic Factor C Glass Factor factor and named this third plasma constituent plasma thromboplastin antecedent (PTA, factor XI). Their finding stemmed from an investigation of a hemorrhagic condition in three patients-‘two sisters-and their maternal uncle. The inability of the plasma of these patients to mutually correct each other confirmed a similar deficiency. Clotting tests performed on Rosenthal's patients indicated inade- quate intrinsic thromboplastin formation, and the ability of their plasmas to correct known factor VIII-deficient and factor IX-deficient plasmas proved that factor XI was not one of these components. Unlike factor VIII and factor IX deficiencies, the absence or grossly reduced level of factor XI in plasma was found to occur in both males and females, with . hemorrhagic manifestations ranging from very mild to moderately severe. It was found that afflicted individuals rarely develop spontaneous bleeding but encounter diffi- culties following trauma or surgical procedures. Factor XI deficiency is inherited as an incomplete Mendelian dominant trait, transmitted by male or female to either male or female progeny. An unusual aSpect of this transmittance was that different degrees of the defect were found within the same family (85,96). A fourth thromboplastin component, the Hageman factor (factor XII), was discovered (90). Although the Hageman factor has been shown to be a participant in the formation 10 of intrinsic thromboplastin, unlike the other thromboplastic factors, its absence from plasma is of no clinical signifi- cance (51). 4. Clotting Factors Involved in the Formation of the Extrinsic Prothrombin Activator a. Factor III (Tissue Thromboplastin) Schmidt (101) first demonstrated the action of tissue (extrinsic) thromboplastin on prothrombin. Morawitz (68) named the agent thrombokinase, believing its activity was catalytic (enzymatic). Howell (42) introduced the name thromboplastin, and suggested that its composition was that of a lipid in association with a protein. Nemerson and Spaet (69) found that the lipid and protein moieties of brain thromboplastin were 2 separable clotting activities, one enzymic and the other a phospholipid. The most active tissue thromboplastins have been pre- pared from lung (20), brain (44), and placenta (28). The active material can be completely sedimented by centrifuging at 10,000 RPM (20). b. Factor V (Accelerator Globulin) Nolf (71) observed that a mixture of tissue thrombo- plastin and calcium rapidly converted the prothrombin in plasma to thrombin but when the tissue thromboplastin- calcium mixture was added to partially purified prothrombin, the conversion took place very slowly. To explain this, he postulated the existence of a thromboplastin plasma cofactor, 11 thrombogen. Quick (82) noticed that the clotting time of oxalated plasma increased on storage and demonstrated that fresh, prothrombin-free plasma corrected the defect. In so doing, he confirmed the existence of Nolf's cofactor, but renamed this plasma component labile factor. Several years later, Owren (76) described a patient with a congenital bleeding tendency caused by the absence of this factor. He called this missing substance factor V, its most accept— able name at present. Ware, Guest and Seegers (121) suggested that factor V, which they called accelerator globulin, enhanced the activation of prothrombin by thromboplastin and calcium. Recently Seegers (108) reported that this factor determines the site at which the prothrombin molecule is cleaved by thrombo- plastin. In the presence of factor V, thrombin is produced; in its absence, a derivative of prothrombin is formed which is incapable of clotting fibrinogen. Both Seegers and Owren suggest that the factor exists in plasma as a precursor and is activated during the course of coagulation. c. Factor VII (Serum Prothrombin Conversion Accelerator) In 1948, Owen and Bollman (75) noted that serum cor- rected the extended clotting time of plasma from patients receiving dicoumarin, a compound known to inhibit the synthesis of prothrombin. Since prothrombin and factor V are not found in serum (both having been utilized in the course of clotting), a second thromboplastin cofactor was 12 postulated. Independently, Alexander et al. (5) also demonstrated the absence of this factor in dicoumarin— treated patients and named the factor serum prothrombin conversion accelerator (SPCA). Koller, Loeliger and Duckert (46) showed that this clotting factor, which they called factor VII, could be removed from normal plasma by filtration through a Seitz (asbestos) pad. Factor VII, the presently preferred nomenclature, has been determined to be a B-globulin present in both plasma and serum (75) from which it can be removed by adsorption onto BaSO4, alumina or asbestos. It accelerates the con- version of purified prothrombin to thrombin in mixtures containing tissue thromboplastin, factor V (AcG) and cal- cium, but apparently is not necessary for producing intrinsic thromboplastin. Factor VII, like factor V, is thought to be formed from a precursor during clotting (74). d. Factor X (Stuart Factor) By reinvestigating the clotting disorder in a patient (R. Stuart) previously diagnosed as hypoproconvertinemic (factor VII-deficient), Hougie, Barrow and Graham (41) discovered a new clotting component that is involved in the formation of thromboplastin. The investigators referred to this substance as Stuart factor. Stuart factor and the clotting component previously proposed by Duckert et al. (27) (Factor X), are now considered identical. Factor X proved to be different from Factor VII (SPCA) when it was 15 found that Stuart's plasma contained the component missing from a factor VII-deficient plasma. Many properties of factor X and factor VII are similar. Both are adsorbed by BaSO4, alumina and Seitz filters and the plasma concentration of both is diminished in coumarin- treated or vitamin K-deficient patients.’ Neither is fully consumed by the coagulation process. 5. Mechanisms Involved in the Formation of the Extrinsic Prothrombin Activator The precise function of the many clotting components involved in the formation of the extrinsic prothrombin activator is unknown, but their place in the clotting scheme has been postulated. Hardisty (55) determined that factors VII (SPCA) and X (Stuart factor) acted as prothrombin conversion accelerators while factor V (AcG) influenced the quantity of thrombin formed. Hjort (58) hypothesized the formation of a complex between factor VII, extrinsic (tissue) thromboplastin and calcium ions. Hougie (40) found that factor VII controlled the speed of formation of the complex (had enzymatic proper- ties) while factor X, acting as a substrate, affected the amount of complex formed. Straub and Duckert (115), in- vestigating the sequence of reactions, demonstrated the existence of two distinct steps; first, the formation of a complex involving tissue thromboplastin, factor VII and calcium, which they called extrinsic reaction product; 14 and second, the union of the reaction product with factor V. A scheme depicting the conversion of prothrombin to thrombin by the extrinsic thromboplastic system may be represented thus: FACTOR III + FACTOR X + CALCIUM (Tissue Thromboplastin) (Stuart Factor) Step 1. FACTOR VII (SPCA) V EXTRINSIC REACTION PRODUCT Step 2. FACTOR v (AcG) EXTRINSIC PROTHROMBIN ACTIVATOR (Extrinsic Thromboplastin) PROTHROMBIN > THROMBIN 6. Mechanisms Involved in the Formation of Intrinsic Prothrombin Activator From the foregoing discussion about extrinsic thrombo— plastin, it can be seen that the term "thromboplastin" implies an activity rather than a chemical entity. Because thromboplastin originally meant a substance extracted from tissue, and still bears this connotation, its use in the intrinsic Clotting scheme, in which tissue thromboplastin is not involved, is misleading. Nevertheless, the prothrombin converting agent derived from plasma constituents is still 15 most commonly referred to as intrinsic thromboplastin. A more fitting title, intrinsic prothrombin activator, was suggested (50), but has not found wide acceptance. Freshly drawn blood or recalcified plasma has no detectable amount of thrombin, but after 5-7 minutes a large amount suddenly appears. Observing this, Biggs (12) sug- gested that plasma contained all of the clotting components required to convert prothrombin to thrombin and that the formation of the substance which activated prothrombin re- quired a series of time consuming intermediate reactions. The nature of many of these intermediate reactions is still a matter of conjecture but sufficient evidence has been accumulated to permit the arrangement of the reactions in a currently regarded logical sequence. _2_vitro, blood clotting is initiated (see Fig. 1) by the adsorption of factor XII (Hageman factor) onto a "foreign" surfacelsuch as glass or kaolin which, by an un- known mechanism, transforms inactive factor XII to an active form (92). When in the adsorbed (activated) state, factor XII activates factor XI (PTA) by what is believed by some to be an enzymatic process (62). In turn, activated factor XI, with calcium as a cofactor, converts factor IX (PTC) from an inactive precursor to an active enzyme (91). 'llt is presumed that i2_vivo, tissue other than normal endothelium acts as the foreign surface. 16 For the next step, Bergsagel (10) suggested that factor VIII (AHG) enhanced an interaction between factor IX and calcium. Later, Bergsagel and Hougie (11) presented evidence that a complex involving factors IX, VIII, X (Stuart factor) and calcium was formed, and that this complex re- acted with platelets to yield what they called "product I," the immediate precursor of the prothrombin activator. The foregoing was resolved into individual steps, as given in Fig. 1, by Lundblad and Davie (56). They showed that activated factor IX, in the presence of phospholipid and calcium, reacted with factor VIII and converted the latter to an active product. Lundblad and Davie (57) also demonstrated that the function of activated factor VIII is to activate factor X. They determined that activated factor X would accelerate the clotting of factor X-deficient plasma but not of factor V (AcG)-deficient plasma. The activation of factor X by activated factor VIII has an absolute requirement for calcium ions. The subsequent reaction in this series of events has not been characterized by means of isolated products and reconstituted systems, but is regarded to involve the trans- formation of factor V to an active enzyme by activated factor X in the presence of platelet-derived phospholipids and calcium (11,57,59). Specifically, it is activated factor V which converts prothrombin to thrombin and which is referred to as thromboplastin. 17 A summary of the reactions-involved in blood clotting by the intrinsic-mechanism was presented by Macfarlane (60). who visualized the scheme as an "enzyme cascade,‘ and by Davie and Ratnoff (24) who, in an identical manner, viewed it as a "waterfall“ sequence. The-series of reactions lead- ing to the transformation of prothrombin to thrombin, as postulated by the latter investigators is presented in Figure 1. For the purpose of comparison, some of the properties of the clotting factors involved in the conversion of pro- thrombin to thrombin by the intrinsic thromboplastin pathway are given in Table II. A final acceptance is not intended for the above clot- ting scheme, for conclusions were based on experiments employing crude preparations of clotting factors—-isolated components are simply unavailable. Other clotting factors and/Or cofactors not yet discovered, or recognized factors present in preparations as contaminants, may have influenced the interpretations. In formulating the pathway many assumptions which have not been experimentally demonstrated were made. In this scheme, all of the clotting factors are considered to be enzymes which are present in blood as in- active precursors, an unproven assumption. Finally, the scheme ignores the proposal (104) that thrombin is of major importance in the conversion of prothrombin by an auto- catalytic process. For these reasons, the proposed mechan- ism must be regarded emphatically as a working hypothesis. "foreign" surface A XII (Hageman factor) Activated XII Fig. 1. A XI (PTA) Activated XI ////////C;;:I\\\\\\\\‘ IX (PTC) Activated IX phOSpholipid VIII (AHG) Activated VIII m X (Stuart factor) Activated X phOSpholipid V (AcG) Activated V ///////’E;::\\\\\\\\ II (Prothrombin) Thrombin A current and tentative mechanism for the con— version of prothrombin to thrombin in human plasma via the intrinsic clotting system (24). ll! gun-I Ounfi IIDI IO‘FII‘ .1. ~ Nas...‘ 9 1 wmmumnu Cmuommmm cmpomwwm Aawv Awav Cmuommmm Cmuomwwm HOHNEDOOHO no moamflOmec uoz uoz COODCOm COODCmm uoz uoz M cflamufl> CH HO>OH mammam Ammv Ammav HHH+HH au>H .HHH au>H .HHH Ammo H coauomum m.ccoo ea docmmmud Comouummp waouummp ucmmmum ucmmmum .CHE on .0 0mm um manmum haumoz haumoz Cmmonummn “oz #02 Ednmm CH wuflHmeumoauwze Anmv Awmv Ammv Admv Ammv ucmcHEOC O>Hmmmomu O>Hmmmomu O>Hmmmomu Awmv COHMMHE O>Hmmmumm muOHmEOOCH ODOHQEOOCH COMCHalxwm mecflalxom O>Hmmmuwm Imcmuu mo OCOS mumuflpmumm Ammv % Ammv aim Ammv d Ammv waldo Ammv mm Ammv >Im coflumumwz Uflumuonmouuumam Aomv 02 Lame mSom Aaev mmw Amv mew Ammo oz Ame OSom vommm ouco penu0mp< Aamoemmumm Aamceom-mm Apmveomumm Acmcemmuo coapchADomup acmmxvmzc “Nov 02 Ammv Oz ”Adv 02 Away 02 Ammv wow Awmv mow coflumasmmoo mcausp COEDOCOO Acmsmmmmo Adamo Auumsumv Loamv Aom Houomm > uouomm muouomm mcflquHO Oflummamonaoune OflmcfluucH mnu mo mmauummoum .HH Danae 20 7. Prothrombin to Thrombin Transformation Through the efforts of Seegers and many associates (105,109), prothrombin represents one of the most highly purified protein clotting factors. Prothrombin has a molecular weight of 68,500, an isoelectric point of pH 4.2, an $20 of 4.84, and electrophoretically migrates like an a2 globulin (50). Seegers discovered that purified prothrombin is trans- formed slowly to thrombin in a 25% sodium citrate solution (106). He submitted this finding as evidence that the pro- thrombin molecule contains all that is necessary for the formation of thrombin. Thrombin formed in this manner has a molecular weight of 8,000, making it the smallest proteo- lytic enzyme yet known (48). However, Alexander (4) pre- pared prothrombin free of factor VII (SPCA) and found that it would not yield thrombin on incubation with citrate, but did when factor VII was added. Although the prothrombin preparation used by Alexander was of higher specific activity than that of Seegers, it still exhibited evidence of con- tamination. Therefore, the formation of thrombin from pro- thrombin in citrate may be similar to the physiological conversion brought about by contaminant clotting factors whose concentrations were too low to detect. Thrombin is not found in normal blood but arises from the proenzyme prothrombin through the action of intrinsic or extrinsic prothrombin activators and calcium: 21 EXTRINSIC OR INTRINSIC PROTHROMBIN ACTIVATOR (Thromboplastin) PROTHROMBI N Ar‘ THROMBI N Opinion is divided still as to whether thromboplastin is a reactant or an enzyme. Mertz, Seegers and Smith (64) favor the reactions as stoichiometric and have shown that the quantity of thrombin produced from an excess of prothrombin is directly proportional to the quantity of added thrombo- plastin. Calcium likewise appears to act stoichiometrically (84). 8. The Formation of the Fibrin Clot Fibrinogen is the natural substrate for the highly specific, proteolytic enzyme, thrombin. Its concentration in normal human plasma has been found to range between 170 and 4001m;/100 ml. Fibrinogen has been isolated in purified form by a number of techniques, including salting out with (NH4)2SO4 at 25% saturation (67), with 11 volumes % ether at 00C (45), and with 6 volumes% ethanol at -20 (21). The molecular weight of fibrinogen has been estimated at 540,000 (18). Its sedimentation constant, Sgo,w, is 7.7-7.9 Svedberg units (100) and its isoelectric point is pH 5.5 (107). From X-ray diffraction studies, fibrinogen ap- pears to be a member of the myosin—keratin-epidermin group of proteins (8). 22 The formation of the insoluble fibrin clot from fibrinogen requires three reversible reactions (99): 1. Proteolysis: FIBRINOGEN ‘Ihrombln‘~ FIBRIN MONOMER . + FIBRINOPEPTIDES. 2. Polymerization: x FIBRIN MONOMERS.;===&F ELONGATED.POLYMER 5. Clotting: y ELONGATED POLYMERS Flégiggse FIBRIN 1’3""? It should be noted that thrombin participates in the firSt step only. Under the influence of thrombin, four peptides (fibrinopeptides) representing two Species of molecular weights 1900 and 2400 are released from the fibrinogen mole- cule (48). Since thrombin has been found capable of hydrolyzing p-toluenesulfonyl-L-arginine methyl ester (111), it was suggested that the cleavage of fibrinogen occurs at an arginyl-glycine bond (49). With the removal of the fibrinopeptide, which contains a negatively charged glutamic acid end group believed by Lorand (55) to impart stability to fibrinogen in solution, the fibrin monomers assume an end to end alignment and polymerize. Scheraga (99) regarded that the polymerization arose by the formation of intermolecular hydrogen bonds between unmasked donor groups and histidyl acceptors assumed to be present in the original fibrinogen molecule. Forma- tion of stronger covalent bonds was ruled out because the reaction readily reversed under relatively mild conditions, such as in concentrated urea solution (47). 25 Finally, a side by side alignment of the linear polymers takes place by means of a transamidation reaction between the N-terminal glycine amino groups of one fibrin monomer and the B—carboxyl group of asparagine of the adjacent fibrin monomer (19,54) to produce the firm fibrin clot. This reaction has been suggested to be mediated by the thrombin-activated enzyme, fibrinase (fibrin stabilizing factor, Laki-Lorand factor) in the presence of calcium ions. 9. Physical and Chemical Properties of the Blood Clotting Components An attempt was made to tabulate the physical and chemi- cal properties of the proteins involved in blood clotting which have been reported in the literature. The result is seen in Table III. It is apparent that much information is absent for most of these proteins, for they have yet to be isolated. It is hoped that this study, and other current investigations, will permit the determination of many of these properties. Ammv .Qoam b Acmammmmv HHx Ammo .Qon a u m Adamo Hx Ammo .mao ..mam H mm .Eumu Z Ammv .QOHm o Ammv ooo.>m Auumspmv x Ammo .noao Amoav .osao e ma do . do Amoav ooo.om AOPEV RH Ammo Aooc mammad cA a as m Aoaav m.¢ .Doao mo ARV ooo.ooa Loads HHH> m Amos lac ooo.mm .Doao o I o -ooo.ma Laudmv HH> Ammo .Qoam A one Amoco > ANOHV.Hmm H mm .Eumu O . Ammv.MHm H mm .EHOH Z Ammv Aimee mammad ca a me A Aome m.e Lowe o.e .Doao do Ammo oom.oo Acflneoucuoudv HH .oseo a m.m “mammad Aooac “was .noao ca a me ooenoea Aeoac m.m o.eue.e a u o lode ooo.oam Acooocflunamc H MOHumwumuumumso Hmnuo Hm_. 3.0mm m.m mm .D unmflmz HOHDOOHOS Houomm mafiuuoao muouomm mcauuoao COOHm Tau mo mmfluummonm HMOHEOSO Cam HMUHmwxm mEom .HHH OHQMB III. EXPERIMENTAL A. Apparatus Temperature Control—-Precision-Freas rectangular constant temperature water bath, Model 160, maintained at 570 1.0.10 C. Aminco 50 gallon constant temperature bath, Model 4-8615, filled with 40% (v/v) ethanol in water, main- tained at 00 1.0.10 C. Centrifugation--International refrigerated centrifuge, Model REF. Continuous flOw Sharples No. 16 refrigerated supercentrifuge- pH Measurement—-Beckman pH meter, Model G, with glass electrode. Absorbance Measurement-—Beckman spectrophotometer, Model DU, with quartz cells. Homogenization--Tenbroeck tissue grinder, 40 ml capacity Kimax Cat. No. 45950. Waring Blendor, single speed, with 1 liter capacity cup and cup cover. 'Speed regulated by a rheostat. 1 Time Measurement--Gallet fifth second stop watch. General Electric 20 second interval timer. Dialysis--Visking cellophane tubing. 25 26 B. Materials and Reagents 1. Chemical Reagents Chemicals--All inorganic and organic chemicals were of reagent grade unless otherwise specified. Diethylaminoethyly(DEAE) Cellulose--Floc DE-50 (Whatman), purchased from H. Reeve Angel and Company. Saline Solutions-—0.9% saline: 9 gm NaCl was dissolved in distilled water and made to 1 liter. 0.05 M NaCl: 2.92 gm NaCl was dissolved in distilled water and brought to 1 liter. 0.15 M NaCl: 8.77 gm NaCl was dissolved in distilled water and made to 1 liter. Calcium Chloride Solutions-~A 1 M stock solution of CaC12 was prepared by dissolving 111 gm in distilled water and adjusting the volume to 1 liter. Solutions of 0.020 M, 0.025 M and 0.050 M were prepared by pipetting the appro- priate amount of M CaClg stock solution into a volumetric flask and bringing the volume to the mark with distilled wat er . Trisodium Citrate Solutions--2%: 22.8 gm trisodium vcitrate°2H20 was dissolved in distilled water and made to 1 liter. For the 4% solution, twice this quantity was used. Potassium Oxalate Solution--1.4%: 14 gm potassium oxalate-H20 was dissolved in distilled water and brought to 1 liter. 27 pH 5.2 Acetate Buffer, Ionic Strength 0.05: 4.1 gm sodium acetate was dissolved in distilled water, made to 1 liter, and the pH adjusted to 5.2 with concentrated hydro- chloric acid. Ionic strength 0.15: The same procedure was followed using 12.5 gm sodium acetate. pH 7.2 Citrate Buffer, Ionic Strength 0.15: A 0.0275 M trisodium citrate solution was prepared and adjusted to pH 7.2 with concentrated hydrochloric acid. pH 8.6 Veronal Buffer, Ionic Strength 0.05: Prepared by dissolving 10.6 gm Barbital (N. F., Fisher Scientific Company) and 0.05 mole NaOH in distilled water and bringing the volume to 1 liter. Acid-Citrate-Dextrose Buffer--N. I. H., formula B. 1.52 gm trisodium citrate-H20, 0.44 gm anhydrous citric acid and 1.47 gm dextrose-H20 were dissolved in distilled water and made to 100 ml. 2. Biologic Reagents a. Calf-Brain Platelet Substitute (P-S) The preparation of calf-brain platelet substitute (P-S) was carried out at.room temperature of about 250 C. The brain matter of a freshly killed calf was ob- tained from a local slaughter house and freed of clots, arteries and encasing membranes. The tissue was washed with distilled water, cut into small chunks, suspended in 500 ml acetone and mascerated in a covered Waring blendor cup until 28 a fine paste-like suspension was obtained. (Caution: Avoid spillage of acetone--it may be ignited by motor). The mas- cerated tissue was recovered by centrifuging at 2,500 RPM for 15 minutes and the residue washed repeatedly with 500 ml of fresh acetone until a negative Liebermann-Burchard reaction (57) on a sample of air-dried residue indicated the absence of cholesterol. The remaining cholesterol-free residue was spread over a large filter paper and air-dried. A 1 gm sample of acetone-brain powder was stirred for 50 minutes with 50 ml chloroform and the extract passed through a Whatman No. 1 filter paper into a 100 ml beaker. The filtrate was evaporated to dryness by placing the beaker on a hot plate set at around 600. With the aid of a rubber policeman, the gummy residue was suspended in 50 ml 0.9% . saline and hand homogenized in a Tenbroech tissue grinder. The homogenized preparation was dispensed in 1 ml aliquots into small shell vials and stored in the deep freeze until needed. b. Purified Factor VIII (AHG) Reagent Factor VIII (AHG) was isolated from fresh normal human plasma according to method 6 of Cohn §£_§l. (21). Per liter of plasma, cooled to 00 C and under constant stir- ring, 177 ml of 55.5% ethanol at -50 mixed with 1 ml 80x concentrated acetate buffer, pH 4.0, was slowly added. The plasma was cooled to —50 during the ethanol addition and stirred for 50 minutes thereafter. The precipitate was 29 collected by centrifuging at -50 in a Sharples No. 16 supercentrifuge. The wet paste, known as'fraction I," was redissolved in 12 ml 0.025 M citrate buffer, pH 6.5, per gram paste, filtered through a Seitz K—5 filter pad and dried by lyophillization. The major protein constituent of fraction I is fibrinogen and this was removed by heat coagulation as follows. Two hundred mg of dried fraction I powder was dis- solved in 15 ml of 0.151d NaCl and the solution was heated in a 560 water bath for 5 minutes. By centrifuging at 2,500 RPM for 5 minutes the coagulated fibrinogen was removed. The supernatant containing factor VIII was dispensed in 2 ml aliquots into small test tubes and stored at —200. c. Purified Factor V (AcG) Reagent Bovine blood was collected without anticoagulant at a local slaughter house and permitted to stand at 250 C for 4 hours. This length of time was necessary for clot formation and retraction and for the destruction of the thrombin produced during the clotting process. The serum was decanted and centrifuged to remove residual red blood cells. Each 100 ml of clarified serum was processed as follows: The sample was batch adsorbed with 4 gm BaCOa powder (Bakers, C.P.) to remove factors II (prothrombin), VII (SPCA), IX (PTC) and X (Stuart factor). No Significant amount of factor V (AcG) was adsorbed upon the barium salt. The BaC03 with adsorbed proteins was removed by centrifug- ing at 2500 RPM for 50 minutes. 50 The supernatant was further treated by addition of freshly precipitated BaSO4. This was prepared by mixing 4 ml 1 M BaCla with 4 ml 1 M Na2804, diluting the mixture with 80 ml distilled water and collecting the precipitate by centrifuging at 2500 RPM for 15 minutes. The BaSO4 precipitate, to which factor V (AcG) is adsorbed, was washed with 200 ml 0.15 M NaCl; then eluted by suspending in 50 ml 2% trisodium citrate solution. The centrifuged eluate was dispensed in 2 ml aliquots into small shell vials and stored frozen. d. Purified Factor IX + X (PTC + Stuart Factor) Reagent Bovine serum was collected and clarified as described- under the preparation of purified factor V reagent. Each 100 ml of clear serum was stirred with 100 mg BaSO4 powder (Baker, C.P.), at 250 C. The BaSO4 with adsorbed protein was centrifuged at 2,500 RPM for 50 minutes, washed twice with 70 ml aliquots of 0.01 M sodium oxalate, and the pro- . tein was eluted with 10 ml 2% trisodium citrate. The eluate, containing factors VII (SPCA), IX (PTC) and X (Stuart factor), was dialyzed against 4 liters, 0.15li NaCl at 2-40 for 24 hours to remove citrate and then to remove the finely dis- persed BaSO4 particles was Centrifuged at 10,000 RPM for 1 hour at 20. The reagent was dispensed in 4 ml aliquots in small shell vials, frozen and stored at -209. 51 e. The Substrate Plasma The substrate plasma supplies prothrombin and fibrinogen for the latter phases of coagulation. A normal ACD anticoagulated plasma was used in preference to an oxalated plasma. On recalcification, the calcium oxalate precipitate which forms in oxalated plasma clouds the solu- tion, making the detection of the end point (the fibrin clot) difficult. When needed, human blood for the preparation of the substrate plasma was donated, a pint at a time, by the Lansing Regional Red Cross Center, Lansing, Michigan. Vacuum sealed bottles (Baxter Laboratories) containing 110 ml ACD (NIH solution B) as the anticoagulant were used for collecting 440 ml blood. The anticoagulated blood was centrifuged 1 hour at 2500 RPM at 2-40 C after which the plasma was siphoned off, care being taken to avoid the inclusion of cellular com- ponents. An average of 250 ml plasma was derived from one unit of blood. The plasma was dispensed in 4 ml aliquots into shell vials, frozen and stored in a deep freeze. C. Methods Employed for Determining Clotting Abnormalities 1. General Considerations A prerequisite to the isolation and characterization of a clotting factor is a means by which the concentration 52 or potency of that factor can be ascertained. It is un- fortunate that of the many reactions which comprise the clotting mechanism, the formation of the fibrin clot is the only visible evidence of coagulation. The appearance of the fibrin gel has been adopted almost exclusively as the indicator of clotting activity in the numerous assays which have been devised for both qualitative and quanti- tative determinations of most of the clotting components. In these systems, therefore, the activity of the component under investigation is studied with respect to its effect on all the other clotting components. Existing assays for the many Clotting factors and for evaluating the various phases of coagulation are too numerous to describe here. However, a brief description of a few of these should be of value as an introduction to the principles upon which most of the assays are based. 2. The Calcium Clotting Time of Plasma An evaluation of the efficacy of an individual's clotting mechanism can be as simple as the observation of the time required for his blood to clot (51). A refined variation of this test is the Calcium Clotting Time. For this determination, 9 volumes of blood is collected in 1 volume of 4% trisodium citrate. The anticoagulated blood is centrifuged at a relatively low speed (1,500 RPM, 5 minutes) to prevent platelet sedimentation and the platelet- rich plasma withdrawn. A 0.1 ml aliquot of this plasma is 55 mixed with 0.1 ml 0.15 M NaCl and coagulation is initiated by the addition of 0.1 ml 0.025 M CaClg. At 570 C, normal plasma will clot between 1.5 and 4 minutes. A clotting time exceeding 4 minutes indicates a complete or partial deficiency of one or more of the clotting components. By this assay, no single clotting factor nor even the errant phase of coagulation can be incriminated. 5. The One—Stage Prothrombin Time Once the presence of a clotting abnormality in a plasma is established, other tests which are more indicative of the specific defect can be employed. The One-Stage Prothrombin Time (Quick Time) (81) is an example. This test consists of adding a mixture containing 0.1 ml tissue thromboplastin (prepared from brain, lung or placenta) and 0.1 ml 0.02 M CaC12 to 0.1 ml of a patient's anticoagulated plasma. ° The addition of tissue thromboplastin and calcium induces coagulation by the extrinsic clotting mechanism, a mechanism which does not involve factors VIII (AHG), IX (PTC), XI (PTA), XII (Hageman factor) and platelet factor 5. Normal plasma will clot between 10 and 20 seconds, dependh ing upon the source of tissue thromboplastin and the con- ditions of assay. Longer clotting times indicate that one or more of factors I (fibrinogen), II (prothrombin), V (AcG), VII (SPCA) or X (Stuart factor) is deficient. On the other hand, if the plasma exhibits a normal One-Stage 54 Prothrombin Time but an abnormal Calcium Clotting Time, one or more of the intrinsic thromboplastic factors (VIII, IX, XI, XII, platelet factor 5) is deficient. 4. The Partial Thrombgplastin Time Test Another type of assay takes advantage of genetically induced clotting abnormalities which are found among the populace. Because of heredity, certain individuals are in- capable of synthesizing a given clotting factor but do possess a full comphément of all other clotting components. The plasma from these individuals is employed in an assay called the Partial Thromboplastin Time Test for determining the concentration of the missing factor which may be present in other plasmas and/or plasma derivatives (70,88). A deficient plasma, such as might be obtained from a hemophiliac, is dispensed in 0.1 ml aliquots into small test tubes. Dilutions (usually 1:10, 1:20, 1:50 and 1:100) of a normal plasma in physiological saline are prepared and 0.1 ml aliquots are added to the tubes containing the deficient plasma. Calcium Clotting Times are then determin- ed by the addition of 0.1 ml 0.025 M CaClg to each tube. When sample dilution vs. clotting time is plotted on log-log graph paper, a "normal" straight-line curve is obtained. In the same manner, dilutions of an unknown plasma or plasma derivative are prepared, added to the deficient plasma, and their Calcium Clotting Times determined. The potency of the unknown is then estimated by comparing a curve of its clotting times with that of the normal plasma. 55 It might appear that this procedure presents an ex- cellent means for assaying a clotting factor. In reality the method has many shortcomings. Plasmas with specific deficiencies are rare, making them difficult to obtain and once procured are not stable. On storage, factors other than the one initially absent deteriorate and the plasma soon responds to more than one clotting factor. 5. The Thromboplastin Generation Test-— Reagents and Procedure The Thromboplastin Generation Test (TGT) as proposed by Biggs and Douglas (15) was designed to differentiate between factor VIII (AHG), factor IX (PTC) and platelet factor 5 deficiencies. Its usefulness as a clinical tool has been evaluated and confirmed by many investigators (112,65,66). This test is conducted such that the first phase of coagulation, the formation of intrinsic thrombo- plastin, is separated from the latter phases; namely, thrombin and fibrin formation. The generation of thrombo- plastin is dependent upon the presence of certain clotting factors in 5 reagents prepared from human blood: i) serum, ii) platelets, and iii) BaSO4-adsorbed plasma. To achieve this and yet avoid complications arising from the presence of tissue thromboplastin, blood is collected by the 2-syringe technique1 and processed into these reagents as follows: lA needle of relatively large bore is inserted into a vein and blood drawn into a syringe. Leaving the needle in the vein, the syringe is removed and discarded and a second syringe attached to the needle for collection of the sample. 56 i. Serum Reagent--A portion of the withdrawn blood is permitted to clot and, on incubation at 570 for 1 hour, the serum which exudes after clot retraction is collected and centrifuged to remove residual red blood cells. The centrifuged serum, diluted 1:10 with 0.9% NaCl, constitutes one of the thromboplastic reagents. ii. Platelet Reagent-~The remaining blood is antico- agulated with 1.4% potassium oxalate (nine parts of blood to one part of oxalate) and low-speed centrifuged to re— move its cellular components. The platelet-rich plasma is recentrifuged at a high speed to sediment the platelets. The platelet pellet is washed twice with 0.9% saline and then suspended in a quantity of saline equivalent to one- third the volume of the centrifuged plasma. The platelet clotting factor is liberated by freezing and thawing the platelet suspension. iii. Adsorbed Plasma Reagent--A portion of the platelet- poor plasma is adsorbed with BaSO4 powder (40 mg BaSO4/ml plasma) to remove prothrombin and factor IX (PTC). after which the BaSO4 adsorbent is removed by centrifuging. This adsorbed plasma, diluted 1:5 with 0.9% saline, comprises the third thromboplastic reagent. The remainder of the platelet-poor, unadsorbed plasma may be employed as the substrate for providing the clotting components prothrombin and fibrinogen for the latter phases of coagulation, but any normal plasma will suffice. 57 The TGT reagents are by no means pure nor even of predictable content, but normal adsorbed plasma can be relied upon to supply factor VIII (AHG); normal serum, factor IX (PTC); and normal platelets, platelet factor 5 whereas hemorrhagic patients could be deficient in any one of these or other factors. A list of all of the clotting factors known to be present in normal plasma and in each of the TGT reagents 515 presented in Table IV. iv. Thromboplastin Generation Test Procedure--An "incubation mixture" consisting of 0.5 ml platelet suspension, 0.5 ml adsorbed plasma, 0.5 ml serum and 0.5 ml 0.025 )4 CaC12 is prepared and incubated at 570. At 1 or 2 minute intervals, 0.1 ml aliquots are withdrawn and added, together with 0.1 ml 0.025 M CaClg, to 0.1 ml warmed substrate plasma and a Calcium Clotting Time determined. With TGT reagents prepared from normal plasma, a minimum Clotting time of 8-14 seconds can be expected after an incubation time of 4-5 minutes, whereas reagents prepared from factor VIII (AHG), factor IX or platelet factor—deficient plasma re- quires a considerably longer time to clot. The minimum clotting times are related to the amount of thromboplastin which is generated in the incubation mixture. When abnormal thromboplastin generation is detected in a patient‘s TGT reagents, the missing factor may be ascertained by substituting normal TGT reagents, one at a time, for the patient's reagents. For example, if a 58 Table IV. Clotting Factors Present in Normal Plasma and in Thromboplastin Generation Test Reagents Prepared from Normal Blood Platelet- Platelet- BaSO4- Clotting Rich Poor Adsorbed Plate- Factor Plasma Plasma Plasma Serum lets I (Fibrinogen) + + + - _ II (Prothrombin) + + — _ _ III (Tissue Thromboplastin) - - - _ - V (Accelerator Globulin) + + + _ + VII (SPCA) + + - + .. VIII (Antihemophilic Globulin) + + + - _ IX (PTc) + + _ + _ X (Stuart Factor) + + — + _ XI (PTA) + + + + _ XII (Hageman Factor) + + . + + _ Platelet Factor 5 + - _ - + 59 patient lacked factor IX (PTC), substituting a normal ad- sorbed plasma for the patient's adsorbed plasma, or a normal platelet suspension for the patient‘s platelet suspension would not lower the clotting times into the normal 8-14 second range, for these reagents do not contain factor IX. When the patient's serum is replaced by a normal serum (which does contain factor IX), the defect is eliminated and normal thromboplastin generation, as indi- cated by a normal minimum clotting time, is observed. An interpretation of the clotting deficiencies which may be determined by using the TGT is presented in Table V. The TGT has definite limitations which restrict its usefulness. Deficiencies of the thromboplastic clotting components other than factor VIII (AHG), factor IX (PTC) and platelet factor 5, namely, factors V (AcG), X (Stuart factor), XI (PTA) and XII (Hageman factor), cannot be de— tected. Furthermore, the test is not quantitative and TGT reagents prepared from the blood of a mildly deficient patient often do not show the defect. D. Development of the Procedure for Assaying Factor XI (PTA) An ideal clotting factor assay might well be one in which all of the required clotting factors, except the one under consideration, are supplied as single components in optimal concentration. At present, few clotting factors 40 Table V. A Summary of the Effect of Thromboplastin Gener- ation Test Reagents on Clotting Time and the Deficiency Indicated TGT Reagents Deficient Adsorbed Clotting Clotting Platelets Plasma Serum Time Factor Normal Normal Normal Normal None Patient Patient Patient Abnormal Patient Normal Patient Normal Factor VIII Patient Patient Normal Abnormal (AHG) Normal Patient Patient Abnormal Patient Patient Patient Abnormal Patient Normal Patient Abnormal Factor IX Patient Patient Normal Normal (PTC) Normal Patient Patient Abnormal Patient Patient Patient Abnormal Patient Normal Patient Abnormal Platelet Patient Patient Normal Abnormal Factor 5 Normal Patient Patient Normal 41 have been purified to this extent. In devising an assay for factor XI (PTA) such a system was kept in mind, but the ideal condition of complete purity could not be achieved. 1. Considerations Concerning the Thromboplastin Generation Test Factor XI (PTA) being predicated in this study as one of the essential components for the formation of intrinsic thromboplastin, it was felt that its determination could best be accomplished by following its effect on the generation of thromboplastin. The Thromboplastin Generation Test (TGT), which isolates the initial phase of clotting (thromboplastin formation), seemed best suited for this purpose. Without modification, the TGT could not be used for assaying many of the intrinsic thromboplastin—forming clot- ting factors because these are found in more than one of its thromboplastic reagents (see Table IV). In order to determine factor XI (PTA), the three reagents--serum, platelets, and adsorbed plasma—-would have to be replaced by improved reagents which supplied the required clOtting factors, but yet were devoid of factor XI (PTA). If this could be accomplished, the assay method would be capable of quantitatively measuring factor XI (PTA) as well as factors V (AcG), VIII (AHG), IX (PTO), and platelet factor. The substitution of purified reagents for the TGT reagents was carried out in this study and their contribution toward the formulation of a factor XI assay system will be discussed. 42 2. Directions for a Modified TGT Procedure A modification of the TGTl which simplified the physical manipulation of the assay was incorporated into this investigation. This consisted of adding sufficient CaClg to the incubation mixture to provide for both thrombo- plastin generation and clot formation when an aliquot of this mixture was transfered to the substrate plasma. This variation eliminated the difficult task of having to add 2 solutions (CaC12 and incubation mixture) Simultaneously to the substrate plasma. The TGT reagents were prepared as described previouSr ly and held in an ice-water bath until needed. Those re- agents which were stored in the frozen state were thawed in a 570 water bath and likewise held at near 00. The incubation mixture was assembled by pipetting into a 15 x 87 mm test tube, 0.5 ml of disrupted platelet sus- pension, 0.5 ml adsorbed plasma (diluted 1:5 in 0.9% saline) and 0.5 ml serum (diluted 1:10 in 0.9% saline). The mix- ture and six 11 x 77 mm test tubes, each containing 0.1 ml substrate plasma, were separately placed in a temper- ature—controlled water bath at 570. Two minutes was allowed for the solutions to reach bath temperature, then 1.0 ml 0.02 M CaClg at 570 was rapidly transferred to the incubation 1This modification was suggested by Dr. W. W. Zeulzer and Dr. J. Rutzky, Children's Hospital, Detroit, Michigan, to whom we are most appreciative. 45 mixture and a clock started. At intervals of 1 or 2 minutes after addition of calcium, 0.5 ml aliquots were withdrawn and quickly blown into a tube of substrate plasma. The con- tents were thoroughly mixed by tapping the bottom of the tube and the tube returned to the 570 bath. Shortly before the anticipated time of gel formation, the tube was taken from the bath and tilted back and forth until a clot was observed. The time elapsing between the addition of the incubation mixture to the substrate plasma and the formation of the clot was measured with a stop watch and regarded as the clotting time (C.T.). That this modified assay in no way altered the characteristics of the TGT was demonstrated by determining the clotting defect in an individual with an hereditary deficiency. Figure 2 is the graph of C.T. vs. incubation time for such a patient (L.S.). The patient's TGT reagents generated little thromboplastin (curve 1). Replacement of the patient's serum with serum from a normal person was of no benefit (curve 2). But the abnormality was fully corrected either by replacing the patient's adsorbed plasma with a normal adsorbed plasma (curve 5), or by the addition of purified factor VIII (AHG) to the patient's TGT reagents (curve 4). With these data the patient was diagnosed as a hemophiliac with a severe factor VIII (AHG) deficiency. 60'- 50'- Seconds 50 — 20 — Clotting Time, —— 44 l 1 J 1 L J I 1 1 J 2 5 44 5 6 7 8 9 10 11 Incubation Time, Minutes Fig. 2. Thromboplastin generation curves resulting from TGT reagents prepared from a patient with a hemorrhagic tendency and the corrective effect of normal reagents. In all curves, constant. Curve Curve Curve Curve (A 01 to P 0.5 ml patient's platelet reagent was held 0.5 ml patient's adsorbed plasma; 0.5 ml patient's serum. 0.5 ml patient's adsorbed plasma; 0.5 ml normal serum. 0.5 ml normal adsorbed plasma; 0.5 ml patient's serum. 0.5 ml patient's adsorbed plasma; 0.5 ml patient's serum, 0.1 ml purified factor VIII (AHG) reagent. Note that the addition of purified factor VIII to the patient's TGT reagenbscorrects the deficiency. 45 5. Replacement of the TGT Platelet Reagent Platelets have been reported as providing 4 factors essential to blood clotting (26). Platelet factor 1 accelerates the conversion of prothrombin to thrombin (120). In this respect, the factor behaves like factor V (AcG). Hjort, Rapaport and Owren (59), have provided evidence that this platelet factor is indeed nothing more than factor V of plasma adsorbed onto the platelet surface. Platelet factor 2 reportedly enhances the formation of fibrin from fibrinogen (120). Many unrelated plasma constituents exhibit similar activity, thus casting doubt on the validity of this factor. Platelet factor 4 has the ability to neutralize heparin, the naturally occurring anticoagulant of plasma. This factor assists clotting in vivo, but is of no conse- quence in in vitro assays. The final constituent provided by platelets, factor 5, is an essential participant in the formation of intrinsic thromboplastin. The chemical nature of this factor has been under extensive investigation (61,72,89,95). Apparently it is a combination of phosphatidyl serine, phOSphatidyl ethenolamine, phosphatidyl inositol and possibly other lipids which is responsible for the clotting activity of platelets. The platelet has been compared to a Sponge upon whose surface many plasma proteins are adsorbed (2). In an assay composed of reagents of known composition with reSpect to 46 clotting factors, the use of platelets with their accompany- ing contaminants would complicate the assay data. Fortunate- ly, Bell and Alton (9) found that an extract of human brain will substitute for platelets in the TGT. This substitute was adopted in the factor XI assay system. Human brain being unavailable, the platelet substitute was prepared from calf brain. These preparations proved to be entirely satis- factory. The optimal amount of P-S added to the incubation mixture had to be ascertained for each preparation because an insufficient quantity reduced the potential thrombo- plastin level and too much was inhibitory. For most prepa- rations, 0.15 ml of a 1:25 dilution in 0.9% saline provided optimal platelet factor activity. The volume of the incubation mixture prior to the addition of calcium was maintained at 0.9 ml. If upon sub- stituting a purified clotting reagent for a TGT reagent the final volume was less than 0.9 ml, the difference was made up with 0.9% saline. Figure 5 demonstrates that the replacement of the TGT platelet reagent by the calf brain P-S did not alter the level of thromboplastin, but significantly reduced the rate at which it was generated (curve 2). This reduced rate ascribed to a partial factor V (AcG) deficiency in the incu- bation mixture--the result of having replaced the platelet ,reagent (which contained factor V) with P-S. The rate of 47 so _ 50 — U) “C c‘. O O (D m 40 _ d E -H E-4 U‘ 50 — c: -r-( 4..) .LJ .2 o 20 t 10 — 0 l I l, 1 I I J 1 2 5 4 5 6 7 Incubation Time, Minutes Fig. 5. Thromboplastin generation curves resulting from the replacement of the TGT platelet reagent with calf brain platelet substitute (P-S) and factor V (AcG). In all curves, 0.50 ml normal TGT adsorbed plasma reagent and 0.5 ml normal TGT serum reagent were held constant. Curve 1: 0.50 ml normal TGT platelet reagent. Curve 2: 0.15 ml P-S diluted 1:25; 0.15 ml 0.9% saline. Curve 5: 0.15 ml P-S, diluted 1:25; 0.10 ml purified factor V reagent, diluted 1:20; 0.05 ml 0.9% saline. 48 thromboplastin generation was returned to normal by the addition of 0.1 ml of purified factor V reagent to the incubation mixture, as seen in curve 5. 4. Replacement of the TGT Adsorbed Plasma Reagent The treatment of oxalated plasma with BaSO4 is essential for the preparation of the TGT plasma reagent as it accomplishes the removal of prothrombin and factor IX (PTC). If these were not eliminated prothrombin would be converted to thrombin by the thromboplastin elicited in the incubation mixture and the clotting times would be a measure of thrombin rather than thromboplastin production. Factor.IX (PTC), an essential thromboplastin component, is found in serum as well as plasma. If it were not removed from plasma, the ability of the TGT to diagnose cases of factor IX-deficiency would be canceled, for either normal plasma or normal serum would be fully corrective. A normal adsorbed plasma reagent will not only supply factors V (AcG), VIII (AHG) and XI (PTA) to the assay but adds its troublesome antifactors antithromboplastin and antithrombin (see glossary) as well. When the TGT platelet reagent is replaced by P-S and purified factor V (AcG) reagents, the necessity for the adsorbed plasma's factor V contribution is eliminated. If serum, which retaifis factor XI (PTA), is utilized as one of the thromboplastic reagents, the adsorbed plasma in this I v. I): () (u 9“", V3. 5A uv “V U '4 I (I) Q I“ v 49 case also need not be depended upon to supply this com- ponent. Therefore, the BaSO4-adsorbed plasma reagent need only be replaced with a purified factor VIII (AHG) reagent to maintain a complete thromboplastin generating system provided the other reagents of the incubation mixture are P-S, purified factor V and serum. That this combination produced a maximum thromboplastin generation can be seen by curve 1 in Figure 4. A decided advantage gained in substituting purified factor VIII (AHG) reagent for the adsorbed plasma reagent is that the aforementioned undesirable plasma antifactors are eliminated. 5. Replacement of the TGT Serum Reagent Serum is defined as the solution of blood proteins remaining after completion of coagulation and removal of the clot. The platelets, factor VIII (AHG), prothrombin and fibrinogen of plasma have been utilized and the thrombin, activated factor V (AcG) and thromboplastin formed during the course of coagulation have been destroyed by the blood's naturally occurring antifactors. Thus the clotting com- ponents which are found remaining in normal serum are factors IX (PTC), X (Stuart factor) and XI (PTA). Because of its factor XI (PTA) content, serum could not serve as a reagent in an assay intended to determine the presence and potency of factor XI. Both the adsorbed 70.. 60*- m 'U c O 8 50 — m d E -a B 40 — U) I: -a 4J ’8 H 50" U 20 - 10 50 *— y..- r h Incubation Time, Minutes Fig. 4. Thromboplastin generation curves demonstrating the effect of replacing the TGT normal plasma and serum reagents with purified clotting reagents. In all curves, 0.15 ml P-S, diluted 1:25, 0.10 ml purified factor V reagent, diluted 1:20, and 0. constant. Curve Curve Curve Curve Curve 20 ml purified factor VIII reagent were held 0.50 ml normal TGT serum reagent; 0.15 ml 0.9% saline. 0.15 ml purified factor IX + X reagent, di- luted 1:5; 0.50 ml 0.9% saline. 0.50 ml BaSO4-adsorbed normal serum, diluted 1:10; 0.15 ml 0.9% saline. 0.50 ml BaSO4-adsorbed normal serum, diluted 1:10; 0.15 ml purified factor IX + X reagent, diluted 1:5. 0.15 ml purified factor IX.+ X reagent, di- luted 1:5, 0.05 ml purified factor XI reagent; 0.25 ml 0.9% saline. 51 plasma and serum TGT reagents would have to be replaced with purified reagents devoid of factor XI in order to assay this component. This was accomplished by preparing an incubation mixture which consisted of P-S, purified factor V (AcG), purified factor VIII (AHG) and purified factor IX + X reagents. Little, if any thromboplastin was produced, as seen in curve 2 of Figure 4, due to the absence of factor XI (PTA). A similar result was Obtained (Fig. 4, curve 5) when BaSO4-adsorbed serum was used in place of the purified factor IX + X reagent. This system contained factor XI, but not factors IX and X, since these were removed from the serum by the BaSO4. The addition of both BaSO4-adsorbed serum and purified factor IX + X reagent resulted in almost optimal thromboplastin generation (Fig. 4, curve 4). Com- plete generation was not attained because of a partial factor XI deficiency, brought about by the adsorption of a moderate amount of this factor onto BaSO4. As seen in curve 5 of Figure 4, full thromboplastin generation was achieved in a totally "synthetic" incubation mixture consisting of: 1) P-S, 2) purified factor V (AcG) reagent, 5) purified factor VIII (AHG) reagent, 4) purified factor IX + X (PTC + Stuart factor) reagent, and 5) purified factor XI (PTA) reagent. 52 6. Identification of Factor XI (PTA) The identity of purified factor XI (PTA) was estab- lished by determining its ability to correct the defect observed with a patient's factor XI-deficient TGT reagents in the modified Thromboplastin Generation Test described previously. Factor XI-deficient plasma and serum were obtained from a patient (0.8.) referred to the Michigan Department of Health for determining the cause of a hemor- rhagic tendency. The absence of factor XI in this patient's plasma was confirmed in the laboratory of the discoverer of this deficiency; Dr. R. L. Rosenthal at Beth Israel Hospital, New York, N. Y. Because a platelet abnormality was not involved, calf brain platelet substitute (P-S) was substituted in place of the patient's platelets. Figure 5 shows that normal adsorbed plasma and serum TGT reagents generate maximum thromboplastin (curve 2), but that similar reagents pre- pared from the factor XI-deficient patient did not (curve 1). A partial correction of the defect could be achieved (curve 5) when normal adsorbed plasma was substituted for the factor XI-deficient adsorbed plasma. However, complete correction was not realized because of the removal of a moderate amount of factor XI from the normal plasma by the BaSO4 treatment. A normal amount of thromboplastin was generated when normal serum was used in place of the deficient serum (curve 4). The same effect was obtained when a purified 55 50 .— X 40 — U) "U C. O U (1) 0') ~ 50 - (D E -H E-I x m \ -5 x x 1 :3 20 _ O H U _ 5 10 .______./. 2 ‘4 3M 4 O I I I (I I I I I I 1 2 5 4 5 6 7 8 9 Incubation Time, Minutes Fig. 5. Thromboplastin generation curves with TGT reagents from a factor XI (PTA)-deficient patient and the corrective ability of normal TGT reagents and purified factor XI re- agent (55). In all curves, 0.15 ml P—S, diluted 1:25, was held constant. Curve 1: 0.50 ml patient's adsorbed plasma; 0.50 ml patient's serum; 0.15 ml 0.9% saline. Curve 2: 0.50 ml normal adsorbed plasma; 0.50 ml normal serum; 0.15 ml 0.9% saline. Curve 5: 0.50 ml normal adsorbed plasma; 0.50 ml patient's serum; 0.15 ml 0.9% saline. Curve 4: 0.50 ml patient's adsorbed plasma; 0.50 ml normal serum; 0.15 ml 0.9% saline. Curve 5: 0.50 ml patient's adsorbed plasma; 0.50 ml patient's serum; 0.05 ml purified factor XI reagent; 0.10 ml 0.9% saline. 54 factor XI preparation to be subsequently described was added to the patients reagents (curve 5). The ability of the purified reagent to supply the clotting factor missing in the factor XI-deficient patient's TGT reagents identi- fied the purified preparation as factor XI. 7. Applications of the Factor XI (PTA) Assay The factor XI (PTA) assay method just described is capable of localizing clinical factor XI deficiencies, but such deficiencies are best detected by noting the corrective effect of a purified factor XI preparation on the patient's reagents in the modified TGT. If the extent of a partial deficiency is to be determined, the factor XI assay should be used. This is accomplished by adding dilutions of the patient's serum to the aforementioned incubation mixture. The assay quantitatively responds to the factor XI content in the serum. Reducing the level (by dilution) of the other clotting factors contained in serum does not influence this assay as it does the modified TGT, since optimal amounts of these components are already present in the in- cubation mixture. The assay of factor XI (PTA) as developed in this investigation was indispensible for following the presence and potency of this clotting factor in the many protein fractions prepared by the various isolation procedures which were performed and are to be described. Without the 55 assay the purification of factor XI would not have been possible. Employing a purified factor XI preparation as a reference standard, a thromboplastin generation curve was developed for each of a number of serial dilutions of the standard ranging from 1:1 (undiluted) to 1:16. Physiologi- cal saline was used as the diluent. The remaining thromboplastic reagents were retained at constant and non- limiting levels. Figure 6 depicts a typical family of curves for the above-mentioned dilutions. The uppermost curve,<fl3tained by omitting factor XI, demonstrates the inability of the incubation mixture to form thromboplastin in the absence of factor XI. For the rest of the curves it is seen that the quantity of thromboplastin which was generated depended upon the concentration of factor XI. The factor XI content of an unknown sample was determined with the assay system developed in this study by adding a known dilution to the incubation mixture and com- paring its thromboplastin generation curve with the refer- ence standard curves. 56 70 _ No Factor XI Seconds Clotting Time, 0 I I I I L I 2 4 6 8 10 12 Incubation Time, Minutes Fig. 6. Thromboplastin generation curves obtained with purified clotting reagents and varying quantities of puri- fied factor XI (PTA) reagent (55). In all curves, 0.15 ml P-S, diluted 1:25; 0.20 ml purified factor VIII, reagent; 0.10 ml purified factor V reagent, diluted 1:20; 0.15 ml purified factor IX + X reagent, diluted 1:5, were held con- stant. The concentration of factor XI reagent was varied by serial diluting the reagent in 0.9% saline. The volume of the incubation mixture was kept at 0.90 ml by adding the appropriate amount of 0.9% saline. 57 E. The Procedure for Isolating Factor XI (PTA) 1. Selection of Some Possible Starting Materials The choice of a starting material for isolating factor XI (PTA) is limited to serum, plasma, or a product known as fraction IV-1 produced from the fractionation of plasma by Cohn's method 6 (21). The selection was influ- enced by one of the objectives of this study: ". . . to devise a method for isolating factor XI in quantities sufficient for clinical use." a. Serum Human serum has limited use and can be procured only by special request and in relatively small amounts. The quantity of serum recovered from clotted blood represents but a fraction of the available plasma proteins since the greater volume of the serum is retained within the matrices of the fibrin clot. For these reasons, and because no pro- cedure has been adopted as yet for recovering useful serum proteins, there was little virtue in employing serum as the starting material for the isolation of factor XI. b. Plasma On the other hand, whole blood and plasma are in con- stant demand. Clinically, these materials are used to replace blood losses and in treating circulatory shock. They are thetherapeutic agents for hereditaryipracquired clotting factor deficiencies where a specific product, such 58 as platelets, fibrinogen, or antihemophilic globulin, is unavailable. At present, whole blood and plasma are used exclusively for controlling a factor XI-deficient patient in episode. Certain disadvantages accompany the administration of whole blood or plasma. Increasing a hemorrhaging‘ patient's.blood volume will aggravate bleeding into joints and soft tissues. When used to supply a missing clotting component, the clinically useful plasma proteins other than the needed specific clotting factor are wasted. Whole blood, such as collected by the American Red Cross, may be administered within 21 days after drawing. If held beyond its expiration date, as set forth by the Division of Biologic Standards of the National Institutes of Health, the blood is centrifuged to sediment the cellu- lar components. The plasma is withdrawn, pooled and frac- tionated to recover medically useful protein components. Among these are fibrinogen, immune serum globulin, albumin, and, when desired, prothrombin, fibrinolysin, transferrin and ceruloplasmin. Certain clotting components such as factors V (AcG). VIII (AHG), and X (Stuart factor) deteriorate in aging plasma, but factor XI (PTA) appears to be unaffected (96). In order to conserve plasma for its maximum clinical applications, it was decided best to start this part of the investigation with the portion of fractionated plasma 59 which contained the greatest concentration of factor XI for the purpose of isolation. c. Cohn's Fraction IV-1 Cohn's method 6 for fractionating normal human plasma has been found to concentrate factor XI (PTA) in fractions III and IV-1, with the greatest amount in fraction IV-1 (96). A diagramatic representation for the preparation of fraction IV-1 is given in Figure 7. Fraction IV-1 was made available for this study through the courtesy of the Division of Laboratories, Michigan Department of Health, Lansing, Michigan. HUMAN BLOOD PLASMA Ethanol 8% Ionic Strength 0.14 pH 7.2-7.4 Temperature -50 C Protein 5.1% II Ethanol 25% Fraction I Ionic Strength 0.09 pH 6.8-7.0 Temperature -50 C Protein 5.0% II Ethanol 18% Fraction II + III Ionic Strength 0.09% ' pH 5.2.: 0.05 Temperature -50 C Protein 1.6% F . IT Supernate Fraction IV—1 Fig. 7. Diagramatic representation of the preparation of fraction IV-1 from normal human plasma by Cohn's method 6. 60 The protein distribution of fraction IV-1 varied from lot to lot. The average composition of several preparations was found to be 64% OEglobulins, 21% B-globulins, 10% albumin, and 5% v-globulins (110). A representative moving boundary electrophoretic pattern of a 1% (w/V) protein solution of a fraction IV-1 lot used in this study is shown in Figure 8. Fraction IV—1 was obtained as a wet paste by centri- fugation at 18,000 RPM in a refrigerated (at -50 C), con- tinuous flow Sharples No. 16 supercentrifuge and then stored in polyethylene bags at -200. The protein content of the wet paste was approximately one—third of its weight, but varied, depending upon the firmness of packing by the centrifugation procedure. Ceruloplasmin and lipoproteins present in fraction IV-1 gave this material its character- istic greenish-yellow color. At present, the only useful protein which has been isolated from fraction IV-1 is ceruloplasmin (98,110). In devising a method for isolating factor XI from fraction IV-1, it was desired to recover ceruloplasmin as a by- product. Such a procedure would eliminate the possible competition of factor XI and ceruloplasmin for the same starting material. 61 /\ I l j I l l 7 B]. + 62 C12 C11 Alb Fig. 8. Free moving boundary electrophoretic pattern of a Cohn plasma fraction IV-1 obtained in a Klett-Tiselius apparatus. Conditions: 1.0% (w/v) protein in veronal buffer of pH 8.6, 0.05 ionic strength, specific conductivity of 0.00247 mhos/cm for 5400 seconds, 18.5 ma current and potential gradient of 10 volts/cm. Table VI. Distribution of Proteins in Cohn's Plasma Fraction IV—1 Analyzed from the Tiselius Free Moving Boundary Pattern of Figure 8. Protein Fraction(s) Percent of Total Albumin 50.4 Q1 52.0 a2 28.5 Bl+52 6.0 y 5.5 62 2. The Effect of (NH4)2SO4 Fractionation of Fraction IV-1 on Factor XI Activity Fifteen grams of fraction IV-1 paste was suspended in 15 ml of 0.05 M NaCl at cryostat temperature of 00 C, and hand homogenized with a 40 ml capacity Tenbroeck tissue grinder (Kimax Cat. No. 45950). The pH of the sus- pension was adjusted from 4.9 to 7.1 with 0.1 N NaOH. Under these conditions practically all of the protein was soluble. The small amount of residue was removed by centrifuging at 00 in an International REF refrigerated centrifuge at 2,500 RPM for 50 minutes. The resulting greenish-yellow opalescent supernate fully corrected the abnormal clotting time of a factor XI (PTA)-deficient plasma in vitro. Also, when used as the source of factor XI in the purified clotting factor assay system described previously, it gave an optimal generation of thromboplastin. The fraction IV-1 solution was further fractionated at 00 by the addition of a 100% saturated solution of (NH4)2SO4. The addition of this solution to 55% salt con- centration caused no precipitate formation. However, approximately one-third of the protein precipitated at 40% saturation, half between 40 and 50%, and the remainder between 50 and 67%. Each of these precipitates was dis- solved in 0.9% saline, dialyzed for 15 hours against 0.9% saline in the coldroom at 2-40, then assayed for factor XI activity. The degree of activity closely paralleled the 65 protein concentration. Hence, the subfractionation of fraction IV-1 by (NH4)2SO4 precipitation did not result in an enrichment of factor XI. 5. Extraction of Fraction IV-1 with 0.05 M NaCl Fraction IV-1 suspended at 00 in 5 volumes of 0.05 M NaCl became almost totally soluble when adjusted to pH 7.1. When the pH of the suspension was not altered from its initial value of 4.9, only 60% of the protein dissolved, but half of factor XI went with the insoluble material. An investigation was made into the possibility that factor XI was more soluble in a 0.05 M NaCl extract of fraction IV-1 at a pH less than 4.9. Aliquots of such a suspension were adjusted to pH's of 4.7, 4.5, 5.9 and 5.5 with 0.1 N HCl, stirred 2 hours and centrifuged. The factor XI content of all of the extracts were similar and represented only half of the activity present in fraction IV-l. Thus pH conditions of less than 4.9 produced no enrichmentof factor XI. Another 0.05DL,NaCl extract of fraction IV-1 was likewise prepared and divided into 2 aliquots. One aliquot was diluted with 4 volumes and the other with 20 volumes of distilled water. Precipitates formed in each case and these were collected by centrifugation. The precipitates were dissolved in 0.9% saline, made to pH 7.1 with 0.1 N NaOH, then assayed for factor XI potency. Of the protein 64 present in the pH 4.9, 0.051% NaCl extract, 54% was pre- cipitated by the 20-fold dilution with water and 16% by dilution with 4 volumes of water. All of the factor XI activity was precipitated by diluting 20 times, but only half by diluting 4 times. These results are shown in Table VII. Table VII. Subfractionation of a pH 4.9, 0.05 M NaCl Extract of Fraction IV-1 by Dilution with ‘Water Precipitated Factor XI in Purifi- Extent of Protein Precipitate cation Dilution n % of Initial % of Initial Factor 0 0.05 0 0 0 4x 0.0125 16 50 5 20x 0.0025 54 100 5 All of the factor XI activity in a third of the protein--a 5-fold purification--was obtained by diluting the 0.05.M NaCl extract 20 times with water. The 50% loss of potency resulting from the extraction of fraction IV-1 with 0.05I4 NaCl, however, overshadows the usefulness of this procedure. These results did show that factor XI re- quired a minimum salt concentration for remaining in solu- tion at pH 4.9. 65 4. Extraction of Fraction IV-1 with Distilled Water The extraction of extraneous water—soluble proteins such as albumin from a suspension of fraction IV-1 by reduction of the salt concentration present in this paste was investigated. A sample of fraction IV-1 paste was homogenized in a Tenbroeck tissue grinder with 5 volumes of distilled water, stirred for 2 hours and centrifuged, all at 00 C. The 5- fold dilution with water did not reduce the ionic strength sufficiently to completely prevent solution of factor XI, for the supernatant contained almost half of the total activity. Further experimentation revealed that by homogenizing fraction IV-1 with 10 volumes of distilled water, very little to no activity was extracted. Depending upon the compo- sition of the fraction IV-1 paste, which varied from lot to lot in moisture, salt and protein content, between 55 and 50% of inert protein was removed. The insoluble material (precipitate 1) and its corresponding supernate, as well as the subsequent products from fractionation procedures which resulted in the best yield of factor XI, are shown in Figure 9 On page 74. As a second purification step, precipitate 1 was re- extracted with 10 volumes of distilled water. An additional 5-10% of non-active material was removed by this procedure. 66 Hand suspension of fraction IV-1 paste in water with a Tenbroeck tissue grinder was suitable for small samples, but larger quantities required a mechanical method. The paste was too rubbery to be finely divided by a propeller or chain stirrer, but a Waring blendor, whose speed was regulated by a rheostat so as to prevent foaming of the protein suspension, proved adequate. Usually 15-50 minutes was required for suspending the fraction IV-1 paste in water. The following is a detailed description of the pro- cedure devised for the extraction of fraction IV-1 with water. Fifty grams of frozen fraction IV-1 paste was placed in a Waring blendor cup and 500 ml of distilled water, at 00 C, was added. This mixture was homogenized for 20 minutes at a Speed slightly below the foaming point. The suspension was transferredtx>a 1 liter beaker and gently stirred for 1 hour at 00. Ten milliliters of stirred sus- pension was withdrawn, solubilized by adjustment to pH 7.1 with 0.1 N NaOH, assayed for factor XI activity and analyzed for protein concentration. The remaining suspension was centrifuged in an Inter- national REF centrifuge at 2,500 RPM for 50 minutes at 00. The tan, opalescent supernate 1 was decanted and its pre- cipitate 1 re-extracted with 500 ml distilled water as described above. Protein and factor XI potency determin- ations on the starting material and subsequent extracts from an average run are presented in Table VIII. 67 Table VIII. Protein Redistribution and Factor XI Activity Resulting from the Extraction of Fraction IV-1 with Water Volume, Proteinl Factor XI Sample ml mg/ml total yield %’ Potency,% Fraction IV-1 540 27.5 14,850 100 100 Supernate 1 470 15.75 6,462 45.5 0 Precipitate 12 70 119.85 8,588 56.5 100 Supernate 2 515 2.52 1,195 8.0 0 Precipitate 22 55 150.78 7,195 48.5 100 lKjeldahl (N x 6.25). 2By difference. 5. Extraction of Precipitate 2 with Acetate Buffer Treatment of the twice-washed fraction IV-1 paste (precipitate 2, see scheme in Fig. 9) with 0.051% NaCl re- sulted in the extraction of only 40% of the available factor XI activity. By increasing the NaCl concentration to 0.15 M, 70% of factor XI was solubilized. In order to control the hydrogen ion concentration and ionic strength of the extraction, citrate, tris (hydroxy- methyl)amunomethane,and acetate buffers were individually investigated in the pH range from 5-7 at various molarities (0.05-0.20) and the amount of factor XI activity that each extracted was determined. A 0.15 M acetate buffer of pH 5.2 was found best. This buffer extracted 90-100% of factor XI in 25-50% (w/V) of the protein from precipitate 2. 68 The extraction, which was conducted at 00 C, was accomplished by suspending precipitate 2 in a volume of 0.15ld acetate buffer, pH 5.2, equivalent to half that of the initial fraction IV-1 suspension. The mixture was homogenized in a Waring blendor for 10-15 minutes and gently stirred for 1 hour at 00. The waxy, yellow-brown precipitate 5 was removed by centrifuging at 2,500 RPM for 45 minutes at 00, leaving a blue-green, slightly turbid supernate 5 which contained factor XI and ceruloplasmin. In addition, supernate 5 contained significant amounts of factors VII (SPCA) and IX (PTC), but no detectable quantity of factors V (AcG), VIII (AHG) or X (Stuart factor). 6. Removal of Ceruloplasmin by Adsorption on DEAE Cellulose Ceruloplasmin was removed from supernate 5 by adsorp- tion on diethylaminoethyl (DEAE) cellulose. To accomplish this, the supernate was diluted with 5 volumes of distilled water at 00 C to lower the ionic strength to 0.05 while maintaining the pH at 5.2. Adsorption was carried out in a coldroom at 2-40. One gram of dry DEAE cellulose was taken for each 50 ml of supernate 5. Under these conditions approximately 50% (w/v) of the supernate 5 protein was re- tained by the cellulose, including all of the ceruloplasmin, and factor XI was not adsorbed. When the ionic strength of supernate 5 was greater than 0.1, ceruloplasmin was not completely retained upon the 69 cellulose. At pH's higher than 5.2, the cellulose ad- sorbed a greater proportion of protein that also included some factor XI. At pH‘s lower than 5.2, ceruloplasmin was not completely withheld. To increase the flow rate in column chromatography, new DEAE cellulose was pretreated to remove the "fines" by preparing a water slurry of the cellulose, permitting the bulk of the cellulose to settle, then decanting the super- natant in which the fine particles were suspended. Usually 5 to 4 decantings sufficed. Used DEAE cellulose was re- generated according to the method of Peterson and Sober (79). Adsorption was carried out by two methods, depending upon the volume of material to be adsorbed. For small quantities, a column was established. For larger amounts-- up to 150 liters of 5-times diluted supernate 5--a batch technique was employed. For column chromatography, the necessary amount of DEAE cellulose was equilibrated with 0.05 M acetate buffer, pH 5.2 and the diluted supernate 5 layered over the cellu- lose. The factor XI-containing effluent 4, which was colorless but slightly turbid, was collected. This effluent was comprised of 8-12% of the original protein and all of the initial factor XI. Two distinct bands were observed on the cellulose. A tan band was situated at the uppermost portion (which subsequently could not be eluted by NaCl solutions) and 70 immediately beneath it, the bright blue ceruloplasmin band. For recovering ceruloplasmin, the column was washed with pH 5.2 acetate buffer, ionic strength 0.005, containing 0.025M NaCl, until the effluent had an absorbance of less than 0.050 at 280 mu. Ceruloplasmin of approximately 55% purity was then eluted with 0.25 M NaCl. The preparation of ceruloplasmin from this stage to 92-96% purity has been reported elsewhere (110). In the batch method for adsorbing ceruloplasmin, DEAE cellulose was equilibrated with 0.05 M acetate buffer, pH 5.2 and collected by filtration under vacuum. The floc was added to diluted supernate 5 in a large tank and stirred for 1 hour. The cellulose was then removed by either fil- tration under vacuum or in a refrigerated, continuous flow Sharpes No. 16 supercentrifuge operating at 18,000 RPM. In this procedure, as well as in the chromatographic method, all of the ceruloplasmin was retained by the cellulose, but none of the factor XI activity. 7. (NH4)2804 Fractionation of Effluent 4 Further purification of factor X1 in effluent 4, ob- tained by step 4 as shown in Figure 9, was continued directly, or when it was desirable to concentrate the pro- tein, was freeze-dried and redissolved in distilled water to approximately a 2% (w/v) protein solution. No detectable loss of factor XI potency was encountered by lyophillization. 71 Effluent 4 was subjected to (NH4)2SO4 fractionation in a 2-40 coldroom. The protein that precipitated at 40% saturation was removed and discarded and precipitate 6, which formed when the (NH4)2804 concentration was raised to 45% saturation, was collected. When redissolved in citrate buffer, pH 7.2, precipitate 6 gave a clear, color- less solution that contained just 4% of the initial protein of fraction IV-1. The recovery of factor X1 in precipitate 6, which varied between 50 and 80%, depended upon the method used for salting out the protein. For large volumes of effluent 4, solid (NH4)2SO4 was added with rapid stirring at 2-40 until 40% saturation was reached. The mixture was gently stirred for 1 hour, then the precipitate was removed by centrifuging at 2,500 RPM for 50 minutes at 00. The (NH4)ZSO4 concentration of the supernatant solution (supernate 5) was increased to 45% saturation by the addition of the appropriate amount of solid (NH4)2SO4. After stirring 1 hour, precipitate 6 was recovered by centrifuging as before. Precipitate 6 was redissolved in a citrate buffer of pH 7.2, ionic strength 0.15. One milliliter of this buffer was taken for each 16 ml of effluent 4 that was fractionated with (NH4hBSO4. The solution was transferredtn dialysis tubing and dialyzed against pH 7.2 citrate buffer, u = 0.15, for 24 hours at 2-40 with 2 changes of buffer. Citrate buffer was chosen to dissolve the final product because it 72 is acceptable for intravenous administration. The dialyzed preparation contained 50-60% of the factor XI activity present in effluent 4. For smaller amounts of effluent, or when a greater recovery of factor XI was desired, (NH4)ZSO4 fractionation was conducted by dialysis. A measured volume of effluent 4 .was transferred to dialysis casings, the casings placed into 8 liter glass cylinders and dialyzed at 2-40 against. 4 volumes of 50% saturated (NH4)ZSO4 for 24 hours with continuous agitation of the dialysis bags. At equilibrium, the solution inside the dialysis tubing was about 40% saturated by (NH4)2804. The precipitated protein was removed by centrifuging at 2,500 RPM for 50 minutes at 00. Supernate 5 was dialyzed against 4 volumes of (NH4)QSO4, made to 46.2% (w/v) satur- ation by the addition of solid (NH4)ZSO4, for 24 hours with constant agitation of the bags. The precipitate was col— lected by centrifuging, dissolved in the previously described citrate buffer and dialyzed against this buffer as described before. This method of fractionation yielded a product which retained 70—80% of the effluent 4 factor XI activity. A comparison of the factor XI distribution in the sub- fractions of effluent 4 obtained by the 2 (NH4)2SO4 fraction- ation techniques that were used is presented in Table IX. 75 Table IX. Redistribution of Factor XI by the 2 Techniques of (NH4)2804 Addition for the Fractionation of Effluent 4 Factor XI Activity, % Fractionation by (NH4)2SO4 Addition of Solid Saturation Fractionation by Dialysis (NH4)2SO4 Precipitate Supernate Precipitate Supernate 0 -- 100 -- 100 40 20-50 70-80 40-50 50-60 45 70-80 0 50-60 0 The stepwise recovery of protein and factor XI activity resulting from the purificatiOn procedure developed in this study and outlined in Figure 9 is summar- ized in Table X. The values are an average of 4 runs start- ing with 100 grams of fraction IV-1 paste. A free boundary electrophoretic pattern of the puri— fied factor XI preparation (solution 7) is shown in Figure 10 and its protein distribution, as analyzed from this pattern, is given in Table XI. It is apparent that the composition of purified factor XI is still quite hetero- geneous. Table XI also compares the protein distribution in a normal human plasma and an average lot of fraction IV-1 with that of the purified factor XI preparation. As com- pared to fraction IV-1, the purified factor XI preparation contains 10 times more y—globulin, 2é-times more Orglobulin, 74 Cohn's Fraction IV-1 10 volumes water Homogenize Step 1 Stir 2 hours 00C Supernate 1 Precipitate l (discard) 10 volumes water Homogenize Step 2 Stir 2 hours 0°C I II Supernate 2 Precipitate 2 (discard) 5 volumes u = 0.15 acetate buffer, \ Step 5 pH 5.2 Homogenize sgir 1 hour 0 C ' l— 7] Supernate 5 Precipitate 5 (discard) 5 volumes water (u = 0.05) Step 4 Adsorb with DEAE cellulose 2 - 40 C Effluent 4 40% sat. (NH4)2SO4 Step5 2-4OC I n Supernate 5 Precipitate 5 45% sat. (NH4)2SO4 (discard) Step6 2‘4OC | I T Supernate 6 Precipitate 6 (discard) 1 volume u = 0.15 citrate buffer, pH 7.2/16 volumes Step 7 effluent 4 2 - 4° C Solution 7 (Purified Factor XI) Fig. 9. The scheme for the purification of factor XI from Cohn's fraction IV-1. 75 .mummm HI>H coauoouw Em OOH Spa? mcHuHmum mcsn w mo wmmum>¢a me a osa.a a.aa ooa .oon .o onnnadaoond ooa m owm.m m.a owed a ucmdammm ooalom ma owm.m m.> mmm m mumcnmmsm o w omN.N m.m maoa m mumcnmmsm o we oom.ma «a mmm a mumcnomsm ooa 00a 00¢.mm mm omoa COHmcwmmDm HI>H .HE R .>DH>HuO< & .%Hm>oowm 0E HE\mE HE uODCOHm Hx uoaomm Camuonm Camuoum HmuOB Camponm OEDHO> H. hpsum many CH UOQOHO>OQ muspmooum coflumoamflusm mzu Eoum anauasmmm >ua>auo¢ HX Houomm Dam camuoum mo >um>oomm .X OHQMB 76 I I I 7 (32+Bl (12+Ql Fig. 10. Free moving boundary electrophoretic pattern of a preparation of purified factor XI obtained in a Klett- Tiselius apparatus. Conditions: 1.0% (w/V) protein in veronal buffer of pH 8.6, 0.05 ionic strength, specific conductivity of 0.00247 mhos/cm for 5400 seconds, 18.5 ma current and potential gradient of 10 volts/cm. Table XI. A Comparison of the Protein Distribution in Normal Human Plasma, Fraction IV-1 and a Purified Factor XI Preparation % Distribution 7 BI+BZ Q1+Oe Albumin Unfractionated Plasma 18 14 15 55 Fraction IV-1 5 21 64 10 Purified Factor XI 54 22 24 0 77 about the same proportion of B‘globulins and albumin is completely absent. 8. Physical and Chemical Properties of Purified Factor XI a. Adsorption Upon BaSO4 The literature contains contradictory reports con- cerning the absorbability of factor XI (PTA) onto BaSO4. Rosenthal (96) stated that a moderate amount of factor XI is removed from plasma by BaSO4 treatment. On the other hand, Ratnoff and Davie (91) commented that this clotting factor is not removed. The ability of BaSO4 to adsorb purified factor XI was therefore investigated as follows. A 1 ml aliquot of purified factor XI solution containing about 12 mg protein/ ml was placed in a 15 ml conical centrifuge tube and 40 mg commercial Basog powder (Baker, C.P.) was added. A fine suspension was produced by thorough mixing with a wooden applicator stick (1/16Eh inch doweling,6 inches long). The suspension was placed in a controlled temperature water bath at 570 for 5 minutes during which time the mixture was agitated intermittently. The BaSO4 was subsequently removed by centrifuging at 2,500 RPM for 15 minutes at room temperature. The supernate was assayed for factor XI activity by the assay system developed and described in this study and found to contain approximately the same activity as the material before treatment. 78 The experiment was repeated, except that this time freshly.precipitated BaSO4 was used as the adsorbent. About a 20 mg batch of BaSO4 was produced by mixing 0.1 ml 1 M BaCla with 0.1 ml 1 M Na2504 solutions and the pre- cipitate collected by centrifugation. The precipitate was redispersed in distilled water to remove most of the NaCl, centrifuged again, decanted and used as the wet paste. This freshly made adsorbent, when mixed with 1.0 ml of the factor XI preparation, removed 50% of the total activity. When the quantity of this BaSO4 was doubled, 75% of the initial activity was lost. Table XII summarizes the adsorption properties of BaSO4. Table XII. The Capacity of Different BaSO4 Preparations to Adsorb Factor XI Quantity of Source of Material Factor XI BaSO4 Adsorbed Adsorbed, % Reference Slight to Undefined Plasma & Serum moderate Rosenthal (96) Baker Plasma None Ratnoff (91) Baker, C.P. Purified Factor XI None This study Freshly pptd. Purified Factor XI 50% This study Freshly pptd. Purified Factor XI 75% This study 79 It was concluded that the adsorption of factor XI by BaSO4 is dependent on both the freshness of the adsorbent and the quantity used. b. Inactivation of Factor XI by Heat Rosenthal (94) determined that the factor XI activity of plasma was partially decreased by heating plasma at 600 for 10 minutes. The thermostability of the purified factor XI preparation of this study was determined at various temperatures. Solutions were heated at 56, 60, 65 and 800 for 5 minutes, then assayed for factor XI activity employ- ing the system developed in this study. The results are summarized in Table XIII. Like factor X1 in plasma, puri- fied factor XI is partially inactivated by heating at 600 for 5 minutes. It is totally inactivated when heated at 800 for this same length of time. Table XIII. Thermostability of Purified Factor XI in pH 7.2 Citrate Buffer, Ionic Strength 0.15 Temperature Time, Residual Factor XI C minutes ActIVIty, % 56 5 90 60 5 67 65 5 10 80 5 0 80 c. Age Stability of Factor XI in the Purified Preparation A purified factor XI solution containing 12 mg protein/ml when held at 250 C, for 5 days or stored in a coldroom at 2-40 for 2 months was found to have lost no potency. Likewise, a freeze-dried preparation kept for 1 year at 2-40 in a stoppered 5 ml bottle displayed its original potency when reconstituted with distilled water and assayed. It is apparent that factor XI, unlike many of the other clotting factors, is extremely stable on storage. IV . DI SCUSSION A. The Development of a Factor XI (PTA) Assay The investigation of factor XI (PTA) has been handi- capped by the lack of a method for isolating this component in purified form, and the major cause for discouraging efforts toward its purification, has been the absence of a reliable assay for measuring its potency. 'An ideal clotting factor assay system would be one in which all participating components--except the one under consideration--are supplied as pure entities (proteins, phospholipids, and metal ions) in optimal concentration. At present, few clotting factors have been purified to this extent. In devising an assay for factor XI, the objective of obtaining such a system was kept in mind, but the ideal conditions of complete purity were not achieved. Since factor XI has been predicated to be an essential reactant in the formation of intrinsic thromboplastin throughout this study, it was felt that its determination could best be accomplished by investigating its effect on the generation of thromboplastin. The already developed Thromboplastin Generation Test (TGT), which segregates the initial phase of coagulation, seemed best suited for adaptation and modification to this purpose. 81 82 Without modification, the TGT does not assay for factor XI because 2 of the 5 reagents (platelet suspension, BaSO4-adsorbed plasma, serum), which provide the necessary clotting components for thromboplastin generation, contain the factor to be assayed. Therefore, these reagents had to be substituted by others which were devoid of factor XI. 1. The Use of Platelet Substitute (P-S) Derived from Calf Brain Although the TGT platelet reagent does not contain factor XI (PTA) (note Table IV), its use is accompanied by certain disadvantages. For one thing, it is a most diffi- cult reagent to standardize. The quantity of platelets invariably differs from preparation to preparation because of the method employed for their isolation (differential centrifugation) and because humans differ in their platelet content (65). Probably the most serious difficulty en- countered by the use of platelets arises from their capacity to adsorb proteins involved in blood clotting (2). Preparations, although twice washed with saline, were seen in this study (Fig. 5) to contain a significant quantity of factor V (AcG). The disadvantages inherent with the use of the plate- let reagent were easily circumvented by replacing it with a lipid extract of calf brain. This platelet substitute (P-S), which is devoid of factor V (as seen'from the results in Figure 5) and other proteins, was readily prepared in 85 quantity, was stable when kept frozen, and its performance in the assay found comparable to that of platelets. 2. The Requirement of Factor V (AcG) in the Assay System When the platelet substitute preparation (P-S) replaced the human platelet reagent, the rate of thromboplastin generation was significantly reduced as seen from the results in Figure 5. This reduction in rate was attributed to the loss of factor V provided by the platelets. Curve 5 of Figure 5 readily shows that the rate of thromboplastin generation was returned to normal by the addition of puri- fied factor V reagent to the incubation mixture. Many ways by which factor V may be obtained in partially purified form have been described in the literature (25, 77, 116, 119). In general, these procedures are long and detailed and the net product is often of transient stability. In this study, to supply the factor V requirement, a simple and rapid method was developed for obtaining this factor in a partially purified, stable form and free from other clotting factors. Bovine serum was selected as the starting material because its factor V content and stability were found to be greater than that from human serum. The pro- cedure takes advantage of the different adsorptive capaci- ties of barium salts. Treating bovine serum with commercial BaC03 powder removes factors VII (SPCA), IX (PTC) and X (Stuart factor), but does not adsorb a significant amount 84 of factor V. The factor V, which remains in the treated serum, can then be adsorbed upon freshly precipitated BaSO4 and subsequently be eluted by a 2% (w/V) trisodium citrate solution. 5. The Incorporation of Purified Factor VIII IAHG) in the Assay System Normal human BaSO4-adsorbed plasma (the TGT plasma reagent) provides the incubation mixture with factors V (AcG), VIII (AHG) and XI (PTA), as shown in Table IV. Of these, factors V and XI were usually present in sub-optimal concentration, since moderate amounts were removed by the BaSO4 treatment. Their reduced concentration does not affect the generation of thromboplastin in the TGT because the platelet reagent's factor V content makes up the partial deficiency of this clotting factor and the serum supplies a full complement of factor XI. In an assay system for determining factor XI, however, the presence of factor XI in any of the reagents is prohibitive. The need for the adsorbed plasma's factor V became unnecessary when purified factor V reagent and P-S were used in place of platelets. Likewise, if serum is retained as a reagent, it will supply the system with an optimal amount of factor XI and thereby make the factor XI present in the BaSO4-adsorbed plasma reagent unnecessary. Thus, maximum thromboplastin generation was retained when a puri- fied factor VIII preparation was used instead of the BaSO4 85 adsorbed plasma. The result of this substitution is seen in Figure 4. The purified factor VIII reagent was fraction- ated from fresh normal human plasma by Cohn's method 6 and the redissolved fraction 1 freed of fibrinogen by heat coagulation. A decided advantage was gained through the use of puri- fied factor VIII in that antithromboplastin, a constituent of plasma, was eliminated. In vivo, the role of antithrombo- plastin is to destroy thromboplastin to prevent the clotting mechanism from getting out of control. When present, it likewise destroys the thromboplastin generated by the assay's incubation mixture. As is observed in curves 5 and 4 of Figure 2, the presence of antithromboplastin is demon- strated by the steady increase in clotting time with increase in incubation time once maximum thromboplastin is generated, attesting to the destruction of thromboplastin. In fact, the minimum clotting time may not represent maximum thrombo- plastin generation, for some thromboplastin may have been destroyed before this point was attained. In the absence of antithromboplastin, the minimum clotting time, once reached, is maintained. 4. Replacement of the Serum Reagent Complete thromboplastin generation was achieved by us- ing an incubation mixture consisting of P-S, purified factor V (AcG), purified factor VIII (AHG), serum and 86 calcium ions. Human serum provides factors IX (PTC). X (Stuart factor) and XI (PTA). In order to asSay for factor XI, serum had to be replaced with a preparation containing factors IX and X, but not factor XI. The biochemical properties of factors IX, and X are strikingly similar (see Table II). Methods for preparing these clotting factors which insure freedom from each other and from other plasma proteins are unavailable. Because only factor XI was under investigation during this study, the separation of these 2 components was not developed. A single reagent containing factors IX and X sufficed and a simple procedure for obtaining this was found. OAS described previously, bovine serum was adsorbed with com- mercial BaSO4 powder; the adsorbate was washed with sodium oxalate, and the clotting factors were eluted with citrate solution. The presence of factor VII (SPCA) in this re- agent (the citrate eluate devoid of factor XI) was unavoid- able, but of no consequence since it is not a component of the intrinsic thromboplastic system. With the elimination of serum, an incubation mixture comprised of purified reagents devoid of factor XI did not generate thromboplastin, as is seen in the uppermost curve of Figure 6. 5. The Determination of Factor XI (PTA) Potency It may be recalled that the secOnd portion of this study involved the isolation and purification of factor XI (PTA). 87 The identity of this preparation as factor XI was estab- lished by its corrective effect on a factor XI-deficient patient's BaSO4-adsorbed plasma and serum TGT reagents in the modified Thromboplastin Generation Test, as shown in Figure 5. The patienfis deficiency, as well as the identity of the purified factor XI reagent as prepared in this study, was tested and confirmed in the laboratory of Dr. R. L. Rosenthal. The requirement of factor XI in the assay system developed in this investigation was easily demonstrated. Curve 5 of Figure 4 shows that rapid and total development of thromboplastic activity occurred when purified factor XI was added to the incubation mixture. A unit expression was not assigned to factor XI activ- ity pending further purification of the reagent. Potency determinations were conducted on a comparative basis. A preparation of purified factor XI was retained and used as a reference standard. Serial dilutions of this standard preparation were assayed by the method developed in this study which employs purified reagents. These dilutions yielded a family of 5 curves as depicted in Figure 6. It can be seen that the quantity of thromboplastin generated by each dilution reflects the factor XI concentration. The factor XI potency of an unknown material was determined by comparing its thromboplastin generation curve with those of the standard factor XI curves. For instance, in the assay 88 of two unknowns containing factor XI, if the thromboplastin generation curve of the first was similar to a standard 1:2 dilution curve, and that of the second to a standard 1:4 dilution curve, the first unknown was regarded to contain twice the factor XI potency of the second. 6. Some Aspects of the Factor XI (PTA) Assay System For the isolation of factor XI (PTA), it was essential that a method for its determination Ina available at all stages of purification. Other procedures which have been reported for the assay of factor XI (88, 94) depended upon the corrective effect of the material to be assayed on the plasma of a factor XI-deficient patient. As discussed previously, plasmas with specific defects--in this instance, factor XI--are difficult to procure. On storage, certain clotting factors, especially faCtors V (AcG) and VIII (AHG), deteriorate and the plasmas are no longer specific in their response. Needed was an assay system which did not involve plasma with a specific deficiency, but instead, could be formu- lated from stable reagents prepared from normal, readily available materials. Such an assay was achieved as the result of this investigation. Its reagents were not diffi- cult to prepare and were stable for at least one year when kept frozen. It was shown that the generation of thrombo- plastin depended upon the presence of platelet substitute, 89 factors V (AcG), VIII (AHG), IX (PTc), X (Stuart factor), XI (PTA) and calcium ions. In the absence of factor XI, the assay system responded quantitatively to the addition of this component, whether it be added as a purified product, plasma or serum. This characteristic permitted determining the extent of the defect in a patient with partial factor XI-deficiency. In this respect, the assay is far superior to the TGT, which cannot localize a factor XI deficiency nor, for that matter, quantitize any of its constitutive components. B. The Purification of Factor XI (PTA) 1. Selection of the Starting Material The blood clotting component, factor XI (PTA), is a normal constituent of human plasma and since it is not totally consumed by the clotting process, is also present in serum. Both plasma and serum, therefore, represent sources from which factor XI may be isolated. It is not customary to collect human blood without anti— coagulant because it cannot be used for intravenous adminis- tration due to the possible presence of thrombin. Further- more, the amount of serum which can be obtained from a given volume of clotted blood is relatively small; the major portion is held within the fibrin clot. On the other hand, anticoagulated blood has many clini- cal uses and its supply barely meets its demand. For want .y- .n« m.u- 90 of a purified product, whole blood, or plasma derived from whole blood is used to treat hemorrhaging factor XI- deficient patients. But when used for this purpose, it Often aggravates bleeding into soft tissues and joints as a result of increasing the patient% blood volume. Because the use of serum or plasma as the starting material for the isolation of factor X1 is accompanied by the aforementioned disadvantages, neither was considered appropriate. The plasma from outdated blood has been fractionated by Cohn's method 6 since 1946 to recover its clinically useful proteins such as albumin, immune serum globulin and fibrinogen. One of these fractions, called fraction IV-1, is usually considered a waste product although it is in this fraction that the copper-bearing protein, ceruloplasmin, has been found. The biochemical role of ceruloplasmin is unknown, but it is thought to be involved in copper trans- port (1). Also, it is in this portion of fractionated plasma that almost all of factor X1 is concentrated. Fraction IV-1 thus provided the ideal starting material for the ultimate isolation of factor XI. From an average of 60.2 gm of protein per liter of plasma, about 5.5 gm are separated as fraction IV-1. Assuming that all of the plasma's factor X1 is present in this fraction, the clotting factor is already concentrated 17 fold. 91 As~a~matter of efficiency and economy it was desirable to devise a method for isolating factor XI which also en- compassed the possibility of separating ceruloplasmin as a byproduct. To accomplish this, procedures which resulted in the destruction or incomplete separation of ceruloplasmin had to be avoided. 2. (NHA)ZSOa Fractionation of Fraction IV-1 The fractionation of whole plasma by (NH4)2SO4 has been found to concentrate factor XI (PTA) in the proteins pre- cipitating under conditions of 25 to 50% (NH4)2SO4 saturation (94). It was of interest to determine at what (NH4)2SO4 concentration factor XI could be precipitated from a solu- tion of Cohn's fraction IV-1, and if this method would af- ford a means of purification. Fraction IV-1 was treated with (NH4)2804 to 55, 40, 50 and 67% saturation and each precipitate was assayed for factor XI activity. Ithas found that the factor XI activity of the dissolved precipitates Closely approximated the protein concentration. _Unlike the (NH4)2SO4 fractionation of plasma, an enrichment of the sought clotting factor was not realized by this procedure. 5. Extraction of Fraction IV—1 Paste With 0.05 M NaCl When the pH of fraction IV-1 was not changed from its initial value of 4.9, a 0.05 M NaCl solution dissolved 60% of the protein but only half of the factor XI (PTA) activity. 92 If it is assumed that pH 4.9 is the isoelectric point of factor XI, a shift from that pH to the lower range, while maintaining all other conditions constant, should result in the extraction of a greater amount of the clotting factor by the 0.05 M.NaCl solution. Decreasing the pH of the 0.05b4 NaCl fraction IV-1 suspension in increments to pH 5.5 did not yield an extract with greater activity. When a 0.05.M NaCl extract of fraction IV-1 at pH 4.9 was diluted 4 fold with distilled water it gave a precipi- tate which was separated and redissolved in 0.9% saline. This solution contained 16% of the protein and half of the factor XI activity of the saline extract (see Table VII). From the same table it can be seen that a similar 0.05 M NaCl extract diluted 20 fold with water yielded a precipitate which contained 54% of the protein and all of the factor XI activity. However, the fact that the initial pH 4.9 0.05 M NaCl extract resulted in a 50% loss of factor XI activity and no degree of purification gave the conclusion that this did not constitute a worthwhile procedure. But the im- portant point of this observation was that factor XI required a certain salt concentration for solution and this fact pointed the way tr) a useful purification step. 4. Extraction of Fraction IV-1 with Distilled Water With the knowledge that factor XI requires salt for its solubility at pH 4.9, and therefore may be a globulin, 95 fraction IV-1 paste was freed of albumins and some globulins by extraction with distilled water. It was found that 10 ml of water per gram of paste removed 50-50% of the protein without loss of factor XI activity in the residue. As shown in Table VIII, 45.5% of the protein was extracted from one lot of fraction IV-1. The rather broad variation in the amount of extracted protein was due to differences in the water, salt, and protein composition of the starting fraction IV-1 material. A second extraction of the washed precipitate with dis- tilled water assured the removal of occluded water-soluble proteins. An additional 5-10% of non-active material was eliminated by this treatment. 5. Extraction of Precipitate 2 with Acetate Buffer The twice-washed fraction IV-1 residue (precipitate 2, shown in the fractionation scheme of Fig. 9) consisted of half of the initial protein and all of the factor XI activity. A solvent was sought which would best extract factor XI from this residue. Different buffers at various ionic strengths were tried over a pH range of 4 to 7. It was found that an acetate buffer of pH 5.2, ionic strength 0.15 was practically ideal. With this buffer, 90—100% of the factor XI activity and 25-50% (w/V) of the protein was extracted (Table X). Like factor XI, no significant quantity of the ceruloplasmin originally present in fraction IV-1 94 was removed by the 2 water washes, but was almost totally extracted, along with factor XI, by the acetate buffer. 6. Adsorption of Cerulgplasmin on DEAE Cellulose An achievement in efficiency and economical use of the starting material, as well as the elimination of a contaminant, was accomplished in separating factor XI (PTA) from ceruloplasmin by adsorption of the latter on DEAE cellulose. Prior to adsorption, the acetate buffer extract (supernate 5, see Fig. 9) was diluted with 5 volumes of distilled water in order to reduce the ionic strength of the extract to 0.05. This was determined by varying the ionic strength of the buffer from 0.05 to 0.15 and the pH from 5.0 to 6.8 to find the optimum conditions for separation. Depending upon the volume of supernate 5 which was to be treated, the adsorption of ceruloplasmin was accomplished by either of 2 methods. For smaller volumes of diluted supernate 5 (up to 1.5 liters), column Chromatography sufficed and for large quantities (up to 150 liters), a batch treatment was the method of choice. Either method afforded an almost complete recovery of factor XI in the effluent that contained 8-12% of the initial fraction IV-1 protein (Table X). To recover the adsorbed ceruloplasmin, the DEAE cellu- lose, in the column or batch, was first washed with pH 5.2 acetate buffer, u = 0.005, containing 0.025bd NaCl, and 95 then eluted with 0.25I4 NaCl. The ceruloplasmin recovered in this manner was 55% pure with respect to the total pro- tein eluted. Further purification of the ceruloplasmin to 92-96% purity has been accomplished and was reported elsewhere (110). 7. (NH4)ZSO4 Fractionation of Effluent 4 When large quantities of fraction IV-1, e.g., 10 kg, were fractionated to obtain factor XI, to reduce the volume of the DEAE cellulose effluent (effluent 4, as shown in the scheme of Fig. 9), the effluent was freeze-dried and re- dissolved in distilled water to approximately a 2% protein solution. No loss of potency accompanied the freeze- drying process. The final purification of factor XI, as shown in steps 5 and 6 of Figure 9, involved the fractionation of effluent 4 with (NH4)2SO4 in either of 2 ways. If maximum recovery of factor XI was of prime concern, fractionation was con- ducted by dialysis as previously explained. With large volumes of effluent 4, or when the saving of time was of importance, fractionation was carried out by the direct addition of solid (NH4)2SO4. The protein precipitating be- tween 40 and 45% saturation constituted the fraction of highest factor XI activity and was retained as the product. Although the direct salt addition method was considerably faster, the factor XI recovery (SO-60%) was less than that obtained by the dialysis method (70-80%, see Table IX). 96 8. The Solvent for the Purified Factor XI Preparation A pH 7.2 citrate buffer of ionic strength 0.15 was chosen for dissolving the factor XI-rich (NH4)2SO4- precipitated protein as it is acceptable for intravenous administration. However, before this preparation, or for that matter, any blood derivative can be used clinically on humans, it must meet certain N.I.H. regulations. For one thing, the preparation must be sterile. To comply with this, clean, sterilized equipment must be used throughout a fractionation process. Because blood deriva- tives are not usually prepared under aseptic conditions, the removal of possible bacterial contamination is assured by sterile filtering the final product. The derivative must also be non-pyrogenic (a matter of maintaining condi- tions of extreme cleanliness to prevent bacterial growth) and safe (non-toxic, non-carcinogenic) to use. Such tests involve animal inoculation and these were not made in the course of this study. 9. Yield and Purification Factor Achieved The protein distribution of the final product, as determined by electrophoresis (Fig. 10, Table 11), was found to be 24% Orglobulins, 22% B-globulins and 54% y-globulins. It has not been ascertained in which of these factor XI resides. 97 The fractionation of one liter of plasma, which averages about 60 gm of protein, by Cohn's method 6, yields an average of 5.5 gm of protein in fraction IV-1. Assuming that most of the factor XI activity in whole plasma is present in this fraction, the factor has been enriched 17 fold. With approximately 75% of the factor XI activity re- covered in 4% of the starting fraction IV-1 protein, as seen in Table X, factor XI has been purified an additional 18 times. The purified preparation therefore represents a 200 fold enrichment over that of plasma. But from the electrophoretic pattern of the purified preparation shown in Figure 10, it is quite apparent that the material is heterogeneous, although devoid of albumins. One of the major accomplishments of the fractionation procedure developed in this study is that it leads to the enrichment of 2 proteins (factor XI and ceruloplasmin) of possible clinical use, from a "waste" product derived from the fractionation of outdated plasma. 10. Adsorption of Factor XI onto BaSOa Contradictory reports have appeared in the literature (91,96) concerning the capability of BaSO4 to adsorb factor XI from human plasma and serum. The adsorption by BaSO4 of factor XI, as prepared in this study, was determined. As shown in Table XII, a commercial source of BaSO4 powder did not remove a Significant amount of factor XI, but a 98 freshly formed BaSO4 precipitate adsorbed from 50 to 75% of the activity. Table XII shows that the quantity ad- sorbed depended upon the amount of adsorbent used. The difference in the adsorptive capacities of a com- mercial BaSO4 powder as compared to the freshly formed precipitate may be due to the fact that the fresh BaSO4, which consists of many finely divided particles, presented considerably more surface area upon which the protein could adsorb. 11. Stability Studies on Purified Factor XI Factor XI in the purified preparation, unlike many of the other clotting factors, was found extremely stable on storage. No loss of activity could be demonstrated in a solution (12 mg protein/ml) held at 250 C for 5 days or after storage for 2 months at 2-40. When freeze-dried and kept in a stoppered bottle for one year at 2-40, the re- constituted solution was likewise found to have lost no activity. On the other hand, Factor XI is not very heat stable. As can be seen from the data of Table XIII, a solution of purified factor XI, when held at 600 for 5 minutes, lost one-third of its activity. When heated at 800, the activity was completely destroyed. V. SUMMARY This study was conducted for the purpose of isolating the human blood clotting component, factor XI (plasma thromboplastin antecedent). In order to accomplish this, it was necessary to devise a means for determining its presence and potency. A. A method for assaying factor XI was achieved through modification of the existing Thromboplastin Generation Test (TGT). In connection with the new assay system, it was found that: 1. The crude TGT reagents had to be replaced with the purified factor XI-free reagents developed in this study to acquire a system depleted of the clotting component under investigation. 2. The purified clotting reagents were easily prepared from readily available biological sources, were repro- ducible and were stable on storage. 5. To generate intrinsic thromboplastin (intrinsic prothrombin activator), a source of factor XI had to be in- cluded together with a mixture of the following biologic reagents: a. Purified factor V (accelerator globulin). b. Purified factor VIII (antihemophilic globulin). 99 100 c. Purified factors IX + X (plasma thromboplastin component + Stuart factor). d. A lipid extract of calf brain (platelet substitute). 4. The assay was capable of detecting factor XI de- ficiencies in patients and quantitatively determining the extent of their defect. 5. The assay responded quantitatively to the presence of factor X1 in plasma, serum or plasma derivatives, thus making it applicable for plasma fractionation studies leading to the isolation of factor XI. B. A method for the purification of factor XI was developed by starting with Cohn's fraction IV-1 and carrying out the following operations: 1. Extraction of albumin and other inert proteins from fraction IV-1 paste by washing twice with distilled water. 2. Extraction of factor XI and ceruloplasmin with acetate buffer from the preceding waterwwashed residue. 5. Preferential separation of factor XI from cerulo- plasmin by adsorption of the latter on DEAE cellulose. 4. Fractionation of the non-adsorbed proteins in the effluent from the DEAE cellulose with (NH4)2SO4. 5. Solution in an isotonic citrate buffer of the pro- teins precipitating between 40-45% (NH4)2SO4 saturation, which were found to yield maximum factor XI activity. 101 C. 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Abbreviation for accelerator globulin (factor,V). Adsorbed Plasma. Plasma treated with an adsorbent such as BaSO4 or alumina to remove prothrombin and factors VII (SPCA), IX (PTC) and X (Stuart factor). Adsorbed Serum. Serum treated with an adsorbent such as BaSO4 or alumina to remove factors VII (SPCA), IX (PTC) and X (Stuart factor). .555. Abbreviation for antihemophilic globulin (factor VIII). Anticoagulant. A chemical, such as trisodium citrate, potassium oxalate or ethylenediaminetetraacetic acid, added to freshly drawn blood to prevent clotting. The anticoagulants mentioned accomplish this by com- plexing calcium. Antihemophilic Globulin. Factor VIII. Antithrombin. Naturally occurring plasma constituent which neutralizes thrombin to prevent the clotting of fibrinogen from getting out of control. Antithromboplastin. A substance in plasma which inhibits or destroys the prothrombin converting activator. 115 114 Cellular Components of Blood. The formed elements in blood. Consists of erythrocytes, leucocytes and platelets. ClottingpFactor. A participant of the blood clotting mechanism. Deficient Plasma. A patient's plasma which is devoid of, or containing a sub-optimal amount of one or more of the clotting factors. Extrinsic Thromboplastin. The substance formed from a tissue factor (tissue thromboplastin, factor III) and plasma factors that effects the conversion of pro- thrombin to thrombin. Extrinsic Thromboplastin Component. One of the clotting factors involved in the reactions which lead to the conversion of prothrombin to thrombin by the extrinsic clotting mechanism. Factor I. Fibrinogen. The precursor of fibrin. Factor II. Prothrombin. The precursor of thrombin. Factor III. Tissue thromboplastin. The tissue component of the extrinsic thromboplastin mechanism. Factor IV. Calcium ions. Factor V. Accelerator globulin. A participant of both the extrinsic and intrinsic prothrombin convertion mechan- isms; believed to be involved in the conversion of prothrombin to thrombin. Factor VII. Serum prothrombin conversion accelerator. A participant of the extrinsic prothrombin conversion mechanism. 115 Factor VIII. Antihemophilic globulin. A participant of the intrinsic prothrombin conversion mechanism; believed to be involved in the activation of factor X. Factor IX. Plasma thromboplastin component. A participant of the intrinsic prothrombin conversion mechanism; believed to be involved in the activation of factor VIII. Factor X. Stuart factor. A participant of both the extrin- sic and intrinsic prothrombin conversion mechanisms; believed to be involved in the activation of factor V. Factor XI. Plasma thromboplastin antecedent. A partici- pant of the intrinsic prothrombin conversion mechan- ism; believed to be involved in the activation of factor IX. Factor XII. Hageman factor. A participant of the intrinsic prothrombin conversion mechanism; believed to be in- volved in the activation of factor XI. Fibrinogen. Factor I. The precursor of fibrin. Formed Elements of Blood. Erythrocytes, leucocytes and platelets. Hageman Factor. Factor XII. Intrinsic Thromboplastin. The substance formed from plasma clotting factors that converts prothrombin to thrombin. Synonymous with intrinsic prothrombin activator. 116 Intrinsic Thromboplastin Component. One of the clotting factors which participates in the reactions which lead to the conversion of prothrombin to thrombin by the intrinsic clotting mechanism. Plasma. The non-cellular portion of blood. To prevent coagulation in drawn blood, it must be anticoagulated. Plasma Thromboplastin Antecedent. Factor XI. Plasma Thromboplastin Component. Factor IX. £15, Abbreviation for plasma thromboplastin antecedent (factor XI). 3293 Abbreviation for plasma thromboplastin component (factor IX). Platelet. A formed element of blood which supplies factors involved in the blood clotting mechanisms and other substances (serotonin, retractoenzyme) necessary for normal hemostasis. Platelet Factor 1. A protein found on the surface of plate- lets (probably adsorbed from plasma) with factor V activity. Platelet Factor 2. A substance found in platelets that accelerates the conversion of fibrinogen to fibrin by thrombin. Platelet Factor 5. A substance or substances (phospholipid(s)) in platelets involved in the intrinsic prothrombin conversion mechanism. 117 Platelet Factor 4. A substance found in platelets which neutralizes heparin, the naturally occurring anti- coagulant of blood. Platelet-Poor Plasma. Plasma deficient in platelets, con- 3 taining 5,000 platelets/mm or less. PlateletmRich Plasma. Plasma containing 50,000 or more platelets/mms. E Prothrombin. Factor II. The precursor of thrombin. Prothrombin Activator. The substance that converts pro- 48‘ 11; i '.~.‘. ~ . -.'.‘-‘_"J‘m4‘"_ \ I. thrombin to thrombin; thought to be activated factor V. - AIL/Hr. Serum. The fluid portion of plasma remaining after com- pletion of coagulation. Serum Prothrombin Conversion Accelerator. Factor VII. 5295, Abbreviation for Serum Prothrombin Conversion 'Accelerator. Factor VII. Stuart Factor. Factor X. Substrate Plasma. Plasma used in an assay for supplying certain clotting factors. In the Partial Thromboév plastin Time test, a patient's plasma, deficient in only one clotting factor, is the substrate plasma. In the Thromboplastin Generation Test, the substrate plasma is used as the source of prothrombin and fibrinogen. Tissue Thromboplastin. Factor III. Thrombin. The enzyme involved in the conversion of fibrinogen to fibrin. 118 Thromboplastin. Is used as a synonym for factor III (tissue thromboplastin) but also carries the connotation of the substance that converts prothrombin to thrombin. Reprinted from noon Vol. 15, No. 3. March. 1960 Printed in U.S.A. The Preparation of Plasma Thromboplastin Antecedent (PTA) and its Assay with Purified Clotting Components By HAROLD CALLICK, L. A. HYNDMAN AND K. B. MCCALL T HAS BEEN apparent from its inception that the thromboplastin generation test"2 is more complicated than the simple interaction, in the presence of calcium, of three factors whose names are given to the reactants of the test: platelets, antihemophilic factor and Christmas factor (plasma thromboplastin component, PTC). The platelet reactant contributes at least two factors active in this test: a protein-linked phospholipid, and accelerator globulin (AcG), the latter probably having been adsorbed from plasma. The antihemophilic factor, supplied as aluminum hydroxide- or barium sulfate-adsorbed plasma, is accompanied by at least three other factors: accelerator globulin (AcG), plasma thromboplastin antecedent (PTA)3 and Hageman factor.4 It may also contain inhibitors. The Christmas factor (PTC), supplied as diluted serum, is accompanied by PTA, Stuart factor,5 Hageman factor, antithrombin, antithromboplastin and variable amounts Of prothrombin. These complexities severely limit the interpretation and understanding of the reactions that are taking place. We wish to describe a system for the assay of certain coagulation factors which replaces the complex reagents of the thromboplastin generation test with purified“ fractions, the concentrations of which can be controlled. We have found that plasma thromboplastin antecedent must be incorporated in the thromboplastin generation system, and this requirement established a laboratory assay for this factor that is not dependent on the use Of PTA- deficient plasma. We have employed this same test system for the semi- quantitative assay Of each Of its components. M ATERIAL AND METHODS Materials Platelet-substitute (P-S).——A lipid extract of calf brain, prepared by the method of Bell and Alton,6 was substituted for platelets. Acetone-dried calf brain was extracted with chloroform, and the chloroform was evaporated from the extract. The residue was emulsified in 0.9 per cent saline. This material may be prepared in quantity; it is stable when kept frozen, and its performance in the assay is comparable to that of platelets. It restored normal prothrombin consumption to platelet-poor normal plasma when the latter was recalcified. Use of the platelet-substitute eliminates the difficulties of interpretation which arise when platelets are used. Accelerator globulin (AcG).—Bovine serum was used as a source of accelerator globulin From. the Division of Laboratories, Michigan Department of Health, Lansing, Mich. This investigation was supported in part by a research grant (H—1463) from the National Heart Institute, National Institutes of Health, U.S.P.H.S. Submitted May 14, 1958; accepted for publication July 31, 1959. 'The term “purified" as used in this manuscript refers to components which have been isolated and freed of other known clotting components. 404 fw-—s—_, ‘. ...~ PMSLVIA THBOL-IBOPLASTIN ANTECEDENT: PREPARATION AND ASSAY 405 because it is readily obtained and its AcG content and stability are greater than that in human serum. Bovine blood, collected without an anticoagulant, was allowed to stand at room temperature for 4 hours. Without disturbing the clot, the serum was decanted, treated with commercial barium carbonate (Bakers, 40 mg./ml.) and centrifuged. The supernatant serum was then adsorbed with freshly precipitated barium sulfate. The barium sulfate was prepared as follows: for each 100 ml. of serum to be adsorbed, 4.0 ml. of 1 M BaCl2 was mixed with 4.0 ml. of 1 M Na2804. The precipitate of barium sulfate which formed was diluted with 80 ml. of distilled water and centrifuged. The barium sulfate precipitate, with adsorbed AcG, was washed once with 200 ml. of 0.9 per cent saline and eluted with 50 ml. of 2 per cent citrate solution (2 Cm. Na3C6H5O7.2 H20 per 100 ml. distilled water). The eluate, containing AcG, was dis- pensed into small vials and frozen. When kept at —-20 C., it remained active for at least one year (based on its ability to normalize the prothrombin time in aged, AcC-free, oxalated, normal human plasma). The preparation was free of thrombin and prothrombin. Antihemophilic globulin (AHG).—Fraction I was separated from fresh normal human plasma by the method of Cohn et al.7 (method 6), within 24 hours of the time the blood was collected. Each kilogram of fraction 1 paste was resuspended in 12 L. of 0.025 M sodium citrate buffer (pH 6.3), filtered under sterile conditions, dispensed in vials and dried from the frozen state. For use, one vial, containing 200 mg. of fraction I protein, was reconstituted with 0.9 per cent saline, heated at 56 C. for 5 minutes, quickly cooled and the coagulated fibrinogen removed by centrifugation. The supernatant liquid, con- taining AHG, was dispensed in 1 ml. aliquots and stored at —20 C. until needed. Plasma thromboplastin component (PTC).—Bovine serum was adsorbed with commercial barium sulfate (Baker, C. P.: 100 mg./ml. sermn). The barium sulfate was washed twice with two-thirds serum volume of 0.01 M sodium oxalate and eluted with one-tenth serum volume of 2 per cent sodium citrate solution. The eluate was dialyzed against 0.9 per cent saline at 2 to 4 C. The dialyzed eluate was centrifuged and the supernatant solution was dispensed in small aliquots and stored frozen at —-20 C. The method is so similar to those published for serum prothrombin conversion accelerator‘3 and PT C9 that the preparation can be expected to contain both components. Plasma thromboplastin antecedent (PTA).—Fraction IV-l was separated from normal human plasma by the method of Cohn et al.7 (method 6). One hundred Cm. of fraction IV-l paste was washed twice with 1 L. of distilled water at 0 C. Each time, a uniform suspension was made in a homogenizer or in a colloid mill. The washings were discarded. The washed paste was resuspended in 500 ml. of 0.15 ionic strength acetate buffer, pH 5.2. The acetate extract was recovered by centrifugation and was diluted to ionic strength 0.05 by the addition of l L. of distilled water. The precipitate which formed was removed by centrifugation and discarded. The supernatant liquid was further diluted to ionic strength 0.015 by adding 3500 ml. distilled water. The precipitate which formed con- tained PTA, and was recovered by centrifugation. The precipitate was resuspended in 100 ml. of 0.3 per cent saline and was then adjusted to pH 2.9 by the addition of l N hydrochloric acid. The solution at pH 2.9 was heated in a 37 C. water bath for two hours. A small precipitate formed; it was removed by centrifugation. The supernatant fluid was adjusted to pH 7.3 to 7.4 by the addition of 1 N sodium hydroxide. The PTA was stored frozen (but will keep for weeks in a refrigerated bath at O C.). The preparation is apparently free from PTC, AcC, AHG, Stuart factor, prothrombin and thrombin. Data supporting the identity of this preparation appear in a later part of this report. Substrate plasma—Fresh normal human plasma, obtained from blood drawn in acid- citrate-dextrose anticoagulant, was dispensed in 2 ml. aliquots and kept frozen at —20 C. until needed. For use, it was thawed quickly in a water bath at 37 C., after which it was kept in a container with crushed ice. Normal oxalated or citrated plasma served equally as well as ACD plasma. The platelet content of the substrate does not affect the test. Calcium chloride—0.020 M solution in distilled water. 406 GALLICK, HYNDMAN AND MC CALL Method of Assay Modified thromboplastin generation test mixture 21.—0.15 ml. platelet-substitute (diluted 1 to 25 in 0.9 per cent saline), 0.30 ml. barium sulfate-adsorbed plasma (1 to 5 in saline), and 0.30 ml. serum (1 to 10 in saline), all made to 0.9 ml. with 0.9 per cent saline. Thromboplastin generation test mixture with purified reagents B.—The amount of each factor required depends upon the potency of the preparation and may vary from lot to lot. The following amounts were typical: 0.15 ml. platelet-substitute (diluted 1 to 25 in 0.9 per cent saline), 0.25 ml. AHC (undiluted), 0.10 ml. AcC (undiluted), 0.15 ml. PTC (diluted 1 to 5 in saline), 0.05 ml. PTA (undiluted), and 0.20 ml. saline, 0.9 per cent, to make total volume to 0.9 ml. Procedure for both test mixture A and test mixture B.—The reagents were kept on ice until mixed. The thromboplastic test mixture, and six tubes of substrate plasma (0.1 ml. in 11 x 75 mm. glass tubes), were placed in a 37 C. water bath. After allowing two minutes for the mixture to warm to 37 C., 1.0 ml. of 0.02 M CaCl2, also at 37 C., was added to it. At intervals of 1, 3, 5, 7, 9 and 11 minutes after addition of calcium, a 0.3 ml. sample of the recalcified mixture was withdrawn and quickly blown into a tube of substrate plasma.’ Sufficient calcium was carried over in the sample that it was not necessary to recalcify the substrate plasma simultaneously, as is necessary in the Biggs and Douglas test. The substrate tube was left at 37 C. until shortly before the expected end point; it was then tilted gently and repeatedly until the liquid was observed to gel. When the reagents were present in optimal amounts, a minimum clotting time of 9 :1: 2 seconds was attained. Omission of any single component resulted in significantly longer clotting times. RESULTS AND DISCUSSION Demonstration of PTA Efiect The requirement for PTA in the thromboplastin generation test was readily demonstrated with the aid of purified reagents. Since PTA is present in both barium sulfate-adsorbed plasma and in serum, according to studies by Rosenthal,3 both of these reagents had to be replaced before the effect of PTA could be shown. The barium sulfate-adsorbed plasma was replaced with our AHC and AcC preparations. This considerably simplified the system, since the purified components were free of PTA, traces of prothrombin and inhibitors normally present in adsorbed plasma. The system was simplified further by replacing platelets with the platelet-substitute. Figure 1 Shows data demonstrating the presence of PTA in serum, and its requirement in the thromboplastin generation test. Rapid development of thromboplastic activity was observed with platelet-substitute, AHC, AcC and normal serum (curve 1). Adsorption of the serum with barium sulfate, thus removing PTC, resulted in marked loss of activity (curve 2). Substitu- tion of the barium-adsorbed material (the PTC preparation) for serum resulted in even greater loss of activity (curve 3). Combination of PT C and barium sulfate-adsorbed serum (curve 4) restored the activity to that of the original system. This demonstrated that the serum contained two ”This method of recalcification, which eliminates the necessity of separately recalcifying the substrate plasma, was observed at Children's Hospital, Detroit, Mich., through the courtesy of Drs. W. W. Zuelzer and J. Rutzky and Miss Ruth Evans. PLASIV'IA THROIMBOPLASTIN ANTECEDENT: PREPARATION AND ASSAY 407 60> 3 50' h .1» +\ ‘23 ho» o .\. U 5 . \.\ i 30- + .— o 19 “ \ 3 + C . \+ 0 20L .1 U A (a \ l l l l l 3 5 7 9 INCUBATION TIME, MINUTES Fig. l.—Demonstration of PTA effect in serum. All curves: P-S, AHC, AcG con- stant. Incubation mixture made to 0.9 ml. with saline. Recalcified with 1.0 ml. CaCl2. Assayed at intervals by transfer of 0.3 ml. to 0.1 ml. substrate plasma. Curve 1, with normal serum, 0.15 ml. of l to 5 dilution. Curve 2, with BaSO4-adsorbed normal serum, 0.15 ml. of l to 5 dilution. Curve 3, with purified PTC, 0.15 ml. of l to 5 dilution. Curve 4, with BaSO4-adsorbed normal serum plus PTC. Curve 5, with purified PTC plus purified PTA. factors: one adsorbable and the other nonadsorbable on barium sulfate. The combination of PT C and purified PTA (curve 5) was also fully active, which showed that PTA could be substituted for barium sulfate-adsorbed serum. Thus we have shown that both PTC and PTA are required to replace normal serum in the thromboplastin generation test. Identification of PTA Using PTA-deficient Plasma The identity of purified PTA was confirmed on the basis of its corrective effect in the thromboplastin generation test, with the use of PTA-deficient 408 GALLICK, HYNDMAN AND MC CALL B\El—-L-i——fl :x.’..——o 5 ¥\9=Q:2/+h CLOTTING TIME, SECONDS W Z 8 o T T I l+./O-O—* INCUBATION TIME, MINUTES Fig. 2.—Thromboplastin generation in PTA deficiency and its correction by PTA. All curves.- P-S, 0.15 ml., in place of platelets. BaSO, plasma, 0.3 ml. of l to 5 dilution. Serum, 0.3 ml. of l to 10 dilution. Incubation mixture made to 0.9 ml. with saline. Recalcified with 1.0 ml. CaClg. Assayed at intervals by transfer of 0.3 ml. to 0.1 ml. substrate plasma. Curve 1, patient’s BaSO, plasma plus patient's serum. Curve 2, normal B2804 plasma plus normal serum. Curve 3, normal BaSO, plasma plus patient’s serum. Curve 4, patient's BaSO, plasma plus normal serum. Curve 5, patient’s BaSO4 plasma plus patient’s serum plus PTA. patient’s plasma and serum“ (fig. 2). With the patient's barium sulfate- adsorbed plasma and serum, abnormal thromboplastin generation was ob- served (curve 1), as compared to the corresponding reagents from a normal individual (curve 2). The deficiency was partially corrected by substituting normal barium sulfate—adsorbed plasma for that of the patient (curve 3), and was fully corrected by substituting normal serum for the patient’s serum (curve 4). The abnormal thromboplastin generation with patient's barium °The PTA-deficient plasma and serum were obtained from a patient whose physician requested our assistance in the study of a bleeding tendency. The diagnosis of PTA deficiency in this patient has been confirmed by Dr. R. L. Rosenthal,10 who also confirmed the corrective effect of our PTA preparation in vitro in the plasma of one of his own PT A-deficient patients. PLASMA THROMBOPLASTIN ANTECEDENT: PREPARATION AND ASSAY 409 70 p- CONTROL - NO PTA 60-- I?“ CLOTTING TIME, SECONDS 8 8 ‘6’ 8 f V I l 2 z z .7; D K/ _. n U ‘ u. o u on o _. o I: _. N INCUBATION TIME, MINUTES Fig. 3.—Thromboplastin generation with purified components and varying quantities of purified PTA. All curves: P-S, AHC, AcG, PTC constant. Variable PTA, and incubation mixture made to 0.9 ml. with saline. Recalcified with 1.0 ml. CaClz. Assayed at intervals by transfer of 0.3 ml. to 0.1 ml. substrate plasma. (PTA, dilutions of stock solution in saline.) sulfate-adsorbed plasma and serum was corrected also by the addition of purified PTA (curve 5). The difference between curves 1 and 5 is the result of the presence of purified PTA in the thromboplastin generation mixture of the latter. Assay of PTA and other Factors with Purified Reagents in Vitro From the standpoint of convenience and practicality, it is desirable that an in vitro assay would not require the use of plasma or serum deficient in a particular factor. Such an assay is obtained when purified reagents are used throughout (see METHODS, Thromboplastin Generation Test Mixture With Purified Reagents). Here, the test system is dependent on the presence of all components, and each component can be varied at will with the others F.“ _* _.._ . .,- 410 GALLICK, HYNDMAN AND MCCALL ' held constant. For each level of the factor studied, a thromboplastin genera- tion curve was determined (plotting clotting time against incubation time). Thus, a family of curves was constructed by using several dilutions of a reference lot for each coagulation factor. One such family of curves for PTA is shown in figure 3. The other com- ponents of the system (P-S, AHC, AcC, PTC) were held at constant, non- limiting levels. Various levels of PTA, from 1:1 (no dilution) to a 1:16 dilution, are shown, compared to the control curve at the top, which shows the activity in the absence of PTA. PTA, purified from fraction IV-l of plasma, was preferred to barium sulfate-adsorbed serum as a source of PTA, because the former does not contain serum factors such as antithromboplastin. We have not attempted to define a unit of PTA activity, pending further study of the test system. At present, it is preferable to assay the PTA content of a test specimen by choosing a dilution of the specimen which will fall on or between two levels of a reference lot of PTA, assayed on the same day as the unknown. The test mixture with purified reagents, as described, does not require the addition of Stuart factor or Hageman factor as specific components. Some Stuart factor is probably supplied by the PTC preparation, since the PT C partially corrects the abnormal thromboplastin generation in a system containing platelet-substitute, barium sulfate-adsorbed normal plasma and Stuart-deficient serum.‘ The possible role of Hageman factor in this system is not clear. Published properties of Hageman factor“-11 are similar to those of PTA, and both factors are reported to be activated by contact with wettable glass.”l3 Our preparation of PTA shortens the recalcified clotting time of silicone-collected plasma in silicone-coated glass. Whether this is a reflection of its PTA activity or of its contamination with Hageman factor will have to be deter- mined. It also remains to be determined whether or not Hageman factor contributes activity to the system which might be mistaken for PTA activity. SUMMARY By replacing the three crude reagents commonly used in thromboplastin generation tests—washed platelets, barium sulfate- or alumina-adsorbed plasma, and serum—with purified clotting factors, many variables and uncertainties were eliminated. It was demonstrated that plasma thrombo~ plastin antecedent (PTA) was required for the generation of thromboplastic activity. A method was developed for the preparation of purified PTA from fraction IV-l of human plasma. Its identity was established by its ability to correct, in vitro, the defect in the plasma of a PTA-deficient patient. Thus, further evidence in support of the belief that PTA is a discrete component, essential °Stuart factor-deficient serum was received through the courtesy of Charles L. Johnston. M.I)., School of Medicine, The University of North Carolina, Chapel Hill, N. C. D 1111‘; PLASMA THROMBOPLASTIN ANTECEDENT: PREPARATION AND ASSAY 411 for blood coagulation, was obtained. More specifically, it was found to be essential in the generation of thromboplastic activity in plasma. A test system for thromboplastin generation was described which was used to assay PTA in vitro and which did not require the use of PTA- deficient plasma or serum. Omission of any one of the components of this system resulted in a marked loss of thromboplastic activity; restoration of activity was proportional to the amount of the component that was added. Thus, with this system of purified components, it was possible to assay any one of them without the use of, or requirement for, plasma or serum specimens from patients with specific coagulation deficiencies. SUMMARIo IN INTERLINGUA Per introducer purificate factores coagulatori in loco del tres crude re- agentes que es communmente usate in tests de generation de thromboplastina —i.e. plachettas eluite, plasma adsorbite a sulfato de barium o alumina, e sero—multe variabiles e multe incertitudes esseva eliminate- Esseva demon- strate que antecedente de thromboplastina del plasma (ATP) es indispensa- bile in la generation de activitate thromboplastic. ' ' Esseva disveloppate un methodo pro 1e preparation de ATP purificate ab fraction IV—l de plasma human. Su identitate esseva establite per su capacitate de corriger in vitro le defecto in le plasma de patientes deficiente in ATP. Assi un nove supporto esseva obtenite pro 1e conception que ATP es un componente discrete que es essential in 1e coagulation de sanguine. Plus specificamente, i1 esseva trovate que ATP es essential in 1e generation de activitate thromboplastic in 1e plasma. Es describite un systema pro 1e effectuation de tests del generation de thromboplastina. Illo esseva usate pro 1e essayage de ATP in vitro. Illo es distinguite per le facto que illo non require le uso de plasma 0 sero deficiente in ATP. Le omission de non importa le qual del componentes de iste systema resultava in un perdita marcate de activitate thromboplastic. Le restauration del activitate esseva proportional a1 quantitate del componente que esseva re-addite. Assi i1 esseva possibile per medio de iste systema de purificate com- ponentes de effectuar tests pro le presentia de non importa le qual de ille componentes sin 1e uso e sin e requirimento de specimens ab patientes con specific defectos de coagulation. REFERENCES l. Biggs, R., and Douglas, A. S.: The hereditary aspects of a new hemo- thrombOplastin generation test. J. philia-like disease. Blood 10:12.0, Clin.Path. 6:23, 1953. 1955. 2. —, —, and MacFarlane, R. C.: The 4. Ratnoff, O. D., and Colopy, I. E.: A initial stages of blood coagulation. J. familial hemorrhagic trait associated Physiol. 122:538, 1953. with a deficiency of a clot-promoting 3. Rosenthal, R. L., Dreskin, O. H., and fraction of plasma. J.Clin.Invest. 34: Rosenthal, N.: Plasma thromboplastin 602, 1955. antecedent (PTA) deficiency: clini- 5. Hougie, C., Barrow, E. M., and Graham, cal, coagulation, therapeutic and I. B.: Stuart clotting defect. I. 412 Segregation of an hereditary hemor- rhagic state from the heterogeneous group heretofore called “Stable Fac- tor" (SPCA, Proconvertin, Factor VII) deficiency. J.Clin.Invest. 36: 485, 1957. 6. Bell, W. N., and Alton, H. C.: A brain extract as a substitute for platelet suspensions in the thromboplastin generation test. Nature 174:880, 1954. 7. Cohn, E. J., Strong, L. T., Hughes, W. L., Jr., Mulford, D. J., Ashworth, I. N., Melin, M., and Taylor, H. L.: Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein com- ponents of biological tissues and fluids. J.Am.Chem.Soc. 68:459, 1946. 8. Alexander, B., Goldstein, R., and Land- wehr, C.: The prothrombin conver- sion accelerator of serum (SPCA): Its partial purification and its proper- ties compared with serum ac-globu- 10. ll. 12. 13. . Aggeler, CALLICK, HYNDMAN AND MC CALL lin. ].Clin.Invest. 29:881, 1950. P. M., Spaet, T. H., and Emery, B. E.: Purification of plasma thromboplastin factor B (plasma thromboplastin component) and its identification as a beta2 globulin. Science 119:806, 1954. Rosenthal, R. L.: Personal communica- tion. Frick, P. G., and Hagen, P. 8.: Severe coagulation defect without hemor- rhagic symptoms caused by a (le- ficiency of the fifth plasma thrombo- plastin precursor. J.Lab.& Clin.Med. 47:592, 1956. Ratnoff, O. D., and Rosenblum, J. N.: The role of Hageman factor in the initiation of clotting by glass. ].Lab. 6: Clin.Med. 50:941, 1957. Rapaport, S. 1., Ames, S. B., and Mik- kelsen, 8.: Evidence that glass in- creases plasma PTA activity. J.Lab.& Climb/led. 52:62.4, 1958.