....... THE EFFECT or KALLIKREIN on PLATE-LU AGGREGATION Thesis for the Begree'oi M. S. mmm STATE UNWERSWY CATHERME ANN. MONCKTON 1 9 7 5 115E318 { J ' r; t . o ' 1};.£‘. )1 o z [a ‘4 1' u' 5" “ BIRBING IV .1? MAR &VSUNS ‘ ‘ 0K MM INC. 1 IanAnv RINDE “~15 ABSTRACT THE EFFECT OF KALLIKREIN ON PLATELET AGGREGATION BY Catherine Ann Monckton A biologically pure preparation of human plasma kallikrein was prepared by glass activation. Contaminating IgG was removed by affinity chromatography. The preparation was found to be free of thrombin and plasmin. Incubation of kallikrein with human platelets prior to addition of adenosine diphosphate (ADP) or epinephrine inhibited the second wave of platelet aggregation. This inhibition was apparently obtained by kallikrein's antagonism of the platelet release reaction. Electron micrographs of kallikrein-treated platelets revealed greatly diminished granule release in stimulated platelets. The enzymatically active site was found to be essential for kallikrein's effect on platelets. Trayslol, a naturally occurring kallikrein inhibitor, was capable of eliminating kallikrein inhibition of the second wave of platelet aggregation. Kallikrein was found to stimulate morphologic changes in plate— lets even in the absence of aggregation. This may cause platelets to be in a refractory state to subsequent activation by conventional aggregating agents such as ADP and epinephrine. THE EFFECT OF KALLIKREIN ON PLATELET AGGREGATION BY Catherine Ann Monckton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1975 FOR MY FAMILY ii ACKNOWLEDGMENTS The author wishes to express her appreciation to the following people: Joan Mattson, M.D., my major professor, for all her support, encouragement and intellectual stimulation given to me throughout my Master's program. Donald Koufman, M.D., for his technical guidance and assistance and the opportunity to do part of my research work in his laboratory in the Department of Human Development. Martha Thomas, M.S., M.T.(ASCP), my academic advisor, for her direction in planning my Master's program. Donna Ladd, B.S., and the Electron Microscopy Laboratory of the Department of Pathology, for the electron micrographs shown in this thesis. Sandra Maryman, of the Department of Human Development, for her technical assistance. ' The Department of Pathology, for the financial assistance given to me to finance my Master's program. iii TABLE OF CONTENTS INTRODUCTION. . . . . . . . LITERATURE REVIEW . . . . . . . . A History of Kallikrein—Kinin Research . The Kallikrein- Kinin System. Biological Activity of the Kallikrein- Kinin System The Role of Platelets in Inflammation. Platelet Aggregation . . The Arthus Reaction. . . . MATERIALS AND METHODS . . . . . . Glass Activation . . . . . . BAEe Assay . . . . . . . . . Kallikrein Units . . . . . . Immunoelectrophoresis. . . . Radial Immunodiffusion . . Affinity Chromatography. Bioassay . . . . . . . . . Isoelectric Focusing . . . Polyacrylamide Gel Electrophoresis SDS Electrophoresis. . . . . Concentration of Kallikrein Solution Assays for Plasmin and Thrombin. Platelet Aggregation . . . Fixation of Platelets. . . Serotonin Uptake . . . . . . RESULTS . . . . . . . . . . . Isolation and Purification of Human Plasma Kallikrein. The Effect of Kallikrein on Platelet The Effect of Bradykinin on Platelet Serotonin Uptake . . . . . . Trypsin Aggregation. . . . DISCUSSION. . . . . . . . . . . . CONCLUSION. . . . . . . . . . . . iv Aggregation Aggregation Page l3 l3 l3 14 14 14 15 16 16 18 18 18 l9 19 20 21 22 22 24 32 32 34 35 37 LIST OF REFERENCES. VITA. . . . . . . . Figure 10 ll 12 LIST OF FIGURES The plasma kallikrein-kinin system . . . . . . . . . Immunoelectrophoresis agar slides. . . . . . . . . . The results of the estrus rat uterus bioassay for kallikrein The effect studies. . The effect The effect inhibition The effect structural The effect The effect The effect actiVity. O O I O O O O O O I O O I O O O of kallikrein on platelets. of kallikrein on ADP aggregation. . . . . of incubation time on kallikrein of kallikrein on ADP aggregation. Ultra- Studies 0 O O O O O O C O O O O O O O O O of kallikrein-Trayslol on ADP aggregation of kallikrein on epinephrine aggregation. of kallikrein on epinephrine aggregation. Ultrastructural studies. . . . . . . . . . . . . . . The effect The effect of bradykinin on ADP aggregation. . . . . of bradykinin on platelets. Ultrastruc- tural studies. . . . . . . . . . . . . . . . . . . . vi Ultrastructural 23 25 26 28 28 3O 3O 31 31 33 33 INTRODUCTION Both plasma kallikrein and platelets participate in the classic . 28 . . . . Arthus reaction. Kallikrein causes increases in vascular permea- . . 28 . . . b1l1ty. The aggregation of platelets is responSible for the formation of the white thrombus which leads to the thrombosis of . . . . 28 vessels and results in the necrOSis and ulceration of the skin. Certain proteolytic enzymes such as trypsin and thrombin are . . . 31 . . . capable of induCing platelet aggregation. Kallikrein and trypSin have esterolytic ability and split the synthetic ester, benzoyl-L- arginine ethyl ester. Bradykinin is generated when kallikrein or trypsin act on kininogen. The occurrence of kallikrein and platelets in certain inflamma- . 30 . . . tory reactions and the ability of other proteolytic enzymes to cause platelet aggregation prompted the following research to investigate the effect of kallikrein on platelet aggregation. LITERATURE REVIEW A History of Kallikrein—Kinin Research The kallikrein-kinin system is an enzymatic cascade which pro- duces a polypeptide of nine to eleven amino acids. The kinin produced is capable of reproducing the four cardinal signs of inflammation, redness, pain, swelling and heat. This system may provide the chemical key to the mediation of inflammation. The work done in discovering the components and describing the function of the kinin system has been done in this century. Some of the highlights are listed below. 33 . . . 1926 Frey and his co-workers observed that human urine contains a large molecular, nondialyzable, thermolabile substance. The substance produced a prolonged fall of arterial pressure in the dog on intravenous injection. 33 . . . . 1928 Frey described a Similar substance in the blood. 25 . . . . 1930 Frey described a Similar substance in the pancreas. He called it kallikrein after the Greek word kallikeas, meaning pancreas. 35 . . . . . . 1937 Werle, working WIth isolated segments of guinea pig ileum, found that human plasma mixed with extracts of the salivary gland promoted sharp contractions. Neither the plasma nor the extract alone were able to bring about contractions. 2 32 . . . . . 1949 Roche e Silva, working w1th guinea pig intestine, found that when trypsin or snake venom were added to plasma, a smooth muscle contracting and vasodilating substance was released. The smooth muscle contraction took seven times as long to reach a peak as when mediated by histamine. Antihistamines had no inhibitory effect on the contractions. He named the substance bradykinin after the words brady, slow and kinein, to move. 7 1954 Keele and Stewart found that on exposure to glass, human plasma formed a peptide. This peptide produced pain in the blister base. 8 . . 1957 Armstrong and co—workers found a pain-produc1ng substance similar to bradykinin in blister fluid and other inflammatory exudates. . . 28 . 1960 Elliot, LeWis, and Horton postulated, by analytical methods, bradykinin as an eight amino acid polypeptide. The synthetic protein was nonfunctional. . 28 . . . . . . 1960 BOissonas syntheSized bradykinin as a peptide containing nine amino acids, and it was functional. . 14 . 4 . . 1970 Kaplan and Austin, and Wuepper and Cochrine purified components of the kallikrein-kinin system. The Kallikrein—Kinin System There is an interaction between the plasma coagulation system and the kallikrein-kinin system. The common point is Hageman factor, plasma clotting factor XII. Hageman factor is activated by surface contact. Glass and antigen-antibody complexes are capable of 4 activating factor XII but the mechanism is not known. It has been suggested42 that adsorption on glass uncoils the molecule exposing the hydrophobic groups which may be important for adsorption of other proteins or may be the enzymatically active sites. Activated Hageman factor is thought to be capable of activating the kinin system. Margolis7 observed that Hageman factor-deficient plasma does not generate kinins with glass contact as normal plasma does. The scheme of the kallikrein-kinin cascade (Figure 1) shows that the generation of active kinins depends on the conversion of kallikreins from an inactive state to an active state. The activated kallikreins then initiate the conversion of the plasma kinin precur- sors, kininogens, into the active peptides called kinins. The conversion of prekallikrein to kallikrein appears to be a proteolytic process that is easily bought about by trypsin. Pre- kallikrein is also activated by change in pH and certain organic solvents.28 Kaplan37 purified prekallikrein by utilizing anionic exchange chromatography, cationic exchange chromatography, and gel filtration. This preparation was shown by gel filtration and functional analysis to have a molecular weight of 127,000. Mobility on disc gel electrophoresis was gamma and the isoelectric point was 2 between 8.5-8.9. Kallikrein was found to have a molecular weight of 108,000, isoelectric point of 8.5—8.9, and mobility as a gammaz. Coleman, Mattler, and Sherry6 worked with human plasma and isolated the enzymatically active fractions which they termed kallikrein I, II, and III. All three kallikreins were biologically active, had similar ratios of hydrolytic activity, a variety of arginine and SURFACE CONTACT , XII i > XII activated PREKALLIKREIN 1 .1 KALLIKREIN KININOGEN 1 . KININ A .1 INACTIVATED PEPTIDES KININASE Figure l. The plasma kallikrein-kinin system. 6 lysine esters and were radioimmunochemically related. Physicochemical characteristics of the three were different. Kallikrein I had a sedimentation coefficient of 5.7, a molecular weight of 99,800, and migrated as a Slow gamma globulin. Kallikrein II migrated as a fast gamma globulin with a molecular weight of 163,000. Kallikrein III had a molecular weight of 124,000, migrated as an alpha globulin, and reacted quite differently with inhibitors. Wuepper and Cochrane38 found their human plasma prekallikrein preparation to have a sedimen- tation coefficient of 5.2 and a calculated molecular weight of 107,000. The isoelectric point was 7.7. After activation of the preparation to kallikrein, there was no observable difference in sedimentation coefficient or molecular weight. Wuepper and Cochrane's38 findings based on analysis of the precursor prekallikrein revealed only one kallikrein in plasma corresponding most closely to kallikrein I of Coleman et al. The molecular weights of prekallikrein and kallikrein are similar, suggesting33 that activation may be due to very minor molecular modification, possibly produced by the removal of a steric hindrance of the active Site for its substrate. Kallikreins are a group of enzymes in an activatable form which are found in the pancreas, blood, lymph, and urine. Kallikreins from different sources are not identical molecules. Plasma kallikrein cleaves one of the kininogens at a lysyl arginine bond, producing bradykinin. Pancreatic kallikrein cleaves the methionyl lysyl bond producing lysyl-bradykinin, also called kallidin. The presently accepted view is that all glandular 7 kallikreins produce a decapeptide from kininogen. The decapeptide is then converted by an aminopeptidase in the plasma into a nonapeptide. Synthetic amino acid esters such as benzoyl-L-arginine ethyl- ester (BAEe) are hydrolyzed by kallikrein. Kallikrein activity is inhibited by diisopropylfluorophosphate (DFP), soy bean trypsin inhibitor (SBTI), and C-1 esterase inhibitor. A natural kallikrein inhibitor has been isolated from various bovine organs.28 The natural inhibitor has a molecular weight of approximately 6,500, contains 58 amino acids, and tends to dimerize. The inhibitor is manufactured under the trade name of Trayslol (FBA Pharmaceuticals, Inc., New York, N.Y.). Kininogens are the natural substrate kallikreins act upon to produce kinins. Kininogens are alpha plasma globulin glycopeptides 2 that have a molecular weight of 70,000 and an isoelectric point of 4.5—4.7.36 Human plasma contains two kininogens with a normal mean value of 6.1 ug kininogen/ml.3 Kinins include bradykinin which contains nine amino acids, lysyl-bradykinin containing ten amino acids, and methionyl-lysyl- bradykinin containing eleven amino acids. The biologic activity of the three kinins is Similar and includes25 vasodilation, enhanced capillary permeability, leukotaxis, pain, bronchoconstrictor effects in the guinea pig, and constriction of certain smooth muscles. Bradykinin concentration in normal subjects is 2.8 ng bradykinin equivalent/m1 of plasma. 8 Biological Activity of the Kallikrein—Kinin System The kallikrein-kinin system has been implicated in various pathologic and physiologic conditions which are described below. When carcinoid tumors metastatize to the liver, spontaneous flushing can occur. Some carcinoid tumors contain very high concen- trations of kallikrein raising the question of the possible role of . . . . . 35 kinins in the carCinOid syndrome. . . . . 5 In the condition of hereditary angioedema, a defect of an inhibitor for C-l esterase and kallikrein has been discovered which results in excessive activity of the kallikrein-kinin system. In the transition from neonatal to adult circulation, the . . . . . 35 . kallikrein-kinin system may play an important role. It is sug- gested that at birth the increased p0 decreased temperature of 2: the umbilical cord, and the increased numbers of granulocytes acti- vate the plasma kallikrein-kinin system. The bradykinin that is released dilates the pulmonary blood vessels while constricting the umbilical cord and the ductus arteriosus. Increased levels of kinins have been observed when synovial fluid exudates from patients with gout have been tested.20 This could suggest that crystalline uric acid deposits activate Hageman factor, which in turn activates the kinin system. Kinins may play a role in migraine headaches.35 When the cerebral spinal fluid of patients suffering with a migraine headache is analyzed, increased levels of kinins are found. 1 . . Graham and co-workers' 1 experiment attempted to delineate the role of the kallikrein—kinin system in inflammation. Bradykinin or 9 activated Hageman factor were injected into a rabbit ear chamber. When activated Hageman factor was injected, a prolonged and prominent leukocyte sticking and emigration occurred. This response was delayed in onset. When bradykinin was injected, the response was immediate, but transient,with leukocyte emigration occurring less frequently. Graham concluded that either activated Hageman factor produced effects by a mechanism other than kinin generation or that bradykinin released endogenously has effects different from a Single injection of exogenous material. Kaufman and Mattson18 injected purified plasma kallikrein into male albino rabbits. Blood and urine samples were collected and assayed for plasma kinins and kininogen and urine kinins. Within one minute after injection, proteinuria was noted, followed by hematuria and oliguria. Decreased plasma kininogen levels with eightfold increases in urinary kinins resulted. When examined by light, electron, and fluorescent microscopy, the rabbit kidneys showed a mild accumulation of polymorphonuclear leukocytes. It was concluded that activation of the kinin system may play a role in the pathogenesis of glomerulonephritis. Kaplan, Kay, and Austin15 showed that purified plasma kallikrein is chemotactic for human neutrophils. Prealbumin fragments of Hageman factor and prekallikrein interacted to convert prekallikrein to kallikrein. The kallikrein formed had both chemotactic and kinin generating activity. When prekallikrein or prealbumin fragments of Hageman factor were tested alone, neither showed chemotaxis. Kallikrein was incubated with diisopropylfluorophosphate, which 10 inhibits the hydroxyl residue at the active Site. No chemotactic ability or kinin generating ability resulted from this mixture. This indicates the essential role of the active site of the enzyme in the expression of its chemotaxis. In subsequent work9 it was found that kallikrein was also chemotactic for mononuclear cells. Diisopropylfluorophosphate inhibited kallikrein chemotaxis and kinin generation. This suggests that the serine active Site is required for chemotaxis of mononuclear cells as well as neutrophils. ' activated non-glass Washed antigen-antibody aggregates2 contacted guinea pig serum. This activated serum enhanced vessel permeability when injected intradermally, contracted the rat uterus, caused hypotension when injected intravenously, and possessed increased esterolytic activity. It was speculated that antigen- antibody aggregates activate Hageman factor, thus initiating the activation of the kinin forming enzymes. The Role of Platelets in Inflammation One of the most intriguing aspects of platelets is their ability to act as an inflammatory cell. Nachman29 performed an experiment that showed that platelets have certain inflammatory promoting processes. From human platelet granules, a cationic protein was isolated which was heat stable and nondialyzable with a molecular weight of 30,000. The protein fraction increased vascular permeability in rabbit Skin. To determine the permeability, 0.1 ml of sample was injected intradermally into a rabbit that had previously received an intravenous injection of Evans blue dye. The permeability fraction 11 was measured for histamine, serotonin, plasmin, bradykinin, and kallikrein. None were detected. There were two phases of permea- bility caused by the platelet factor. An acute phase (15 min) of enhanced permeability was found to be dependent on endogenous histamine release presumably due to tissue mast cell degranulation. The delayed (3 hr) increase in vascular permeability was non- histamine dependent and was characterized by tissue leukotaxis, presumably secondary to direct or indirect in vivo chemotaxis. Platelet Aggregation Platelets are essential components of the hemostatic mechanism. They function in hemostasis24 by occluding the opening of blood vessels by adhering to the exposed collagen at the site of disruption and aggregating to form the platelet plug. Following platelet aggregation, platelet factor 3, a phospholipid essential for plasma coagulation, is released. Platelets contain intracellularly 30-50%42 of the total blood content of plasma coagulation factor XIII. Plasma coagulation factors V, VIII, and XI are associated with the platelet membrane. Various substances are capable of causing platelet aggregation and include: adenosine diphosphate (ADP)1; particulate materials13 such as collagen, aggregated gamma globulin, and antigen-antibody complexes; certain proteolytic enzymes31 such as thrombin and trypsin; biogenic amines42 such as serotonin and epinephrine. Studies of in vitro platelet aggregation40 have Shown that ADP—, epinephrine-, serotonin-, thrombin-, and trypsin-induced aggregation can occur in two waves. The first wave is due to the addition of 12 exogenous aggregating agent and the second wave is due to the release of endogenous ADP from the platelet. This phenomenon is known as . l7 . . the platelet release reaction and includes the extru51on of not only endogenous ADP but also ATP, serotonin, catecholamines, potassium, calcium, and platelet factor 4 into the surrounding plasma. The granules and dense bodies contain adenine nucleotides, serotonin, catecholamines, and platelet factor 4. Electron micrographs of . 41 platelets harvested at the height of the second wave reveal greatly reduced numbers of platelet granules and dense bodies. The Arthus Reaction Maurice Arthus noted in 1903 that horse serum injected into the Skin of rabbits induced a mild local inflammatory reaction.28 How- ever, on subsequent injections, a violent local inflammatory reaction developed that was so severe that thrombosis of vessels occurred resulting in necrosis and ulceration. The presence of platelets and kallikrein is essential for the occurrence of the Arthus reaction. Kallikrein causes increases in vascular permeability and platelets cause the formation of the white thrombus. MATERIALS AND METHODS Glass Activation Fresh frozen human plasma (donated by the American Red Cross, Detroit, Michigan) was dialyzed for three days in phosphate buffered saline (PBS) at pH 7.4. Contact with glass was avoided. Glass powder (Sigma Chemical Co., St. Louis, Missouri) was prepared accord- ing to Henriques.12 Equal amounts of plasma and glass powder (1 m1 plasma/1 gm glass powder) were swirled for 30 seconds at 4°C. The glass powder was washed with 0.95 NaCl three times. Kallikrein was eluted off the glass powder with a solution of 0.1 M glycine—0.1 M NaCl, pH 10 in the proportion of 1 ml buffer for each 2.5 gm glass powder. The eluate was collected and dialyzed in PBS for three days at 4°C. The dialysate was frozen for further use. BAEe Assay Kallikrein's ability to hydrolyze benzoyl-L—arginine ethyl ester (BAEe) (Sigma Chemical Co., St. Louis, Missouri) to benzoyl—L— arginine and ethanol was assayed on a Beckman Spectrophotometer at a wavelength of 253 nm. The assay was performed by standard methods according to the Handbook of Experimental Pharmacology.8 The increase in absorbance at one and five minutes was recorded. The controls that were used were: 0.22 mg/ml trypsin (Worthington l3 l4 Biochemicals, Freehold, New Jersey) and 0.14 mg/ml Hog kallikrein (National Biochemical Corporation, Cleveland, Ohio). Kallikrein Units One kallikrein unit iS defined as that amount of enzyme that . . 8 . . hydrolyzes one umole of BAEe in one minute at 25°C. It is obtained by multiplying the change in absorbance by the extinction coefficient for benzoyl arginine which is l x 103. Immunoelectrophoresis Immunoelectrophoresis was carried out in a Shannon apparatus (Shannon Electronics Systems, Surrey, England) according to the method of Scheidegger34 with 1% agar Slides. Veronol buffer, ionic strength 0.075, pH 8.6 was used as the electrolyte buffer. Current was applied for 45 minutes, allowing 4 ma/Slide with an upper limit voltage of 100v. Goat antiserum to human IgG, IgM, IgA, and whole serum (Hyland, Costa Mesa, California) were placed in the agar trough. Precipitin arcs were allowed to develop for 48 hours. After development the Slides were washed in at least four changes of saline over a 24-hour period to remove extraneous protein. The Slides were stained with 1% Ponceau stain for 2 hours and then rinsed in 2% acetic acid until the background was clear. The final rinse was made in 2% acetic acid containing 50 ml glycine/1. Radial Immunodiffusion The amount of IgG in the kallikrein solution was quantitated . . .19 . . . . . . by performing ManCini radial immunodifquion uSing Behring Diagnostic LC Partigen Low Level IgG plates (Hoechst Pharmaceuticals, 15 Inc., Somerville, New Jersey). Kallikrein solution was placed in the wells of plates containing antisera Specific for human IgG in buffered agar gel. After 60 hours' incubation at 25°C, the diameter of the precipitin rings was read. Affinity Chromatography Contaminating IgG was removed from the kallikrein solution by performing affinity chromatography in which rabbit anti-human IgG was attached to cyanigine bromide activated Sepharose 4B gel. The chromatography gel was prepared by washing 50 ml Sepharose 4B (Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey) with 500 ml distilled water and dissolving 7 gm cyanigine bromide (Aldrich Chemical Co., Milwaukee, Wisconsin) in 50 ml distilled water. In a hood, the Sepharose 4B and cyanigine bromide were mixed and the pH raised to 11 by adding 3 N NaOH. The mixture was stirred continuously while maintaining the temperature at 20°C by using an ice bath and the pH at 11 by adding 3 N NaOH. The reaction was determined to be complete when protons ceased to be released. The Sepharose 4B- cyanigine bromide mixture was then washed with 500 ml of cold 0.1 M NaHCO3, pH 10.0. Immediately following, 248 mg of rabbit anti-human IgG was added to the gel and stirred for 25 hours at 4°C. The Sepharose 4B-cyanigine bromide-anti-human IgG gel was washed with 500 ml of 0.1 M NaHCO3, pH 10. A 10x 300 mm glass column was packed with the gel and washed with PBS. The kallikrein solution was added to the column, eluted, and fractions collected and assayed for kallikrein. l6 Bioassay The kallikrein solution was tested for biological activity as demonstrated by its ability to release bradykinin from kininogen or heat-inactivated plasma as measured by contractions of the estrus rat uterus. Three—week-old female virgin rats were injected subcu— taneously with 50 pg stilbestrol (Sigma Chemical Co., St. Louis, Missouri)/100 gm body weight 48 and 24 hours before performing the assay. The rats were anesthetized with ether and one horn of the uterus was removed. The uterine segment was placed in an organ bath containing Hank's solution at 37°C with 95% 02-5% C02 gas bubbling gently through it. Contractions were detected by connecting the uterus in the Schultz-Dale apparatus to an Offner Dynograph recorder. Bradykinin standards (Sandoz Pharmaceuticals, Hanover, New Jersey) were used to find the contraction threshold. Equal amounts of kallikrein and heat-inactivated plasma or kininogen prepared according to Brockelhurst and Zeitlen3 were incubated together for 15 minutes at 37°C. A 0.1 m1 aliquot was placed in the organ bath and contractions recorded. Heat-inactivated plasma was prepared by heating fresh plasma at 60°C for 30 minutes, thus destroying any kallikrein present. Trypsin (1 mg/ml) was used as a control. Isoelectric Focusing Gels composed of 4% acrylamide were pre-electrophoresed for 45 minutes with ampholines using an upper voltage of 100v with a current of l ma/gel. The gel tops were then washed with a solution of 2% ampholines-5% sucrose. Kallikrein, to which 2% ampholines-10% sucrose were added, was placed on top of the gels (0.4 ml/gel) and 17 a 1 ma/gel current with an upper voltage limit of 100v was applied for 15 hours. Gels were then removed from their tubes and either stained in 0.2% Coumassie blue (Sigma Chemical Co., St. Louis, Missouri) or fractionated. Gels composed of 4% acrylamide were prepared in the following manner: Stock Amount Stock/Gel l. Acrylamide, 14% 3.5 gm 1.14 ml (Canalco, Inc., Rockville, Maryland) 25 ml H20 N"N'Methylenebisacry1a- 0.1 gm mide, 0.4% (Canalco, Inc., Rockville, Maryland) 2. Sucrose, 40% 10 gm/25 m1 H20 1.0 m1 (Mallinckrodt Chemical Works, St. Louis, Missouri) 3. Ammonium Persulfate, 57 mg/lO m1 H20 0.4 ml 0.57% (Canalco, Inc., Rockville, Maryland) 4. Distilled water 1.26 ml 5. N,N,N',N', Tetramethylene- 0.005 ml diamine (Canalco, Inc., Rockville, Maryland) 6. Ampholines, 40% (Sigma 1.4 m1/3.5 m1 H 0 0.2 ml Chemical Co., St. Louis, 2 Missouri) A 0.01 N NaOH in 5% sucrose solution was used for the cathode chamber buffer and a 0.01 N H2804 in 5% sucrose solution was used as the anode chamber buffer. Fractionation was performed by freezing the gels for 10 minutes and then cutting them in 1 mm sections. The gel sections were placed 18 in PBS, mashed, and put in a 37°C water bath with gentle Shaking for 1 hour. To insure complete elution of protein from the gels, the gels were stored overnight at 4°C. Kallikrein activity was tested for by using the BAEe assay on the eluateS of each gel section. One gel was cut in 1 mm sections and placed in 1 ml water overnight. The pH of each section was obtained. Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed on 7.5% gels using the method obtained from Fundamental Techniques in Virology.21 SDS Electrophoresis SDS electrophoresis was performed on 5% polyacrylamide gels containing 1% Sodium Dodecyl Sulfate (Pierce Chemical Co., Rockford, Illinois). The electrophoresis buffer was 0.1 M phosphate, 0.1% SDS, pH 7.1. Protein samples consisting of 100-250 ug protein/gel were electrophoresed at 5—6 ma/gel for l to 1-1/2 hours. The gels were stained with 0.25% Coumassie blue and destained with 15% acetic acid. The gel and bands were measured for amount of migra— tion in millimeters. The molecular weight of kallikrein was obtained from a Standard curve. The standard curve was drawn on semi-log paper from points of marker proteins (i.e., IgG, albumin, etc.) plotting molecular weight against migration in millimeters. Concentration of Kallikrein Solution The kallikrein was concentrated by placing it in dialysis bags and covering the bags with G 200 gel powder (Pharmacia Fine Chemicals, l9 Piscataway, New Jersey) until the desired volume was reached. Assays for Plasmin and Thrombin The kallikrein solution was assayed for plasmin and plasminogen using the caseinolysis method.l6 Thrombin was determined by its . . . . . 42 ability to Split fibrinogen. Platelet Aggregation Blood from human donors who had abstained from drugs for at least five days preceding donation was anticoagulated with one part 3.8% sodium citrate to nine parts whole blood. Plastic labware was used throughout the procedure. Platelet rich plasma (PRP) was pre- pared by centrifuging the blood for five minutes at 1,300 rpm. Platelet-poor plasma (PPP) was prepared by centrifuging at 2,600 rpm for 10 min. A Chrono-log aggregometer (Chrono-Log Corporation, Broomall, Pennsylvania) that constantly stirred the platelets at 37°C was used. To document platelet aggregation, 10% and 90% transmission were set using the PRP and PPP. The increase which occurred in the transmission of light through the platelet rich plasma as aggregates formed was followed on a continuous recorder and was used as an index of platelet aggregation. The following aggregating agents, in concentrations giving a two wave curve of aggregation, were used: the disodium salt of adenosine 5' phosphate (Sigma Chemical Co., St. Louis, Missouri) and epinephrine (Parke, Davis & Co., Detroit, Michigan). The ADP stock solution was prepared by dissolving 11.8 mg of ADP in 10 m1 immidazole (Sigma Chemical Co., St. Louis, Missouri) 20 buffered saline, pH 7.4. Working solutions were prepared by diluting 0.1 m1 Stock solution with 1.9 m1 saline. Twenty lambda working solution was added to 0.5 m1 platelet rich plasma giving a final concentration of 5 x 10.—6 M. An 0.1 m1 aliquot of a 1 mg/ml epinephrine solution was added Ito 0.5 m1 PRP resulting in a final concentration of 5.9 x 10'.2 M. Glass-activated kallikrein, bradykinin, or Trayslol- (FBA Pharmaceuticals, Inc., New York, N.Y.) inactivated kallikrein were incubated in the aggregometer with PRP for at least 5 minutes prior to activation with aggregating agents. All studies were performed within three hours from the collection of blood. Fixation of Platelets Platelets were fixed for transmission electron microscopy using a double fixation technique.22 An equal volume of 0.1% glutaraldehyde (Fisher Scientific Co., Fair Lawn, New Jersey) in 0.1 M cacodylate buffer, pH 7.4 (Electron Microscopy Sciences, Fort Washington, Pennsylvania) is added to platelet rich plasma with constant stirring to prevent coagulation of plasma proteins. After ten minutes' fixation the platelets were Spun at 2,600 rpm for 10 minutes to form a pellet. The 0.1% glutaraldehyde was removed and 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, was added carefully to avoid disturbing the platelet pellet. This second fixation was carried on for at least 3 hours to cause hardening of the platelet pellet. The platelet pellet was then post-fixed with osmium, dehydrated, embedded, stained with uranyl 21 citrate and lead citrate, and viewed on a Phillips 201 electron microscope. Serotonin Uptake Serotonin uptake studies were performed according to standard methods.10 The S-Hydroxy (G-3H) Trypamine Creatine Sulfate (Amersham/Searle Corporation, Arlington Heights, Illinois) was diluted with sterile saline to give a working solution of 25 uCi/ml. Of this solution, 0.15 ml was added per milliliter of PRP. Immediately after, 30 kallikrein units were incubated with 0.5 ml PRP and the platelets were stimulated with ADP. The samples were incubated for 60 minutes at 37°C. The samples were then washed with ice cold saline centrifuging for 10 minutes at 3,000 rpm at 4°C. The supernatant was discarded and the platelet button was dissolved in 2 ml Protosol (New England Nuclear, Boston, Massachusetts) and 1 m1 Methanol (Mallinckrodt Chemical Works, St. Louis, Missouri). Ten milliliters of Toluene Counting Fluid, prepared by adding 12 gm PPO (Research Products International Corp., Elk Grove Village, Illinois), 0.3 gm dimethyl POPOP (Research Products International Corp., Elk Grove Village, Illinois), and 3 liters Toluene (Mallinckrodt Chemical Works, St. Louis, Missouri) were added to the samples. Samples were counted for 2 minutes in a Packard Model 3003 Tri-Carb Scintillation Spectrophotometer. RESULTS Isolation and Purification of Human Plasma Kallikrein The kallikrein isolated from fresh frozen human plasma by glass activation was found to be contaminated with IgG, but not IgA or IgM by immunelectrophoresis. The amount of contaminating IgG was determined to be 10.9 mg/dl by radial immunodiffusion. Affinity chromatography successfully removed the IgG from the kallikrein. Figure 2 Shows immunoelectrophoresis agar plates of kallikrein before and after affinity chromatography. The purity of the preparation was further demonstrated by a Single band in the gamma region on 4% polyacrylamide disc gel electrophoresis and by SDS gel electrophoresis which Showed two bands: one 82 glycoprotein MW 30,000, a common contaminant of the purified preparation, and a second band, kallikrein with a MW of 100,000. A Single band was noted on acrylamide disc gel electrophoresis. The kallikrein was found to be free of thrombin, plasmin and C—1 esterase activity. The kallikrein was checked for biological activity by performing a bioassay using the estrus rat uterus. The threshold of the uterus to bradykinin was found to be 2 ng bradykinin/m1. Kallikrein, trypsin—LBTI (Lima bean trypsin inhibited), heat-inactivated plasma, and kininogen alone were not able to cause contraction of the uterus. 22 23 IMMUNOELECTROPHORESIS HORMAL HUMAN SERUM t T KALLIKREIN (PRE-AFFINITY CHROMATOGRAPHY) .-,.-~ »' é'ANTI IGG HORMALLHUMAN SERUM V é-ANTI IGG T KALLIKREIH (POST-AFFINITY CHROMATOGRAPHY) Figure 2. Immunoelectrophoresis agar slides. 24 At the time of kininogen testing the uterus was experiencing spastic contractions as seen by the fact that the peaks always returned to the base line and were evenly Spaced. Trypsin was used as a control because it can generate bradykinin from kininogen. Lima bean trypsin inhibitor (LBTI) was used to stop the action of trypsin after incu- bation with kininogen or heat-inactivated plasma. The kallikrein possessed biologic activity as demonstrated by its ability to generate bradykinin which causes contractions of the estrus rat uterus (Figure 3). The isoelectric point of kallikrein was obtained by performing isoelectric focusing. The pH of the gel in the area that the band stained was 7.6 :_.3,38 which was comprable to that reported in the literature. Kallikrein activity, as measured by the ability to split BAEe, was not obtained from any of the gel sections. Something in the process appeared to be inactivating the enzymatic active Site. The kallikrein solution was concentrated until a 0.5 ml aliquot contained 50 Kallikrein Units when tested by the BAEe assay. The Effect of Kallikrein on Platelet Aggregation The addition of 30 Units of Kallikrein to PRP did not result in aggregation. Specimens were taken for transmission electron microscopy (TEM) 2 sec and 3 min after the addition of 30 Kallikrein Units to platelet rich plasma (Figure 4). The electron micrographs Showed platelets with pseudopod forma- tions and Sphering. This indicates that although aggregation had not occurred, kallikrein had altered the disc-like morphology of 25 Figure 3. The results of the estrus rat uterus bioassay for kallikrein activity. 26 Figure 4. The effect of kallikrein on platelets. Ultra- structural studies. 27 resting platelets. Normal resting platelets are flat smooth discs with randomly dispersed organelles. The effect of kallikrein on platelet aggregation initiated by other agents was then studied. Kallikrein was Shown to influence both ADP and epinephrine aggregation. ADP and epinephrine both cause two waves of platelet aggrega— tion. The first wave of aggregation is due to the exogenous aggre— gating agent; the second wave is due to the release of endogenous ADP from the platelet granules. This phenomenon is known as the release reaction and includes the extrusion of not only endogenous ADP but also ATP, serotonin, catecholamines, potassium, and platelet factor 4 into the surrounding plasma. When 20-50 Units of Kallikrein were added to PRP prior to acti- vation with ADP, the second wave of platelet aggregation was inhibited (Figure 5). This was not dependent on the length of time PRP and kallikrein were incubated together prior to the addition of ADP (Figure 6). Samples were taken for TEM after the addition of ADP to platelets incubated with 30 Kallikrein Units. ADP alone causes dramatic internal changes to platlets as aggregation proceeds.23 At 5 seconds the platelets take on a Spherical shape with deeply convoluted surfaces. The microtubules move centrally, encircling the granules. The other organelles are also displaced to the center. 'Degranulation of platelets begins at 1 minute. By 3 minutes the platelets are in large fused aggregates with the center of the aggregate composed of degranulated empty platelets. 28 EFFECT OF KALLIKREN ON ADP ,- . I 50 UNITS ADP 5x10-6M Figure 5. The effect of kallikrein on ADP aggregation. EFFECT CF INCUBATION TIME ON KALLiKBElN. INHIBITION Figure 6. The effect of incubation time on kallikrein inhibition. 29 Platelets that have been preincubated with 30 Kallikrein Units and then stimulated with ADP Show some different structural changes (Figure 7). At 5 seconds the platelets Show central migration of granules, organelles, and microtubules. At 1 minute there are few aggregates and no degranulation. At 3 minutes the platelets have not formed fused aggregates and degranulation still has not occurred. Trayslol, a natural kallikrein inhibitor, was incubated with kallikrein at 37°C for 30 minutes. The ability of kallikrein to split BAEe after incubation was found to be reduced by 75%. Kalli- krein, incubated with Trayslol prior to addition to PRP, had no effect on platelet aggregation (Figure 8). Kallikrein added to platelets in concentrations of 10-30 Kallikrein Units inhibited the second wave of platelet aggregation due to epinephrine (Figure 9). Epinephrine itself has little effect on the ultrastructure of platelets.23 At 5 seconds after the addition of epinephrine, platelets appear plump but do not have marked convolutions of the surface. The organelles remain randomly dispersed inside the plate- let. This morphology is Similar to that of the unstimulated platelet. At 1 minute platelets are sphered with the majority still containing randomly dispersed organelles. Some platelets Show central migration of organelles and deep surface convolutions typical of ADP stimula- tion indicating that the release of ADP has occurred. At 3 minutes the large epinephrine aggregates are identical to those seen in ADP- stimulated 3-minute samples. Figure 7. The effect of kallikrein on ADP aggregation. Ultrastructural studies. EFFECT OF KALLIKREIN TRAYSLOL ON .ADP AGGREGATION ADP 313x10"6 M Figure 8. The effect of kallikrein-Trayslol on ADP aggregation. 31 EFFECT OF KALLIKREIN ON EPINEPHRINE AGGREGATION MM” ou~rrs 7. / - 1.1.11.1...” 10 UNIT * _ 30 UNITS -_. _.- EPINEPHRINE 59x10‘2mglml Figure 9. The effect of kallikrein on epinephrine aggregation. ,4... Figure 10. The effect of kallikrein on epinephrine aggre- gation. Ultrastructural studies. 32 Platelets incubated with 30 Kallikrein Units and then stimulated with epinephrine (Figure 10) show extensive pseudopod formations at 5 seconds. At 1 minute platelets are still showing pseudopod forms and randomly dispersed granules. The 3 minute sample shows very few loose aggregates with a small degree of centralization of organelles and microtubules indicating that some slight degree of ADP release has occurred. However, fused aggregates and complete degranulation are absent. The Effect of Bradykinin on Platelet Aggregation Bradykinin's effect on platelet aggregation was examined. Synthetic bradykinin, incubated with PRP in concentrations of 1 mg- 25 ng bradykinin/ml prior to stimulation with ADP, had no effect on the two-wave curve of platelet aggregation (Figure 11). Samples were taken for TEM 15 seconds and 3 minutes after the addition of brady- kinin to platelets giving a final concentration of 25 ng/ml (Figure 12). Both samples showed flat discoid platelets with a small amount of pseudopod formation. Serotonin Uptake The results of each group are averages of triplicate samples. The results are given in counts per two minutes. A B C Platelets—ADP 21,100 22,000 23,700 Platelets-Kallikrein-ADP 22,400 26,100 24,300 Platelets-Kallikrein 23,800 26,400 23,900 Platelet control 23,600 23,500 19,600 33 -7 EFFECT OF BRADYKININ ON ADP AGGREGATION Ono/ml n 1"”? I 11 5 ng/ml .31--. i ..... - 1 1;- 1 -93 11.1111! 1 ,- f. 111 111 25nglml O: i _ 1.11.“. 11-11. -1 v. ; f ‘ . m?— ' 1 t : I . - +_-...-._.__--_._‘¥ _-_-- 1! .__. _. . . . . ~39 ; (J. _, 11111111111 _ ? :-;/. _ _ ,; , P: 5x10‘6 M 9' t 4. 111111111--. Figure 11. The effect of bradykinin on ADP aggregation. Figure 12. The effect of bradykinin on platelets. Ultra- structural studies. 34 The anticipated results were that kallikrein would protect the serotonin storage organelles as demonstrated by transmission electron microscopy studies and give higher counts for platelets incubated with kallikrein prior to stimulation with ADP than platelets just stimulated with ADP. Because the platelet controls which should have had the highest counts did not work out, no conclusions based on these results can be drawn. Unstimulated control platelets do not aggregate and therefore are lighter than stimulated platelet aggregates. In the washing procedure the lighter control platelets could more easily be lost resulting in the low values. Trypsin Aggregation Aggregometer studies were done with trypsin. A two wave curve identical to the two wave curve of ADP was obtained by adding 0.025 ml of a lmg/ml solution of trypsin to 0.5 m1 PRP resulting in a final concentration of 0.0176 mg/ml trypsin. Trypsin in concentrations resulting in BAEe hydrolysis equal to the hydrolysis of BAEe caused by 50 Kallikrein Units did not result in aggregation and had no effect on ADP two wave aggregation. DISCUSSION A biologically pure preparation of kallikrein with a molecular weight of 100,000 and isoelectric point of 7.6 was prepared by glass bead activation. The preparation was found to be free of plasma and thrombin. The effects of the kallikrein solution on platelet aggre- gation are not due to C-1 esterase. Trayslol does not inhibit C-l esterase activity in the concentrations used to eliminate kallikrein inhibition of the second wave of platelet aggregation. Trypsin in concentrations giving esterase activity of BAEe similar to that obtained by the kallikrein had no effect on ADP and epinephrine- induced platelet aggregation. Kallikrein stimulates platelets in the absence of aggregation as seen when electron micrographs of kallikrein-treated platelets are examined. The platelets are sphered with pseudopods and appear to be in a refractory state to subsequent activation by conventional aggregating agents such as ADP or epinephrine. Platelets that have been incubated with kallikrein prior to addition of ADP or epinephrine fail to produce second wave aggrega- tion. This is most likely due to the diminished granule release seen in electron micrographs of 1 minute and 3 minute samples of kallikrein- treated platelets stimulated with ADP or epinephrine. The 5 second sample of kallikrein-treated platelets stimulated with epinephrine 35 . .. '14th :- mnr'v... 5"?" 36 demonstrated pseudopod formation and sphering similar to platelets This is quite different than the incubated with kallikrein alone. somewhat plump smooth disc-like morphology of platelet samples 5 seconds subsequent to addition of epinephrine alone. Platelets incubated with bradykinin appear as normal resting Aggregation due to platelets with very few pseudopod formations. ADP proceeds through the normal two waves even when platelets are This causes some doubt about whether incubated with bradykinin. bradykinin generation in PRP is the mediator of kallikrein's effects on platelets. Increasing the incubation time of platelets and kallikrein prior to addition to PRP points out the necessity of the active site of the enzyme for the inhibition of the second wave of platelet aggregation. These experiments suggest that kallikrein may have an inhibi- tory effect on platelet aggregation at a time when many agents are This action of being generated to cause platelet aggregation. kallikrein may serve as a safeguard against the formation of excessive thrombi in inflammatory processes. CONCLUS ION The enzymatically active site of a biologically pure prepara- tion of human plasma kallikrein inhibited the second wave of human aggregation. This was apparently obtained by kallikrein's antagonism to the platelet release reaction as seen by the inability of kalli- krein—treated platelets to release their granules when stimulated. The exact mechanism of kallikrein's actions on platelets is not known. The inability of bradykinin to affect platelets causes doubts about bradykinin generation as the mediator of kallikrein's effects. Kallikrein stimulates platelets to a certain degree, as seen by the sphering and pseudopod formation of kallikrein-treated platelets. This may cause platelets to be in a refractory state to subsequent activation by conventional aggregating agents such as ADP and epinephrine. 37 LIST OF REFERENCES 10. 11. LIST OF REFERENCES Born, G. V. R. Aggregation of Blood Platelets by Adenosine Diphosphate and Its Reversal. Nature 194:927-928. 1962. Booyse, F., and Rafelson, Jr., M. Regulation and Mechanism of Platelet Aggregation. Annals New York Academy of Sciences 37-60. 1972. Brocklehurst, W., and Zeitlin, I. Determination of Plasma Kinin and Kininogen Levels in Man. J. Physiol. 191:417-426. 1967. Cochrane, C., and Wuepper, K. The First Component of the Kinin- Forming System in Human and Rabbit Plasma. Journal of Experi- mental Medicine l34:986-1004. l97l. Colman, R., Mason, J., and Sherry, S. The Kallikreinogen- Kallikrein Enzyme System of Human Plasma. Ann. Int. Med. 71:763-773. 1969. Colman, R., Mattler, L., and Sherry, S. Studies on the Prekallikrein (Kallikreinogen)-Kallikrein Enzyme System of Human Plasma. 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The author attended Edgewood College in Madison, Wisconsin, and completed a medical technology internship at St. Mary Hospital Medical Center in Madison, Wisconsin. She graduated with a Bachelor of Science Degree in Biology and Medical Technology in May, 1972. The author worked for eighteen months at Central Dupage Hospital in Winfield, Illinois, as a staff technologist before beginning a course of study at Michigan State University in the Clinical Labora- tory Science program in the Department of Pathology in January, 1974. 42 MICHIGAN STATE EUNI IVERSITY LIB RAR IIIILI IIIL II ILIIIIILI II III II