" ‘ "w ‘ OVERDUE FINES: 25¢perdqyperiten RETURNIMS LIBRARY MATERIALS: Place in book return to remove charge from circulation records THE PROSTAGLANDIN SYNTHESIS PATHWAY OF PLATELETS IN MINK AFFECTED WITH THE CHEDIAK-HIGASHI SYNDROME BY Wayne R. Patterson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1979 ABSTRACT THE PROSTAGLANDIN SYNTHESIS PATHWAY OF PLATELETS IN MINK AFFECTED WITH THE CHEDIAK-HIGASHI SYNDROME By Wayne R. Patterson The possibility of a defect in prostaglandin synthesis in Chediak-Higashi mink was studied, and it was concluded that no dif- ference exists between Chediak-Higashi and non-Chediak—Higashi mink as concerns prostaglandin synthesis. Chemiluminescence, platelet aggregations, and malonyldialdehyde assays were utilized to challenge this hypothesis. Significant differences were not observed (P<0.0l) between the two test groups. It appears that, at least in mink, the primary defect responsible for prolonged bleeding associated with the Chediak-Higashi syndrome is the decreased amount of secretable (storage) pool adenine nucleotides. The possibility that a defect exists in prostaglandin synthesis in other species affected with Chediak-Higashi syndrome is doubtful due to the homology of the disorder between species; however, more research in this area is needed in order to make this conclusion. TO DEBBIE AND MEGHAN the most important assets in my life ii ACKNOWLEDGEMENTS Any verbal or written expression of gratitude to Dr. George A. Padgett seems inadequate. As my major professor and chairman of my graduate committee, he supplied continual and concerned support throughout my research; but in my opinion, most importantly he taught me to understand the importance of the educational experience beyond the classroom, which undoubtedly will prove to be a valuable asset throughout my career. For his invaluable advice and insight, I am eternally grateful. Special thanks are deserving to the other members of my committee also. Dr. Thomas Bell, Dr. Robert Bull, Ms. Kathy Colando, and Mrs. Martha Thomas provided continual support, which especially with Dr. Bell was at times more psychological than technical. Kathy and Martha, who I guess would be considered as dual academic advisors, first helped me adjust to my return, after five years, to the academic environment, which was totally unlike that I had ever experienced before. Ms. Irene Brett was very helpful by providing technical advice and assistance whenever it was needed, and a well deserved expression of thanks is extended to her. Although I have dedicated this thesis to my wife and daughter, I am especially grateful for the patience and understanding that my wife, Debbie, showed during difficult times. She deserves as much of the credit as I for the accomplishments made during my graduate program. iii TABLE OF CONTENTS INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O LITEMTUE “VIEW 0 O O O 0 O O O O I O O O O O O O O O O 0 General Aspects of the Chediak—Higashi Syndrome . . Platelet Anatomy. . . . . . . . . . . . . . . . . . Platelet Production . . . . . . . . . . . . . . . . Platelet Aggregating Agents . . . . . . . . . . . . Platelet Function . . . . . . . . . . . . . . . . . Prostaglandin Synthesis . . . . . . . . . . . . . . Control of Prostaglandin Production and Aggregation Measurement of Prostaglandin Production . . . . . . Chemiluminescence . . . . . . . . . . . . . . . . . Drug Effects on Platelets . . . . . . . . . . . . . MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . Animals . . . . . . . . . . . . . . . . . . . . . . Blood and Platelet Collection . . . . . . . . . . . Platelet Aggregations . . . . . . . . . . . . . . . Collagen Preparation. . . . . . . . . . . . . . . . Arachidonic Acid Preparation. . . . . . . . . . . . Chemiluminescence Measurements. . . . . . . . . . . Malonylaldehyde Assay . . . . . . . . . . . . . . . Platelet Particulate Fraction . . . . . . . . . . . Aspirin Preparation and Injection . . . . . . . . . Statistical Analysis. . . . . . . . . . . . . . . . mSI-JLTS. O O O O O O O O O O O O O O O O O O O O O O O O 0 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . VITA iv Page 21 21 21 22 23 23 24 24 26 26 27 28 42 45 52 Figure LIST OF FIGURES Page Possible fates of arachidonic acid released from membrane phospholipids. . . . . . . . . . . . . . . . . . l3 Chemiluminescence response plotted as cpm x 103 vs. time. 0 O O O O O I O O I O I O O O O O O O O O O O O O 0 30 Mean malonyldialdehyde assay results produced by non-C-HS and C-HS mink platelets during chemi- luminescence induced with arachidonic acid. . . . . . . . 32 Mean malonyldialdehyde assay results produced by non-C-HS and C-HS mink platelets during platelet aggregation induced by arachidonic acid and collagen. . . 34 Mean malonyldialdehyde assay results produced by non-C-HS and C-HS mink platelets during platelet aggregation induced with arachidonic acid and collagen 1 hr after aspirin injection . . . . . . . . . . 37 Platelet aggregation curves for non-C-HS and C-HS mink in response to arachidonic acid (8 uM) and collagen (0.05 ng/ml) . . . . . . . . . . . . . . . . . . 39 Platelet aggregation curves for non-C-HS and C-HS mink in response to arachidonic acid (8 uM) and collagen (0.05 ng/ml) 1 hr after aspirin injection. . . . 41 ADP ATP BHT c-AMP C-HS cpm EDTA 5-HT HBSS LASS MDA PGD PGE PGE PGF PGG PGH PGI PPP PRP LIST OF ABBREVIATIONS Adenosine diphosphate Adenosine triphosphate Butylated hydroxytoluene Cyclic-adenosine monophosphate Chediak-Higashi syndrome Chemiluminescence Counts per minute Deoxyribonucleic acid Ethylenediaminetetraacetic acid 5-Hydroxytryptamine Hank's balanced salt solution Labile aggregation stimulating substance Malonyldialdehyde Prostaglandin D2 Prostaglandin E Prostaglandin E Prostaglandin F Prostaglandin G Prostaglandin H Prostacyclin Platelet poor Platelet rich plasma plasma vi TBA Thio-barbituric acid TxA2 Thromboxane A2 vii INTRODUCTI ON The Chediak-Higashi syndrome (C-HS) is a genetic disease that . . . . l . 2,3 is known to occur in 6 different speCies: man, mink, cattle, 4 . 5 . 6 . . . cats, mice, and killer whales. The syndrome is inherited as an autosomal recessive trait7 and has several striking defects which affect all species in much the same way. One of the readily observed defects is the formation of abnormally large intracyto— plasmic granules in neutrOphils and other cells which normally produce such structures. Affected individuals of all species display a partial oculocutaneous albinism which can be seen in the hair, skin, and eyes10 due to the presence of giant melanin granules in melanocytes.ll In every species affected with C-HS, there are reports of increased susceptibility to infection.12 All species show a marked increase in frequency and severity of infections when exposed to agents that cause either minor infections or no infection at all in individuals not affected with C-HS.13'14'15 Lastly, another defect in C-HS individuals is an abnormally prolonged bleeding time. Prothrombin times, partial thromboplastin times, coagulation factor assays, and platelet counts all are within the normal ranges.16 This then indicates a defect in the platelet itself.l7 Several investigators subsequently reported a storage pool defect characterized by decreased stores of serotonin (5-HT) 17,18,19 and non-metabolic adenine nucleotides, which are stored in the dense granules of the platelet.20 It is the release of the storage pool adenine nucleotides, namely adenosine diphosphate (ADP), that is responsible for the second wave of aggregation that is observed during platelet aggregation induced with epinephrine and ADP.21'22 It is well accepted that the cause of the prolonged bleeding time in C-HS individuals is the reduced amount of "storage pool" constituents. However, the possibility that there may be a defect in the arachidonic acid to prostaglandin pathway, which recently has evolved as "the other mechanism" of platelet aggregation, has not been studied in C-HS. The pathway has been elucidated and the interactions of by-products of the pathway have been studied in depth.23'24'25 Several intermediates and by-products of the pathway are known to be potent inducers of aggregation. The purpose of this research project was to compare the prosta- glandin pathway of C-HS affected mink to non-C-HS mink. The methods used to study the prostaglandin pathway activity and viability were chemiluminescence, platelet aggregations, and measurement of a stable by—product of the pathway, malonyldialdehyde (MDA). After correlating the results, a useful comparison was made and evaluated in order to determine if any defect existed in the prostaglandin pathway of C-HS mink. LITERATURE REVIEW General Aspects of the Chediak-Higashi Syndrome The first published reports of the Chediak-Higashi syndrome were those of Bequez-Cesar (1943),1 in which he observed abnormal granules in the leukocytes of children from the same family. In addition to the abnormal granule formation, he also described partial albinism, and an increased susceptibility to infection, in these C—HS children. Several years later, Chediak (1952)26 and Higashi (1954)27 reported on the same syndrome, and in 1954 Sato28 named the condition the Chediak-Higashi syndrome. In the years to follow, several investigators described the occurrence of C-HS in other species. Leader et al. (1963)2 reported finding abnormally large intracytoplasmic granules in mink leukocytes. Padgett et al. (1964)3 were the first to report the occurrence of the Chediak-Higashi syndrome in mink and cattle, with particular reference to a familial (auto- somal recessive) occurrence in these species. Abnormal granules in the leukocytes of the beige mouse were reported by Lutzner et al. (1967)5 and Taylor and Farrell (1973)6 reported the same finding in killer whales. Kramer et al. (1974)4 found abnormal melanin and leukocyte granules in blue smoke colored Persian cats and compared their results to those reported in other species affected with C-HS. Reviews have been published7'8 that compare each species as to clinical and morphological findings. There seem to be 4 characteristics 4 common to all species affected with C-HS, with only minor variations between species. The first of these characteristics is the develop- ment of abnormally large intracytoplasmic granules in cells that normally produce these structures. The two most commonly studied cells exhibiting this defect are neutrophils and melanocytes. Electron microscopic and biochemical studies have determined that the large granules in neutrophils are aberrant primary granules, some of which contain various acid hydrolases. Normally, proteins synthesized by polyribosomes on the endoplasmic reticulum move through the endoplasmic reticulum to the Golgi complex, where they are concentrated, surrounded by a membrane, and ultimately released into the cytoplasm as primary granules. Any theory that explains the abnormal formation must then encompass all cells that produce granules and not be specific for one cell type, i.e., in all cells the formation of the granules is Golgi mediated.7 Lutzner et al. (1965)29 suggested that the Golgi involvement could be one of the following: a defect in the transfer of material from endoplasmic reticulum to Golgi bodies, a defect in the process of granule assembly, or a defect in granule membrane formation, any of which could be caused by a mutant gene. Another feature of C-HS is a partial oculocutaneous albinism secondary to fusion of melanocytic granules. Melanocytes are cells that are reSponsible for skin pigmentation. In C-HS, the pigmentary dilution is caused by an abnormal fusion of melanocyte granules into large melanin-containing structures with the albinism occurring because of an abnormal melanosome distribution. This defect can be seen in the hair, skin, and eyes of affected individuals of all . 10,11 speCies. 5 A third characteristic common to the Chediak-Higashi syndrome is the increased susceptibility to infection. Affected individuals of all species seem to have more frequent and severe infections than non-C-HS individuals. In all species there have been reports of severe pyogenic infections in C-HS individuals, caused by agents that also affected non-C—HS members of each species. The non-C-HS indi- viduals, however, were not stricken as frequently or as severely as their counterparts. Chediak-Higashi syndrome cattle exhibited greatly increased bacterial infection rate, while non-affected cattle exposed to the same infectious agents often had no infection at all.12 In mink, the findings are similar. Chediak-Higashi syndrome mink can exhibit an almost chronic periodontitis and seem to be more susceptible to pyogenic abscesses (personal observation). In addition, in mink there is a particular susceptibility to a disease of viral etiology (i.e., Aleutian disease). All mink may contract Aleutian disease; however, in C-HS mink the disease follows a more severe and rapid course with the life expectancy of C-HS mink being much shorter than 12,13 non-C-HS mink. There are several reports that indicate that neutrophil dysfunction may be the primary cause of the increased 31,32,33,34 susceptibility to infection. It appears that the defect lies in impaired intracellular killing of bacteria by neutrophils in C-HS and not in normal individuals.15 Several investigators have implicated an abnormal microtubular assembly to account for defective 35,36 chemotaxis, which is also present in C-HS individuals, and one researcher has reported a correction of the defective chemotaxis by administering ascorbate, which is said to lower the intracellular level of cyclic-adenosine monophosphate (c-AMP).37 6 Lastly, a defect common to all species affected with C—HS is an abnormally prolonged bleeding time and hemorrhagic tendency which was recognized relatively early in the study of the disease. Several investigators attempted to determine the cause of this hemorrhagic disorder, employing many different methods. Prothrombin times, partial thromboplastin times, platelet counts, and clotting factor assays were done, and all results were found to be within normal limits.16 However, the template bleeding times were in some cases as much as 10 times normal.17 These results suggested an intrinsic platelet defect in C-HS. Platelet aggregations of C-HS individuals revealed a marked reduction in the extent and rate of aggregation in response to collagen and an absence of secondary aggregation in response to critical concentrations of ADP and epinephrine.17 Bio- chemical analysis of platelet-rich plasma (PRP) yielded data showing a marked decrease in adenine nucleotides (ATP and ADP), an increase in the ATP/ADP ratio, and a reduction in the amount of stored sero- 7 . . 1 ’19 Several investigators tonin as well as total serotonin uptake. have reported that serotonin and the nonmetabolic adenine nucleotides ATP and ADP are stored in the same granules (the dense granules) of the platelet.18'20 All of the above-mentioned studies suggest a "storage pool" disorder which leads us to a discussion of platelet anatomy and function. Platelet Anatomy In the peripheral circulation of normal individuals, the platelet is disc-shaped and is about 2.5 pm in diameter. One of the most remarkable characteristics of platelets is the capability to change shape in reSponse to a number of stimuli.38 The shape change involves 7 an increase in surface area with little increase in volume. The changes can be seen microsc0pically by the formation of pseudopodia 39,40 as it transforms into a "spiny sphere." This change of shape appears to be the first response of platelets to almost any stimulus,41 including exposure to cold; but more importantly it is achieved by the addition of various aggregating agents which will be discussed later. The outer layer of the platelet is a relatively thick glyco- protein coat which may play a role in platelet adhesion. Inside this coat is a trilaminar platelet membrane containing platelet specific proteins.42 Two types of granules have been described in the platelet cytosol: primary (alpha) granules and dense bodies. The primary granules are the most numerous and have been shown to contain various acid hydrolases. The dense bodies are less numerous and contain adenine nucleotides, serotonin, and calcium. The adenine nucleotides in the dense bodies are ADP and ATP, which are metabolically inert 40’42 Platelets and are considered to be "storage pool" constituents. are capable of carrying out the glycolytic, the tricarboxylic acid cycle, and the hexose monophosphate shunt reactions for the production of ATP needed in energy requiring reactions.39 One of the energy consuming reactions that utilizes the metabolic ATP is shape change, which was previously mentioned, and is mediated by the action of the microtubular system. Platelet Production Normal bone marrow contains giant multinucleated cells, cells 39 called megakaryocytes, which arise from multipotential stem cells. The nucleus of the megakaryocyte divides without division of the 8 . . . 39,43 cytoplasm, and in a mature megakaryocyte the nucleus is polyplOidy. The cytoplasm increases in volume during this process and is baso- . . . . . 43,44 philic, but becomes aCidophilic as the megakaryocyte matures. The mature megakaryocyte cytoplasm, by invaginations of the plasma membrane, forms demarcation membranes encapsulating cytoplasm.43'44 The platelets are then separated from the megakaryocyte and, when the cytoplasm is depleted, the remaining nucleus is promptly disposed of by macrophages.43'44 The regulation of megakaryocyte proliferation and, ultimately, of platelet production is very strictly controlled, and there is recent evidence for the existence of thrombopoietin, a substance similar to erythropoietin, that regulates platelet produc- tion on a hormonal 1evel.45'46 Normally, platelet production is approximately equal to platelet destruction, with a normal platelet count being between 150,000 and 450,000, with the mean of about 250,000/mm3 in the peripheral circulation. The life span of the platelet is about 10 days.44 Platelet Aggregating Agents As a prelude to discussing platelet function, it would probably be helpful to review some of the agents that cause visible changes in the platelet, i.e., platelet aggregation. Platelet aggregation can be induced in vitro in normal individuals by numerous compounds. Among these are low molecular weight molecules (ADP, epinephrine, serotonin), proteolytic enzymes (thrombin, trypsin, snake venom), particulate matter (collagen, fatty acids, latex particles, endotoxin, viruses, antigen-antibody complexes), strongly positive charged ions (poly- lysine), and an antibiotic (Ristocetin).39 The most commonly used aggregation inducers, for the assessment of a bleeding diathesis that 9 is platelet related, are ADP, epinephrine, and collagen. Some of the other agents are useful if measurement of released products is desired but may not provide insight into aggregation problems. Platelet Function Normally, platelets circulate in the peripheral blood in a discoid shape for approximately 10 days, which is their normal life span. However, when vascular trauma occurs which exposes certain tissues in vessel walls to circulating platelets, a rapid chain of events takes place that stops the bleeding and allows for healing of the wound. There are several reactions that occur in platelets that allow them to arrest bleeding by "plugging" the hole from which bleeding is occurring. The series of events that takes place in the order that they occur is as follows: adhesion to exposed tissue and subsequent shape change, release of granule contents, and aggrega- tion caused by released products.42 Each of these reactions will be discussed further in subsequent pages. Trauma to a blood vessel that disrupts the endothelium exposes subendothelial layers to which the platelets can adhere. Of all the components of the subendothelial layers, platelets have the greatest 42'47 The affinity for collagen is very affinity for collagen. specific and dependent on the prOper quaternary structure in order to elicit the required reactions. The helical portion of the collagen molecule is necessary to initiate the platelet response.48 Platelets moving in the blood stream recognize the exposed collagen and immedi- ately adhere to it. The exact mechanism of adhesion is unknown at present. The next reaction to occur is shape change. As previously mentioned, shape change is the first reaction of the platelet to most 10 stimuli and is a sign of platelet stimulation. A small and reversible amount of aggregation directly to collagen fibers takes place and lays the groundwork for the following reactions necessary for the formation of a stable hemostatic plug. The release mechanism is actually a complex set of reactions which causes the expulsion of the platelet granules. There are actually two release reactions. Release I is the release of dense body contents, i.e., ADP and serotonin,49 and Release II is the release of the alpha granule contents.SO Release I can be induced by ADP and epinephrine,48 and Release II is induced by thrombin or collagensoand occurs later than Release I. After adhesion of platelets to collagen, the platelet is stimulated by an unknown mechanism to release its granule contents into the surrounding medium. One of the constituents of the dense bodies, ADP, has been shown to be a potent inducer of platelet aggre- . 40,42,47,51 gation. The released ADP then causes other platelets which are flowing by to aggregate, and the process continues until the platelet plug is formed. The formation of the platelet plug is called primary hemostasis and without release is reversible, and the aggregation of platelets by released ADP is irreversible second wave aggregation. Controls on the mechanism of primary hemostasis will be discussed later. Prostaglandin Synthesis Another mechanism which occurs simultaneously with and causes release was not discussed in the preceding section because of its complexity, but should not be thought of as separate. This mechanism is the production of prostaglandins from arachidonic acid. The investigation of the conversion of arachidonic acid into biologically 11 active compounds is relatively young, with most of the research starting in the early 19705. In 1973 a biochemical intermediate in prostaglandin synthesis, labile aggregation stimulating substance (LASS), was isolated and proven to act as a physiologic intercellular 52,53 The messenger that promoted platelet aggregation and release. relationship between prostaglandin synthesis, the platelet release reaction, and second wave aggregation was recognized fairly early. It has been known for some time that the platelet release reaction is associated with second wave aggregation (aggregation caused by the release of endogenous ADP from platelets). Several investigators blocked prostaglandin production with the use of aspirin, and found 24,54 that this also blocked second wave aggregation. It was then suggested that, because of their potent aggregating abilities, the prostaglandin endoperoxides, prostaglandin G (PGGZ) and prostaglandin 3,55 2 . . . 2 H (PGHZ), played an important role in platelet aggregation and 2 are the first products that are biologically active in the prosta- 56 57 . . . ' These endoperoxide intermediates, however, are glandin pathway. very short-lived and degrade to more stable and measurable end- products. Figure l is a representation of the prostaglandin pathway of active platelets, showing the ultimate fate of arachidonate and the endoperoxides. The arachidonic acid to prostaglandin pathway was first eluci- 58'59 and later found to be true for dated in sheep vesicular glands platelets. Platelet membranes contain phospholipids which are made up of fatty acids; the most important, as far as the platelet is concerned, is arachidonic acid. When platelets are exposed to collagen and thrombin, a specific phospholipase (phospholipase A2) is acti- 6 . . . . - vated O which cleaves arachidonic aCid from the membrane phosphatides, 12 Figure 1. Possible fates of arachidonic acid released from membrane phospholipids. Cyclooxygenase is the key enzyme for platelet aggregation and is irreversibly acetylated by aspirin. Prostaglandin Gz (PGGZ), PGH2 and thromboxane A2 are potent inducers of aggregation; PGEz acts synergistically with biological aggregating agents to potentiate aggregation; and PGIZ is a potent inhibitor of platelet aggregation by stimulating adenylate cyclase. 13 Thrombin Membrane Collagen Phospholipid Epinephrine I «1r Phospholipase A2 lZ-hydroxy- Lipooxygenase Arachidonic Acid perOXidase 4 Cyclooxygenase HETE PGG2 Prostacyclin PGI2 PGFla“*”,/’gf” u§~\\\\‘\“. ® I m R d t PGH2 so erase PGE2 e uc ase._PGF2a MDA / PGD Thromboxane 2 Synthetase Thromboxane A2 Thromboxane 82 Figure l 14 42'61'62 The available phosphatidylcholine and phosphatidylinositol. arachidonic acid then is converted, through the prostaglandin endo- peroxides, to prostaglandin E2 and an (PGE2 and PGan) by the action of cyclooxygenase, prostaglandin synthetase, and other enzymes.61'63'64 The end-products of the pathway, PGE2 and PGan, have been shown to act synergistically with aggregation inducers, but in low concentra- tions are not actually aggregation inducers per se.55 It has been shown that the formation of prostaglandin endo- peroxides is associated with the release reaction of platelets, namely second wave aggregation, but the exact association is unclear. Most investigators believe that endoperoxide formation precedes and is the cause of release of platelet granules. A recent article, however, draws conclusions that are perhaps slightly divergent. Charo et al. (1977)65 suggest that there may be two different methods of inducing release, one being aggregation dependent and the other being aggregation independent. Their data show that release and second wave aggregation are parallel events and that one does not necessarily cause the other. Because of these data, the theory that second wave aggregation is caused by released ADP, etc., may in fact be incorrect. Two other articles suggest the possibility that the released substances, adenine nucleotides and serotonin, may act as cofactors for prostaglandin synthesis.22'25 In one article by Willis and Weiss,22 storage pool deficient platelets were shown to have defective prostaglandin production. It should be obvious, then that there is much more research needed in this area. 15 Control of Prostaglandin Production and Aggregation In any biological process, the control of that process is tanta- mount in preventing overstimulation or overreaction of that process, and platelet function is not an exception. Normally the response to vascular injury is rapid and complete in attaining hemostasis, and the control of the mechanisms involved is taken for granted unless thrombotic episodes occur. Numerous drugs, to be discussed later, have been used in an attempt to augment the biologic control, but we will now consider some of the physiological control mechanisms. Until 1971, not much was known about the blockade of prostaglandin synthesis. At that time it was pr0posed that variations in the oxygenation system of prostaglandin synthesis could be a rate-limiting step.66 In 1974, a previously considered physiologically inert prostaglandin, PGD , was found to be very portent as an inhibitor of 2 . 7 . . platelet aggregation.6 In fact, it was found to be more than tWice as potent as PGE as an inhibitor of platelet aggregation in Vitro. l Prostaglandin E1, which is a derivative of the metabolism of dihomo- y-linolenic acid,68 was shown to be an inhibitor of platelet aggre- 69,70 gation. Holmsen (1977)63 reported a positive feedback mechanism that could account for the rapid hemostatic reaction of platelets to vessel trauma. He stated that exogenous substances, the platelet membrane, and released substances act synergistically to potentiate platelet aggregation and hemostasis. He made no mention, however, of a negative feedback system. Recently a series of articles appeared that stress the importance 68,71,72 of prostacyclin, PGIZ. Prostacyclin was first characterized by Moncada et al. (1976) to be derived from PGH and, among other 2 properties, is an inhibitor of platelet aggregation. In platelets, 16 the major metabolite of prostaglandin synthesis is thromboxane A2 (TxAz),68 a potent inducer of platelet aggregation.73 It is suggested that a delicate balance exists between TxA2 and PGI2 production, and it is this balance that regulates platelet aggregation.68 Prosta- 68,71 PGH2 cyclin production is confined mainly to vascular tissue. produced by the platelet might migrate to vessel walls and be converted to PGIZ.71 Thromboxane A2 and PGI2 have opposite effects on c—AMP levels, which may be the mechanism by which regulation of aggregation occurs (TxA2 decreases c-AMP levels and PGI2 increases c-AMP levels).68'71 The process of regulation of thrombosis is interesting and intriguing and is now a frequent topic of discussion; however, exact mechanisms of regulation will require more research before they are generally accepted. Measurement of Prostaglandin Production In order to ascertain the activity and viability of any enzymatic pathway, measurement of intermediates, end-products, or by-products of that pathway is necessary. Measurement of intermediates of the prostaglandin pathway, however, is particularly difficult due to the short half—life of the endoperoxides, PGG and PGH .74 Equally diffi- 2 2 cult to quantify are the end-product prostaglandins, PGE2 and PGFZa' Measurements of PGE2 and PGFZa in the past have been done by thin- 22 . . 25 . layer chromatography or radiOimmunoassay procedures, which are not inherently difficult but, due to the small and sometimes variable amounts of these prostaglandins that are produced, give results that . . 74 . are oftentimes equally variable. An often used and more reliable method for assessing prostaglandin synthesis is measurement of a stable by-product of the PG pathway, malonyldialdehyde (MDA).75 l7 Malonyldialdehyde is produced in quantities directly proportional to the amount of prostaglandins that are produced,64 and is biochemi- 74'75'76 The method cally stable and measurable by direct methods. involves reacting an acid extract containing MDA with thiobarbituric acid, and then reading the absorbance at 532 nm on a spectrophotometer. Chemiluminescence A recently published method being employed to assess the activity of the arachidonic acid to prostaglandin pathway is chemiluminescence 77,78 (CL). Chemiluminescence was initially employed with human neutrophils79'80'81 81’82 and later with monocytes to measure phago- cytic activity. There is some disagreement as to the cause of the chemiluminescence. Some investigators suggest that is is caused by a decay of a singlet oxygen from an excited state to a more relaxed state, or perhaps by electronically excited compounds formed in secondary reactions between a singlet oxygen and other molecules. For instance, carbon-carbon double bonds could react with singlet oxygen to form dioxetanes which, in turn, cleave to form aldehydes and ketones.80 Nelson et al. (1978)82 suggests that, since CL can be reduced by the addition of superoxide dismutase, a possible source of CL is the non-enzymatic conversion of superoxide (a product of oxidative metabolism) to singlet oxygen, which then relaxes to a ground state. This might explain the reports that the decay of singlet oxygen is not the major emitter of CL.79'80 It is agreed, though, that an electronically excited intermediate, which by the emission of photons measurable as CL, relaxes to a more steady state with the amount of CL being prOportionate to the number of excited species produced. Chemiluminescence has also been demonstrated in l8 platelet suspensions stimulated by the addition of arachidonic acid. It has been used as a measure of prostaglandin pathway enzyme activity, and the results correlate well with MDA production.78 This could be a useful tool in determining whether any differences in platelet aggregation exist between normal controls and abnormal subjects caused by defects in prostaglandin production. For the purposes of this project, CL was used to compare PG production between non—C—HS mink and C-HS mink. Most investigators feel that the bleeding defect in C-HS individuals is caused by the decrease in "storage pool" adenine nucleotides; however, no work has been done to date that specifically evaluates the prostaglandin pathway in these subjects. Drug Effects on Platelets Another prime area of interest in the study of platelet function is the evaluation of drugs as anti-thrombotic agents, considering that a major medical problem for our present population is occlusive vascular diseases such as atherosclerosis. There has been increasing recognition of the platelet's role in thrombus formation, especially in arteries; therefore, there is an active search for true platelet . 83,84,85 suppreSSive agents. Several drugs have been tried in clinical situations with varying results; e.g., aspirin, sulfinpyrazone, . . . . 8 dipyridamole, clofibrate, and others (non-clinical) 6 show some . . . 84,85 potential as anti-thrombotic agents. There are several ways in which platelet aggregation could be affected by drugs: 1) direct inhibition of release, 2) blockage of enzyme pathways, or 3) creating an increase in a cellular constituent that inhibits some part of platelet function are possible mechanisms 19 to be considered. An example of the latter is supported by reports indicating that drugs that cause an increase in c-AMP cause an inhi- 87'88'89 Increases in c-AMP may occur bition of platelet aggregation. by either stimulating adenyl cyclase or inhibiting phosphodiesterase, enzymes which regulate c—AMP levels.90 Cyclic-AMP has been shown to be a potent inhibitor of platelet aggregation.87 Another approach to inhibiting platelet aggregation is to block the prostaglandin pathway. Perhaps the most ideal method of achieving blockade is by inhibition of one of the enzymes in the pathway; however, the blockade must occur before the production of endo- peroxides, since PGH can be utilized in the production of vaso- 2 active compounds. There are two important steps in the pathway that occur before endoperoxide production: the mobilization of arachidonic acid by phospholipase A2 and the oxidative transformation of arachidonic acid by cyclooxygenase. Blocking arachidonic acid mobilization might have deleterious effects in vivo, since it is an essential fatty acid; however, if the action of platelet cyclooxygenase could be blocked, the physiologic effects would probably be minimal. The inhibition of platelet cyclooxygenase is the mode of action of aspirin and other non-steroidal anti—inflammatory agents,61'91 whereas clofibrate appears to inhibit arachidonic acid release from phospholipids.61 Aspirin inhibition of aggregation occurs due to an 85’91 Due to the irre- irreversible acetylation of cyclooxygenase. versible inhibition of platelet cyclooxygenase, inhibition of aggregation remains even after the drug has been completely excreted. It is this fact that makes aspirin very attractive as a possible anti-thrombotic agent. In this study, aspirin was employed to 2O determine if the quantity or activity of platelet cyclooxygenase differs in C-HS mink as compared to non-C-HS mink. MATERIALS AND METHODS Animals The mink utilized for this project were C-HS and non-C-HS males and females ranging in age from 6 months to 1 1/2 years. Some were obtained from a commercial breeder (Phil Clemmons Fur Farm, Barryton, Michigan) and maintained at the mink research facility at Michigan State University. Specimen collection and some testing were done on location at the farm. All mink were determined to be free of Aleutian disease prior to testing, since the presence of the disease could interfere with the results. When the identity of C-HS mink was questioned, a peripheral blood smear was made and examined for the presence of abnormal neutrophil granules. Blood and Platelet Collection Blood was collected directly from the heart of the mink while under ether anesthesia. The blood was drawn through a 21-gauge, 1 1/2 inch needle into a 10 cc syringe containing 3.8% trisodium citrate solution with the ratio of citrate to blood being 0.8 ml:9.20 ml. Several ratios, including 1 part citrate to 9 parts blood, were tested for citrate inhibition, and the 0.8:9.20 ratio was found to be optimum for mink. The specimens were transferred to 15 ml plastic centrifuge tubes and centrifuged for 10 min at 200 g to obtain platelet rich plasma (PRP). If the specimen was to be used for platelet aggregations, 0.5 ml aliquots of PRP were transferred to 21 22 siliconized glass aggregation tubes, and the remaining blood specimen was recentrifuged at maximum rpm to obtain platelet poor plasma (PPP). Each mink specimen yielded from 1.5 to 2.0 ml PRP, which allowed 3 to 4 aggregation tracings on each mink. Platelet counts on the PRP were done using the unopette method (Becton—Dickinson and Co., Rutherford, New Jersey). Aliquots for chemiluminescence were collected and PRP prepared as previously described. Washed platelet suspensions were then pre- pared by adding 1 part 10% EDTA (0.343 M, pH 7.4) to 9 parts PRP, centrifuging at 600 g for 10 min at 4°C, discarding the plasma, and resuspending the platelets in 1 ml Hank's balanced salt solution (HBSS). Platelet counts were done on the resuspended solution and adjustments to platelet suspensions were made to equalize the counts to 0.8-1.2 x 109 plateletsfinl. Plateletggggregations Platelet aggregations were done on a Payton Dual Channel Aggregometer, with a Houston Instruments dual pen recorder. The aggregometer tracing range was adjusted to 90% of the possible range using PPP as 100% transmittance and PRP as approximately 5% trans- mittance to allow maximal pen movement to show shape change. Platelet rich plasma was continuously stirred at 900 rpm by a metal rod in the aggregation tube, allowing maximum platelet-platelet contact, and was simultanedusly incubated at 37°C during the entire procedure. Aggregation was induced by adding 0.030 ml collagen (0.05 mg/ml as determined by the Bio-Rad protein assay) or 0.050 ml arachidonic acid (8 uM) to the PRP. Incubation at 37°C occurred for 2 min prior to and 4 min after the addition of the aggregating agent to allow for maximum 23 aggregation. The plasma was immediately centrifuged in an Eppendorf microcentrifuge for 2 min and the supernatant fluid frozen for later malonyldialdehyde measurements. Collagen Preparation Collagen was prepared using Sigma bovine collagen (Sigma Chemical Co., St. Louis, Missouri). One gram of collagen was added to 100 ml of 83.5 mM acetic acid. The collagen-acetic acid mixture was mixed using a tissue homogenizer to break up the collagen fibers and make an even suspension of collagen in the acetic acid. This homogenation was done for 30 sec while keeping the mixture as close to 0°C as possible by means of an ice water bath. After achieving an even suspension of collagen, the volume was adjusted to 1 liter with 16 mM acetic acid. Approximately 1 ml aliquots were pipetted into small vials and frozen for later use. The assayed value of collagen concentration was determined to be 0.05 mg/ml. When needed for platelet aggregation, a vial was thawed, mixed vigorously on a vortex mixer to insure an even suspension of collagen, and kept in an ice bath during use. Arachidonic Acid Preparation Arachidonic acid (>99% pure, Sigma Chemical Co., St. Louis, Missouri, 50 mg vials) was made up as a solution of the sodium salt by the addition of 0.164 ml 1 N NaOH and then adding Tris buffer (0.015 M, pH 8.5) to a final volume of 1 ml. After the addition of Tris buffer, the vial was shaken vigorously for approximately 1 min to allow portions of the fatty acid that had crystallized to go into solution. The final concentration of fatty acid was calculated to be 0.16 mM/ml. The solution was kept frozen until immediately 24 before use and was then refrigerated and protected from eXposure to light during use to prevent autooxidation. Excess arachidonate was refrozen and used later with no degeneration in effectiveness observed. Chemiluminescence Measurements All scintillation vials for this procedure were dark adapted for at least 1 hr prior to testing, and were not exposed to light until after testing in order to prevent elevated CL results. Three milliliters phosphate-buffered saline (pH 7.4) was added to the 1 ml washed platelet suspension in the scintillation vials under red illumination. After obtaining a background count which was later subtracted from the final results, 50 ul (8 uM) arachidonic acid was added and the solution hand-agitated vigorously for 2 sec. The specimens were then counted for 2 min at 0.1 min intervals. Counting was accomplished on a 8 scintillation counter (Model LS-9000, Beckman Instruments, Inc., Fullerton, California) at room temperature with the counter adjusted out of coincidence and the window Opened wide to count the entire measurable light spectrum emitted. The results were plotted as counts per minute (cpm) vs. time. Malonylaldehyde Assay The measurement of MDA was accomplished by reacting an acidi- fied protein-free filtrate of the reacted platelet suspension with thiobarbituric acid (TBA) in the following manner. The sample was decanted into an equal volume of 20% trichloroacetic acid in 6 N HCl, mixed by gentle inversion, and allowed to stand at room temperature for 10 min to allow complete protein precipitation. The acid solu- tion was centrifuged at 1500 g for 15 min or in a serofuge for 5 min. 25 The supernatant was then filtered through glass wool to remove any remaining protein precipitate. Thiobarbituric acid was prepared by mauoo coeuomum oumasofiuumm pom .mmnonso: .mmlu How m n c uo>uso amen: .Emo ooo.wv um mm3 xmwm some cofluomum wumasowuumm uwHoumHm m can Emu ooo.Hh on» How m n my .GBOcm mm powwomo um mmcommwu unmomcflfidaflEwno xmmm some m cm: muoamumam RCHE mmnolcoz new Emu ooo.vh um oncommmu ucmomocHEoHfiEmno xmmm some n on: anon mumaoumHm xcHE mmlo can muoaoumam amaom .oEHu .m> moa x 590 mm omuuon oncommou wocoomwcflesaflamnu .N musmwm 3O mamasofluumm unamumam m muoofim . E LIL]... i N; 3 muons“: cw wsaa a“ cu an 94 if on 31 Am n :V .omsomwon unwommcwfioHHEan now oon>amcm Hmfl>\muoawumam 00H x N.H ou m.o Scum omcmu ou owuomnuou mums mmooum anon Mom mucsoo umamumam .xcHE mmlo Mom Ha\m: o.m H m.m can xGHE mmuuuco: Mom HE\mc m.m H m.m oum3 mmoam> some one .oflom oasooflnomnm cues voodoo“ mocoommcfieoaflamso ocfluso muoamumam xcflE mmlu ocm malolso: >n Umosoonm muHSmoH mommm mp>£moamwoa>coamfi cow: .m musmflh 1F 32 10t C-HS Non- C-HS Figure 3 33 S n 5 .>Ho>fluoommmn .coomaaoo can ofiom UACOpflnomum >3 ooosocw xcfle mmsu How HE\mc m.o H H.m men 6.0 H 9.8 cam sans mmuouco: you Hexm: m.o A m.~ can H.H H m.o mums mmzam> some are . Ecmmmzoo ocmgoflom cacoownomuo .3 poooorfi cofivmmoummm uoamumam 93.436 mumamumam xcHE mmlo pom masoncoc >9 couscoum muaomou >mmmm moanooamfioaxconE coo: .v whomflm 34 r p P MDA ng/ml \\\ \ \ \ \ \ \ \\\\\\\_\\\\\\ \\\\\\\\\\\\\ \\\\\\\\\\‘\‘\\ P C-HS Non— C-HS Figure 4 35 arachidonic acid and collagen and then assayed for MDA. The non-C-HS mink values were 6.8 i 1.1 and 2.5 :_0.9 ng/ml, and the C-HS mink results were 6.9 i 0.8 and 3.1 :_0.9 ng/ml using arachidonic acid and collagen, respectively, as inducing agents. The MDA assay results after aspirin injection and platelet aggre- gation are illustrated in Figure 5. The mean values for non-C-HS mink were 5.1 :_1.0 and 2.3 :_0.6 ng/ml and, for C-HS mink, 4.4 :_0.9 and 1.7 :_0.4 ng/ml using arachidonic acid and collagen, respectively, as aggregating agents and correcting platelet counts as previously described. Figures 6 and 7 represent typical aggregation curves of non-C-HS and C-HS mink in response to arachidonic acid and collagen used as inducing agents before (Figure 6) and after (Figure 7) the injection of aspirin. A marked decrease in the extent and rate of aggregation is seen after aspirin injection. 36 Am n :V .>Hm>wuoommmn .commaaoo tam ofiom cacopflzomum >Q omUDUCA xcflfi mzuo “Om HE\mc v.0 H >.H can m.o H v.v new goes mmao Icon HOw HE\mc 6.0 H m.m ocm o.H w H.m mums mosam> some one .coHuomncfl cfiuflmmm umuwm as H \\\\ cmmmfloo ocmgoflom cacoofisomum fiflz condone :oHummoummm umHmumam 95.3w muoaoumHm xcwE mmno ocm mmlolco: >n ooooooug muHSmmH wommm moxnooamfioaaconE coo: .m musmfim 37 \\\\\\\\ \\\\\\\\ \\\\\\—\\ OOOOOOOOCOOCOOOOOOOOOOO MD ng/ml Non- C-HS Figure 5 38 .iae\mc mo.ov cmmmaaoo can 12: we chum cacocnnomum ou oncommou CA xcflE mmlu pom maloncoc you mm>nso coflummoummm uwaoumHm .o whomfim 39 m whomflm coaumwonwg cmmmzoo cowumwwuwm" 384 3:03:03,» $70 1602 mmlo 1:02 $70 mzlo momma Q 25 4O .sofluooncw sfluflmmm umumm u: H AHE\mc mo.ov :mmmHHoo now As: my oflom oesoofinomum ou oncommmu CH xcflE mmao new mmuunco: new mo>uso :ofiummoummm uoaoumam .h madman h muzmfim couumwmuwwc. ammeHoo coauwwwumw’w 39¢ oesoownomuc m:-o -coz Afl momma 4 . $70 .162 41 were mmlu v 1 mafia DISCUSSION It has been shown in the past that C-HS affected individuals of all species studied have an abnormally prolonged bleeding time. The prolongation of bleeding times is related to the platelet count and/or platelet function.92 Similar findings are also observed to a lesser extent in patients who have ingested aspirin. In vitro studies illustrate defective platelet aggregation, which is directly related to primary hemostasis, and in C-HS was determined to be caused by decreased amounts of "storage pool" ADP released by platelets during aggregation with collagen. Collagen normally causes a release of storage pool ADP during the platelet release reaction of aggregation and the released ADP causes aggregation. Like ADP, the proper concen- tration of epinephrine first has a direct effect on platelets in initiating aggregation and is followed by a second wave of aggrega- tion (biphasic response) caused by released ADP. It is this second wave of aggregation which is absent in C-HS individuals. In summary, a decreased or absent pool of releasable ADP characterizes itself functionally as a reduction in response to collagen and a loss of second wave aggregation in response to ADP and epinephrine. These defective responses are seen both in storage pool disease (C-HS) and in patients who have ingested aspirin. Interestingly, non-primate species, except for cats, do not exhibit a biphasic response to ADP and epinephrine even with normal amounts of storage pool adenine 42 43 nucleotides present. Therefore, a storage pool deficiency is some- what more difficult to characterize in these species. Failure to produce a secondary wave of aggregation may also be due to a defect in prostaglandin synthesis. Certain myeloprolifera- tive disorders exhibit decreased prostaglandin synthesis,64 suggesting that defects in platelet aggregation previously attributed to storage pool deficiencies could also have underlying defects in prostaglandin synthesis. In this study, using C-HS mink, the possibility of prosta- glandin synthesis defects in addition to storage pool deficiencies was explored. Assays measuring the chemiluminescent response of non-C-HS and C-HS mink platelets found no significant differences between the two groups (P<0.01). Peak CL values were, for all practical purposes, the same. The rates of relaxation to a more steady state depicted by the decrease in counts per minute over time were also basically equal. Malonyldialdehyde values after chemiluminescence were nearly equal for both groups (P<0.01) and no significant differences were detected. Another approach, the measurement of MDA after platelet aggre- gation, was also undertaken using 2 different aggregating agents, arachidonic acid and collagen. As with the chemiluminescence results, no significant differences were detected between non-C-HS and C-HS mink (P<0.01) with either aggregating agent. Measurement of MDA after aspirin injection and platelet aggre- gations revealed no significant differences between the two test groups (P<0.01). However, there were significant differences between the non-aspirin treated and aspirin treated values. Theoretically, treatment of platelets with aspirin would completely stop MDA and prostaglandin production. Since this was an in vivo injection of 44 aspirin, however, problems in the concentration injected and/or absorbance from the peritoneal cavity could prevent all platelets from being exposed to aspirin and the non-exposed platelets could account for the MDA that was produced. The one hour delay between injection of aspirin and specimen collection was possibly not adequate for complete absorption of the drug. The minor difference between C-HS and non—C—HS mink after aspirin treatment (P<0.01). although not significant, might be sup- ported by two different, relatively recent studies. First, it has been shown that the effects of drugs both in vivo and in vitro are influenced by the concentration of red cells present in PRP.93 It is difficult to obtain PRP completely void of red cells, and it is entirely possible that differences in red cell concentrations in PRP contributed to the minor difference observed. Secondly, the concen- tration of divalent cations, especially calcium, is important in platelet aggregation and the inhibitory effects aspirin and other drugs have on platelet aggregation are enhanced as the calcium con- centration decreases.94 Any one or combinations of the above possi- bilities could account for the minor observed differences in MDA production between the test groups after aspirin injection. Considering the previously presented data, the author concludes that no difference exists between C-HS and non—C-HS mink as concerns the prostaglandin synthesis pathway. 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Buchanan MR and Hirsh J: A comparison of the effects of aspirin and dipyridamole on platelet aggregation in vivo and ex vivo. Thromb Res 13:517—529, 1978. VITA VITA The author was born in Houlton, Maine, on November 22, 1949, and as the son of a dairy farmer, he grew up in the small town of Sherman, Maine. His elementary and secondary education was attained in Sherman and culminated with graduation from Katohdin High School in 1968. He then entered the United States Air Force Academy located near Colorado Springs, Colorado. Graduation from the service school occurred on June 7, 1972, when he received a Bachelor of Science degree in Life Sciences and a commission in the United States Air Force. From the academy he was assigned to Wilford Hall USAF Medical Center in San Antonio, Texas, to attend a 12-month clinical laboratory internship. Upon completion of the internship, he tested for and received a registry as Medical Technologist from the American Society of Clinical Pathologists. His next assignment was to USAF Hospital Loring, Loring AFB, Maine, as Director of Laboratory Services, which began in August 1973. In the fall of 1976, the author received a scholarship for the Air Force Institute of Technology (AFIT) to obtain an advanced degree beginning the subsequent school year. The author chose Michigan State University as his first choice of schools and matriculated in September 1977. Upon completion of his degree requirements, Captain Patterson will be assigned as Chief of Hema- tology and Coagulation at Keesler AFB Medical Center, Keesler AFB, Biloxi, Mississippi. 52 53 The author married Debbie McAvoy on June 17, 1972, in Benedicta, Maine. Their daughter, Meghan, was born on March 9, 1976, at Loring AFB in Maine.