'1 0 a- L-m-t‘::- .'.‘:.‘7 This is to certify that the dissertation entitled Platelet Function In Basset Hound Hereditary Thrombopathy presented by Wayne.Robert Patterson has been accepted towards fulfillment of the requirements for Pathology mC/KJ/ Major professor Ph ° D ' degree in Date April ’4, 1986 0-12771 MS U is an Affirmative Action/Equal Opportunity Institution MSU LlBRARlES _—:—- RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. TM..- PLATELET FUNCTION IN BASSET BOUND HEREDITARY THROMBOPATHY BY Wayne Robert Patterson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1986 1$2 4&8- ABSTRACT PLATELET FUNCTION IN BASSET BOUND HEREDITARY THROMBOPATHY BY Wayne Robert Patterson An inherited intrinsic platelet aggregation defect has been identified in Basset Bounds which clinically resembles the human disease, Glanzmann's thrombasthenia. The defect was named Basset Hound Hereditary Thrombopathy (BHT) and initial studies revealed variably prolonged bleeding times, normal blood coagulation parameters, normal platelet count and morphology, normal whole blood clot retraction, and an abnormal platelet aggregation response to adenosine diphosphate (ADP). An evaluation of the specific platelet defect included simultaneous measurement of dense granule release and aggregation, two dimensional electrophoretic and crossed immunoelectrophoretic techniques in order to determine if the defect was associated with platelet membrane glycoprotein abnormalities, and measurement of radiolabeled fibrinogen binding to ADP stimulated platelets. Affected Basset Hound platelets release storage pool adenosine triphosphate in quantities not significantly 514 diffwrent from normal controls when stimulated with leO’ ADP. However. the release occurs so rapidly that it is complete in approximately one—sixth of the time required for release from wormal control platelets. An analysis of two- dimensional and crossed-immunoelectrophoretic gels revealed no abnormalities in platelet protein/glycoprotein content when affected Basset Hound platelets were compared to normal dog platelets. Finally, the amount of radiolabeled fibrinogen bound by the thrombopathic platelets after stimulation with ADP was not significantly different than that bound by normal dog platelets. It is clear from the results that this syndrome is not an animal homologue of Glanzmann's thrombasthenia. Further, the results suggest that the binding of fibrinogen is not sufficient for platelet aggregation and other factors such as receptor mobility and protein-lipid interactions may play a critical role in platelet aggregation. TO DEBBIE AND MEGHAN ii ACKNOWLEDGEMENTS Any verbal or written expression of gratitude to Dr. Thomas Graham Bell would be inadequate. As chairman of my guidance committee and my research advisor, he was continually there with encouragement, advice, and new ideas. He showed me all the aspects of what a PhD means in terms of thinking through the problem, performing the research, and achieving my research goals. For his invaluable advice and insight I am eternally grateful. Special thanks are also deserving to the other members of my committee. Dr. George Padgett, Dr. Ken Schwartz, and Dr. Doug Estry provided advice, encouragement and constructive criticism which helped keep the project moving forward and me on the right track. My sincere thanks to Mr. and Mrs. Kovalic for their support in the project by providing normal and heterozygote dogs for the study. Even though I have dedicated this dissertation to my wife and daughter, I would like to say that I am especially grateful to my loving wife, Debbie, who had tremendous patience and understanding throughout my program. She deserves much credit for this degree. iii TABLE OF CONTENTS LIST OF TABLES AND FIGURES O O O O O 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 .v LIST OF ABBREVIATIONS O O O O O O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O OVi CHAPTER 1 : IMRODUCTION O I O O O O O O O O O O O O O O O O O O O I O O O O O O O C O O O O 1 CHAPTER 2: ABNORMAL RELEASE OF STORAGE POOL ADENINE NUCLEOTIDES FROM PLATELETS OF DOGS AFFECTED WITH BASSET HOUND HEREDITARY THROMBOPATHY.....8 AbStraCt O O O O O O O O O O O O O O O O O O O O O O O O O O O O C I O C O O O O O I O O O O O C 8 IntIOdUCtiono O O O O O O O O I O O O O O I O O O O O O O O O O O O I O O O I O O O O O O .8 Materials and ”ethOds. O O O O O O O O O O O O O O I I O O O O O I O O O O O C O .9 ReSU1ts.O.O...O....0.OOOOOOOOOOOOOOOOOOOOO0.000.000.10 DiSCUSSiODOOOOOOOOOOOOOOOOICOOOOOOOOOOOOOOOOOOO0.0.013 Legends for Electron Hicrographs....................14 ReferencesooooO0.000000000000000.0.0000000000000000017 CHAPTER 3: TWO-DIMENSIONAL ELECTROPHORETIC STUDIES OF PLATELETS FROM DOGS AFFECTED WITH BASSET HOUND HEREDITARY THROMBOPATHY: A THROMBASTHENIA-LIKE AGGREGATION DEFECT........19 AbstractOOOOOOOOCO0.0.00.0...0.00.00.00.00...00.0.0019 IntrOdUCtione O O I O O I O D O O O O O O O O C O O O O O O O O O I O O O O O O O O O O O .19 materials and Methods 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O .20 Resu1ts.COO...O...0.0.00...O0.0.0.0.000000000000000022 DiBCUBSiOnOOOOOOOOO0......00......OICCOOOOOOOOOOOOOOZS ReferenceSOOOOCOOOOOOOOOOOOOOOOOOOOO00.00.000.00000026 CHAPTER 4: FIBRINOGEN BINDING TO PLATELETS FROM DOGS WITH BASSET HOUND HEREDITARY THROMBOPATHY A THROMBASTHENIA-LIKE AGGREGATION DEFECT......29 AbstraCtOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.30 IntIOductionOOOOOOOOOOOO0.0.0.0.0...0.00.00.00.0000031 "ethOdSOOOOOOOO...OO0..OI0.00.0.00.00.00.0000000000033 ResaltSOO...0.00.0..OOO0.0..0.0.0.0.000000000000000035 DiBCUBSiOnOOOOOOOOOOCOOOOOOOOOOOOOOO00.000.000.0000037 ReferenceSOOOOO...OCO.I.0.0...0.0.0.0000000000000000‘6 CHAPTER 5: SUWYOOOOO0.0...OOOOIOOOOOOOOOOOOOO0.00000053 LIST OF REFERENCESOOOOOOOOOOOOOOOOOOOOOOOOO00.0.00000000057 iv ‘ LIST OF TABLES AND FIGURES Table Platelet ATP release and time to maximal 1uminescence...................ll Comparative fibrinogen binding in BBT.................44 Figure ATP release to varying ADP concentrations.............9 Chrono-Lume potentiation of aggregation...............10 Platelet ATP release..................................ll Electron micrographs of BET and normal dog platelets..l4 Normal and BBT platelet aggregation...................21 Crossed-immunoelectrophoresis gels....................22 Nonreduced-reduced 2-D gels...........................22 O'Farrell gels........................................23 Aggregation tracings..................................42 Competitive inhibition of fibrinogen binding..........43 LIST OF ABBREVIATIONS ADP................Adenosine Diphosphate ATP................Adenosine Triphosphate BHT................Basset Bound Hereditary Thrombopathy or Basset Hound Thrombopathic BSS................Bernard-Soulier Syndrome CBR................Coomassie Blue R CIE................Crossed Immunoelectrophoresis c-AMP..............Cyclic Adenosine Monophosphate GP.................Glycoprotein GT.................Glanzmann's thrombasthenia NR-R...............Nonreduced-Reduced PRP................P1atelet Rich Plasma SDS-PAGE...........Sodium dodecyl sulfate - polyacrylamide gel electrophoresis VWD................von Willebrand's Disease VWF................von Willebrand's Factor vi CHAPTER 1 INIBQDQQIIQN In normal hemostasis, the blood vessel wall, platelets, and plasma clotting factors all interact to arrest bleeding. A dysfunction of any of the three components can cause a hemorrhagic diathesis which may or may not be clinically evident until the hemostatic mechanism is stressed. Although they have not always been recognized as such, platelets now appear to play a central role in normal hemostasis. They adhere to injury sites, recruit more platelets by releasing their internal granules, and aggregate with other platelets to form the primary platelet plug, and in the course of events, they help to accelerate the normal coagulation cascade. Also, some of the released granule components and synthesized products aid in the hemostatic process due to their vasoactive nature. Due to their central role in hemostasis, platelets which are quantitatively or qualitatively abnormal can cause severe bleeding problems. Qualitative platelet defects can be classified into defects of adhesion, release, or aggregation, and each type of defect can be investigated separately. Disorders of adhesion include Bernard-Soulier Syndrome (BSS) and von Willebrand's Disease (VWD). In BSS there is an absence of platelet membrane glycoprotein Ib (GP lb) (1), and in VWD there is an absence of a plasma factor which is part of the l INIBQDQQIIQN In normal hemostasis, the blood vessel wall, platelets, and plasma clotting factors all interact to arrest bleeding. A dysfunction of any of the three components can cause a hemorrhagic diathesis which may or may not be clinically evident until the hemostatic mechanism is stressed. Although they have not always been recognized as such, platelets now appear to play a central role in normal hemostasis. They adhere to injury sites, recruit more platelets by releasing their internal granules, and aggregate with other platelets to form the primary platelet plug, and in the course of events, they help to accelerate the normal coagulation cascade. Also, some of the released granule components and synthesized products aid in the hemostatic process due to their vasoactive nature. Due to their central role in hemostasis, platelets which are quantitatively or qualitatively abnormal can cause severe bleeding problems. Qualitative platelet defects can be classified into defects of adhesion, release, or aggregation, and each type of defect can be investigated separately. Disorders of adhesion include Bernard-Soulier Syndrome (BSS) and von Willebrand's Disease (VWD). In BSS there is an absence of platelet membrane glycoprotein Ib (GP lb) (1), and in VWD there is an absence of a plasma factor which is part of the l 2 Factor VIII molecule called von Willebrand Factor (VWF)(2). It is now believed that VWF forms a bridge between GP Ib and the subendothelial tissue after an injury and is the molecular event responsible for adhesion (1). An absence or dysfunction of either GP lb or VHF would result in defects in adhesion and a hemorrhagic diathesis. Inherited defects in platelet release are probably quite rare and would include storage pool disease and cyclo- oxygenase deficiency. However, acquired release defects are common and are usually caused by aspirin and other non-steroidal anti-inflammatory agents which are irrevers- ibly antagonistic to platelet membrane cyclo-oxygenase and thromboxane synthesis (3). Some other drugs may affect release by other mechanisms, but the effects are usually reversible. Aggregation defects are recognized by an 1n__x1;;9 failure of platelets to aggregate in response to various stimuli. Perhaps the most common inherited aggregation defect in humans is Glanzmann's thrombasthenia (GT). In GT there is an absence or reduction of platelet membrane glycoproteins IIb and IIIa (GP IIb-IIIa) (4) which are normally present as a calcium-dependent heterodimer in the platelet membrane (5,6). Since GT platelets have both decreased GP IIb-IIIa and fibrinogen binding, the platelet membrane fibrinogen receptor is presumably located on the GP IIb-IIIa complex (7). It has long been known that fibrinogen and calcium are required for platelet aggregation, and investigators now believe that the dimeric fibrinogen molecule forms a bridge between adjacent platelets by binding to GP IIb-IIIa which is exposed by stimulation with various aggregating agents (8). It follows then that defective or absent fibrinogen, absent or reduced GP IIb-IIIa, or abnormal exposure of the fibrinogen receptor would all be potential causes of aggregation defects. An animal model with an inherited platelet aggregation defect has been identified and clinically the bleeding diathesis resembles the human disease Glanzmann's thrombasthenia (GT)(9,10). The defect was found in a colony of line-bred Basset Hounds and is named Basset Hound Hereditary Thrombopathy (BHT) (10). These Basset Hounds were found to have variably prolonged bleeding times, normal blood coagulation parameters, normal platelet numbers and morphology, normal whole blood clot retraction, but an abnormal platelet aggregation response to adenosine diphosphate (ADP) and collagen (10). The aggregation defect was similar to that seen with thrombasthenic platelets but the presence of normal clot retraction was a factor which immediately distinguished BHT from GT. It was decided at this point to investigate the possibility of storage pool deficiency, platelet membrane glycoprotein abnormalities, and platelet membrane fibrinogen binding in an attempt to ascertain the molecular defect responsible for the aggregation defect. The following chapters (11-13) are reprints of articles submitted and/or published which 4 attempt to systematically define the pathogenesis of BHT. BEEEBENQES l. Caen JP, and Nurden AT. Inherited Abnormalities of Platelet Glycoproteins. Surv Synth Path Res 1:274, 1983 2. Senogles SE, and Nelsestuen GL. Von Willebrand Factor. A Protein Which Binds at the Cell Surface Interface Between Platelets. J Biol Chem 258(20):12327, 1983 3. Vane JR. Inhibition of Prostaglandin Synthesis as a Mechanism for Aspirin-Like Drugs. Nature New Biology 231:232, 1971 4. Herrmann FH, Meyer M, and Ihle E. Protein and Glyco- protein Abnormalities in an Unusual Subtype of Glanzmann's Thrombasthenia. Haemostasis 12:337, 1982 5. Kunicki TJ, Pidard D, Rosa JP, and Nurden AT. The Formation of Ca++-Dependent Complexes of Platelet Membrane Glycoproteins IIb and IIIa in Solution as Determined by Crossed Immunoelectrophoresis. Blood 58:268, 1981 6. Howard L, Shulman S, Sadanandan S, and Karpatkin S. Crossed Immunoelectrophoresis of Human Platelet Membranes. J Biol Chem 257:8331, 1982 6 7. Gogstad GO, Brosstad F, Rrutnes MB, Hagen I, and Solum NO. Fibrinogen—Binding Properties of the Human Platelet Glycoprotein IIb-IIIa Complex: A Study Using Crossed-Radioimmunoelectrophoresis. Blood 60:663, 1982 8. Kornecki E, Niewiarowski S, Morinelli TA, and Kloczewiak M. Effects of Chymotrypsin and Adenosine Diphos— phate on the Exposure of Fibrinogen Receptors on Normal Human and Glanzmann's Thrombasthenic Platelets. J Biol Chem 256:5696, 1981 9. Johnstone I, and Lotz F. An Inherited Platelet Function Defect in Basset Hounds. Can vet 20:211, 1979 10. Bell TG, Leader RW, Olson PM, Padgett GA, Penner JA, and Patterson WR. Basset Hound Hereditary Thrombopathy: An Autosomally Recessively Inherited Platelet Dysfunction. Qng_fled1ging, (Ryder/Byrd, eds). Springer-verlag, pp 335- 344, 1984 11. Patterson WR, Padgett GA, and Bell TG.(Chapter 2) Abnormal Release of Storage Pool Adenine Nucleotides From Platelets of Dogs Affected with Basset Hound Hereditary Thrombopathy. Thromb Res 37(1): 61, 1985 12. Patterson WR, Kunicki TJ, and Bell TG. (Chapter 3) Two-Dimensional Electrophoretic Studies of Platelets From Dogs Affected with Basset Hound Hereditary Thrombopathy: A Thrombasthenia-like Aggregation Defect. Thromb Res (Accepted Jan 1986) 13. Patterson WR, Estry DW, Schwartz RA, and Bell TG. (Chapter 4) Fibrinogen Binding to Platelets From Dogs with Basset Hound Hereditary Thrombopathy: A Thrombasthenia-like ” Aggregation Defect. Blood (Submitted Feb 86) CHAPTER 2 ABNORMAL RELEASE OF STORAGE POOL ADENINE NUCLEOTIDES FROM PLATELETS OF DOGS AFFECTED WITH BASSET HOUND HEREDITARY THROMBOPATHY Wayne R. Patterson, George A. Padgett and Thomas G. Bell Department of Pathology, Animal Health Diagnostic Laboratory, Michigan State University East Lansing, Michigan 48824, U.S.A. ABSTRACT Platelets from dogs affected with Basset Hound Hereditary Thrombo- pathy (BHT), have a thrombasthenia-like aggregation defect but re- lease storage pool ATP in quantities not significantly different from normal controls or BHT heterozygotes when stimulated *with 1x10'5M ADP and 0.22 U/ml thrombin. However, the release occurs so rapidly in the BHT platelets stimulated with ADP that it is complete in approximately one-sixth of the time required for release from normal control and heterozygote platelets. Sequential electron micrographs reveal early release of BHT dense body constituents 30 seconds after stimulation with 1x10’5M ADP while resting BHT morp- hology is indistinguishable from normal control animals. INTRODUCTION Recent characterization of a group of closely related Basset Hounds experiencing hemorrhagic diathesis has revealed a defect in intrinsic primary platelet aggregation (1). This syndrome has been labeled Basset Hound Hereditary ThromboPathy (BHT), and has been shown to be inherited as an autosomal recessive trait (1). Data on various hemostatic parameters revealed variably prolonged bleeding times, a normal blood coagulation mechanism, nor- mal platelet numbers and morphology, normal whole blood clot retraction, and a defective platelet aggregation response to adenosine diphosphate (ADP) (1,2). Except for normal clot retraction, this syndrome closely resembles thrombas- thenia in humans in which there is absent or reduced clot retraction (3). In general, failure of platelets to arrest bleeding may be attributed to defects Keywords: platelets; adenine nucleotides; dense body release: thrombasthenia- like thrombopathia. 9 in adherence, aggregation, or release of platelet storage pool granule consti- tuents (4), all of which require interaction between plasma and platelet com- ponents. In BHT, the plasma component appears to be normal (1,2) but previous data suggested a failure of ADP induced aggregation. The analysis of platelet aggregation data in BHT dogs revealed an inordinately large and prolonged shape change and the loss of dense body granules on electron micrographs: further, mixtures of normal and BHT platelets appeared to have enhanced aggre- gation. This paper documents a difference of platelet nucleotide release in BHT and compares it to normal and heterozygote dogs. Also included is a sequen- tial electron microscoPic study of BHT and normal dog platelets after stimula- tion with ADP. MATERIALS AND METHODS The experimental subjects were grouped into three categories. The BHT or affected group consisted of four Basset Hounds previously studied (1) which had no platelet aggregation in response to 0.2x10'5M ADP, a concentration that caused maximal aggregation in normal and carrier dogs. Normal controls con- sisted of two Basset Hounds proven to be normal when tested for platelet aggregation, and two unrelated Golden Retrievers previously characterized (1). The carrier group consisted of four heterozygote Basset Hounds that had pro- duced BHT affected offspring, but exhibited normal platelet aggregation. Blood for platelet aggregation was collected in trisodium citrate, plate- let rich plasma (PRP) prepared, platelet counts performed, and platelet counts standardized to 3x105 platelets/ul as described previously (1). Platelet aggregation studies were performed within 4 hours of blood collection. Storage pool adenine nucleotide release was monitored using the Lumi- Aggregometer (Chrono—Log Corp., Havertown, Pa) according to Charo et al. (5). The Lumi-Aggregometer uses a firefly luciferin-luciferase system to detect released adenosine triphosphate (ATP), and simultaneously measures release of ATP and platelet aggregation. Monitoring was performed at 37°C with a teflon- coated stir bar rotating at 900 rpm within the sample. Results were recorded cu: a dual-pen Houston Omniscribe Chart Recorder (Houston Instrument Division of Banach a Lombe, Inc., Austin, TX). Aggregation cuvettes contained 0.45 ml. of PRP (3x105 platelets/ul) and 0.05 ml of Chrono-LumeR No. 395 luciferin- luciferase reagent (Chrono—Log Corp., Havertown, PA) added to the PRP imme- diately prior to incubation at 37°C. After temperature equilibration for 2 minutes with stirring, either ADP or thrombin was added to a final con- centration of 1x10’5M and 0.22 U/ml respectively (ADP was obtained from Sigma Chemical Company, St. Louis, MO; thrombin was a generous gift from Dr. Houria Hassouna, Department of Pathology, Michigan State University prep #174; Activity=5664 u/ml: 99.6% Activity). Aggregation and release were monitored until the luminescence caused by the released storage pool ATP had started to subside. At this point a standard solution of ATP (final concentration of 4x10'5M) was added. The amount of ATP released from the platelets was then calculated by using the peak luminescence point from both the platelet release and the addition of the standard concentration of ATP. The standard ATP solu- tion was run with all samples to avoid slight changes due to reagent and/or platelet degradation, and all reagents were kept on ice until added to the aggregometer cuvettes. The time from injection of the aggregating agent to maximum luminescence was measured directly from the chart using the time increments and running the recorder at a speed of one inch per minute. 10 Platelets from each of the control animals were used to determine the Optimum concentration of ADP to be used to stimulate release from platelet granules. This was performed by measuring platelet ATP released in response to five concentrations of ADP; 0.1x10‘5M, 0.2x10‘5M, 0.6x10'5M, 1.0x10'5M, and 2.0x10'5M. Since maximum release of storage pool ATP was desired, the con- centration beyond which there was no significant increase in ATP release was chosen as Optimum. Platelets used for electron microscopic studies were stirred at 900 rpm for two minutes at 37°C and then ADP (1.0x10’5M final concentration) was added to all samples except the resting samples from BHT and normal control dogs. At various times after addition of ADP, 0.5 ml of 0.1t glutaraldehyde solution was added and the sample allowed to stir for an additional one minute. The Specimens were then centrifuged for 15 seconds at 12,800 x g, the supernatant discarded, and 1.0 ml of 3% glutaraldehyde added. The specimens were capped and allowed to fix for a minimum of thirty minutes at which time they were transferred to a 3% buffered glutaraldehyde solution and processed for electron ndcrosc0py as described by Mattson, et a1. (6). This procedure was followed for unstimulated platelets, and for samples at 30, 100, and 200 seconds after addition of ADP to BHT and normal control platelets. RESULTS Effect of ADP concentration on the quantity of ATP released. Since the objec- tive of this investigation was to determine the availability and release of storage pool adenine nucleotide in BHT platelets, a concentration of ADP causing maximal release was desired. As can be seen in Fig. 1, a signifi- cant increase in the amount of ATP released occurs when the ADP concentration T? T. 3.0. A 2 O I 0 db " 2.0» “ X V C .- ‘< B 1.0 - d) '( I“ J “I C: 0.10.2 0.0 1.0 2.0 ADP cowcewrnandw (x 10"» FIG. 1 Platelet ATP release during aggregation from four normal dogs (dupli- cate determinations) in response to varying concentrations of ADP used as the aggregating agent. 11 is raised from 0.1x10'5M to 1.0x10'5M, but the amount of ATP released does not increase significantly at higher concentrations of ADP. Therefore, 1.0x10'5m ADP was used to stimulate platelet aggregation and release. Varying the con- centration of thrombin from 0.22 - 0.44 U/ml did not cause significant dif- ferences in the amount of ATP released, however thrombin concentrations below 0.22 u/ml showed significant specimen to specimen variation in ATP release. Potentiation of aggregation by Chrono-LumeR. While monitoring aggregation, an apparent potentiation of aggregation was observed in samples containing Chrono—LumeR and stimulated with ADP similar to that observed by Mehta, et a1. (7)(Fig.2). Control 1 <-— lncrease in Optical Transmission Control 2 Time (I——-—4) 1 minute FIG. 2 Platelet aggregation tracings in response to 1.0x10‘5M ADP. Normal dog platelets were collected from two normal Basset Hounds and two unre- lated Golden Retrievers. BHT platelets were collected from four BHT affected Basset Hounds. In all cases the slope and extent of aggregation were increased in samples containing Chrono-LumeR (BHT 2 and Control 2). Release of ATP and time to maximum luminescence. The data from these measure- ments are shown in Table 1 and Fig 3. It can be observed from the data in Table 1 that the quantity of ATP that is released and the time to maximum luminescence in response to thrombin are not significantly different when all three groups are compared by the student T test (p<.001). This rules out a storage pool abnormality as a cause for the aggregation defect. However, the time to maximum luminescence in response to ADP is significantly different in the BHT group when compared to normal and proven heterozygous dogs (p<.001). 12 BHT platelets reach maximum luminescence, indicating maximum release of storage pool ATP, in approximately one sixth of the time required for normal platelets. TABLE 1 Platelet ATP release and time to maximal luminescence in BHT, normal, or dogs heterozygous for BHT‘1) Normal Heterozygous BHT ADP* Thrombint ADP * Thrombinf ADP‘ Thrombint RELEASED ATP (x 10'6M) 2.64 2.83 2.94 2.47 3.00 2.75 amiss" _+_o.60 10.79 10.49 10.35 10.42 10.85 TIME TO MAXIMUM LUMINESCENCE (MINUTES) 2.64 3.12 2.20 3.87 0.41 2.97 uran1s0** 19.34 19.47 19.44 19.47 19.06 19.64 (1)*1x10'5M: + 0.22 u/ml; ** n = 10 PLATELET ATP RELEASE N . P ‘ATP Standard (4x10’6M) (3.00x1o'6M) BHT (2.64x10'6M) Control Increase in luminescence ADP (1x10'5M) Time (s A.) 1 minute FIG. 3 Platelet ATP release in response to 1x10'5M ADP. Release of ATP occurs significantly faster from BHT platelets than from controls. Numbers in parentheses are peak values representing the mean values from BHT and control platelets (Table 1). 13 Electron microscoPic findings. Sequential electron micrographs of BHT and normal platelets taken at 30, 100, and 200 seconds after stimulation with ADP, morphologically support early release of dense granules from BHT platelets. Unstimulated (resting) platelets appear similar in both groups. At 30 seconds after stimulation the BHT platelets display shape change, centralization of granules, lack of aggregation, and absence of dense granules. In contrast, the normal platelets show shape change, but no centralization of granules, no microtubule formation, presence of dense granules, and partial aggregation. The 100 and 200 second BHT samples showed a lack of platelet aggregation. With the 200 second sample, an apparent dissociation of mdcrotubules was also noted. The 100 and 200 second sainples from controls displayed significant platelet aggregation and dense granules were still present in a few platelets. DISCUSSION Normal hemostasis is accomplished through a complex set‘of interacting fac- tors that involve platelets, the vascular wall and the coagulation factors (8). The platelet's hemostatic role depends on the ability to change shape, adhere to subendothelial tissue, release storage pool granule constituents, and to aggregate into viable and stable plugs (9,10). To do this, there must be a functional receptor on the platelet membrane, and the stimulated receptor must transmit to the platelet interior; 'Then. microtubule mediated con- centration and release of platelet granules occur. Finally, surface fibrino- gen receptors are exposed and react with fibrinogen, followed by platelet- platelet interaction and evolution of a stable plug (11-13). This investigation has documented that BHT platelets change shape in response to an aggregation stimulus, and also release normal amounts of storage pool granule ATP. Further, citrated resting BHT platelets appear ultrastructurally normal on transmission electron microscopy. Previous stu- dies have documented normal BHT plasma factors and platelet adhesion (1,2). Although ADP stimulated BHT platelets change shape and release normal amounts of storage pool granule constituents, the rate of release is abnormal and they do not aggregate. Thrombin stimulated BHT platelets, however, re- lease in a normal fashion but still do not aggregate. Lack of aggregation may point to a defect of the fibrinogen receptor, which has been shown by several investigators to be a calcium dependent complex of two specific glyc0proteins: glyc0proteins IIB and IIIa (GP IIb-IIIa) (14,15) or to defective fibrinogen receptor exposure. The inability of BHT platelets to bind fibrinogen or to expose fibrinogen receptors may, in some way, be related to rapid ATP release after ADP stimulation. One may be the cause of or be involved in causing the other. A possible mediator in both of these reactions could be Ca++. It has been shown that ADP induced platelet activation increases surface bound Ca++ and the exchange rate of Ca++ into the intracellular pool without increasing the amount of intracellular Ca++ (16). An abnormality in either the rate or quantity of calcium exchange might explain the rapid ATP release and aggrega- tion defects in BHT. The fact that BHT platelets support normal clot retrac- tion but fail to aggregate seems to be an enigma. However, there are distinct receptors for fibrinogen, required for aggregation, and for fibrin, required for clot retraction (17). Lastly, platelet ectosialyltransferase activity has been shown to be involved in cell adhesion and platelet aggregation (18). The receptor for the enzyme has been shown to be glchprotein 11b (19). Altered platelet ecto- l4 sialyltransferase activity could be responsible for defective aggregation in BHT, since GP IIb is part of the fibrinogen receptor complex and the receptor for the enzyme. Further understanding of this platelet defect will require specific study of membrane fibrinogen receptors, fibrinogen binding, calcium binding, and ectosialyltransferase activity. By using this animal model to examine plate- let fibrinogen interaction, calcium involvement, and aggregation, the impor- tance of platelet involvement in thrombosis may be extended. LEGENDS FOR ELECTRON MICROGRAPHS Figure 4. Citrated resting platelets from. a normal control dog(x 9000). Platelets display normal discoid shape and evenly dispersed dense granules (closed arrow). Figure 5. Citrated resting platelets from a dog affected with BHT(x 9000). These platelets are similar to the normal platelets in Fig. 4 and also show discoid shape and evenly distributed dense granules (closed arrow). Figure 6. Normal dog platelets (x 9000) 30 seconds after stimulation by ADP show shape change (pseudOpodia, open arrows), no centralization of granules, no mdcrotubule formation, partial aggregation, and dense granules are still present (closed arrow). Figure 7. BHT affected platelets (x 9000) 30 seconds after stimulation with ADP show shape change (pseudOpodia, Open arrows), centralization of granules by microtubules (closed arrow), lack of aggregation, and no dense granules which is in agreement with release data. Figure 8. Normal dog pflatelets (x 9000) 100 seconds after stimulation with ADP show significant aggregation and the presence of non- centralized dense granules (closed arrows). Figure 9. BHT platelets (x 9000) 100 seconds after stimulation with ADP are similar to the 30 second BHT sample with definite centralization of granules by microtubules, no dense granules, no aggregation, and shape change (pseudOpodia, open arrows). Figure 10. Normal dog platelets (x 9000) 200 seconds after stimulation with ADP show total aggregation and the presence of fewer dense granules (closed arrow) than previous samples (Figures 6 and 8). Figure 11. BHT platelets (x 9000) 200 seconds after stimulation with ADP show no aggregation and an apparent dissociation of microtubules. Pseudopodia are still evident (open arrow) and there are no dense granules present. 3. IU'mn: PK .7 . up“ E. “e. U? l7 ACKNOWLEDGMENTS The authors would like to thank Stoneybluff Basset Hounds for supplying phenotypically normal dogs for testing, Jan Grace for expert care of affected dogs, Donna Ladd Craft for electron micrographs preparation, Maggie Hoffman for professional graphics preparation, and Gerry White for preparation of the manuscript. This work was supported by NIH Grant number 3 P40 RR01173-03S1 AR, The American Heart Association of Michigan, and the Animal Health Diagnostic Laboratory at Michigan State University. 10. 11. 12. 13. REFERENCES BELL, T.G., LEADER, R.W.,- OLSON, F.H., PADGE’I‘T, G.A., PENNER, J.A., PATTERSON, W.R. Basset Hound Hereditaryv ThrombOpathy: An autosomally recessively inherited platelet dysfunction. One Medicine,(Ryder/Byrd), Springer-Verlay, 335-344, 1984. JOHNSTONE, I., LOTZ, F. An inherited platelet function defect in Basset Hounds. Can Vet, 3_0_,211-215, 1979. HERRMANN, F.H., MEYER, M., IHLE, E. Protein and glchprotein abnor- malities in an unusual subtype of Glanzmann's Thrombasthenia. Hemostas, l2_,337-344, 1982. MALPASS, T.W., BARKER, L.A. Acquired disorders of platelet function. Sem. Hematol, _1_'_7_(4),242-258, 1980. CHARO, I.F., FEINMAN, R.D., DETWILER, T.C.' Interrelation of platelet aggregation and secretion. J. Clin. Invest., _§_0_,866-873, 1977. MATTSON, J.C., BORGERDING, P.J., CRAFT, D.L. Fixation of platelets for scanning and transmission electron microsc0py. Stain Technology, 32(3),151-158, 1977. MEHTA, P., MEHTA, J., OSTROWSKI, N., AGUILA, E. Potentiation of platelet aggregation by Chrono-Lume®. Thromb Res, _3_2_(5),509-s12, 1983. SEEGERS, W.H. Basic principles of blood coagulation. Sem. Thromb. Haemostas, 1,180-198, 1981. MUSTARD, J.F., PACKHAM, M.A. Factors influencing platelet function: adhesion, release, and aggregation. Pharm. Rev., 3;,97-185, 1970. MARCUS, A.J. Platelet function. Part I. N. Engl J Med, 280,1213-1220, 1969. BORN, G.V.R., RICHARDSON, P.D. Activation time of blood platelets. JO We 3101., 2387-90, 19800 FEINSTEIN, 14.8., mm, J.J., SHA'AFI, R.I., WHITE, J.G. The cytoplasmic concentration of free calcium in platelets is controlled by stimulators of cyclic AMP production. Biochem. BiOptys. Res. Oomm., 113(2),S98-604, 1983. BENNETT, J.S., VILAIRE, G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J. Clin. Invest., _6_4_,1393-1401, 1979. a! .1. l.c “ A‘D 14. 15. 16. 17. 18. 19. 18 NACHMAN, R.L., LEUNG, L.L.K. Complex formation of platelet membrane gly- coproteins IIb and IIIa with fibrinogen. J. Clin. Invest., §_9_,263-269, 1982. GOGSTAD, 6.0., BROSSTAD, P., KRU’I‘ES, M.B., HAGEN, 1., SOLUM, N.O. Fibrinogen—binding properties of human platelet glyc0protein IIb-IIIa complex: A study using crossed-radioimmunoelectrOphoresis. Blood, 62,663-671, 1983. BRASS, L.F., SHATTIL, S.J. Changes in surface-bound and exchangeable calcium during platelet activation. J. Biol. Chem., 257,14000-14005, 1982. NIEWIAROWSKI, S., LEVY-TOLEDANO, 8., CAEN, J.P. Platelet interaction with polymerizing fibrin in Glanzmann's Thrombasthenia. Thromb. Res., BAUVOIS, B., CARTRON, J.P., NURDEN, A., CAEN, J. Glycoproteinsialyltrans- ferase activity of normal human, thrombasthenic, and Bernard Soulier pla- telts. Vox. Sang., 18.71-78, 1981. BAUVOIS, B., CACAN, R., NURDEN, A.T., CAEN, J., MONTREUIL, J., VERBERT, A. Membrane glyc0protein IIb is the major endogenous acceptor for human platelet ectosialyltransferase. FEES letters, 125(2),277-281, 1981. CHAPTER 3 TWO-DIMENSIONAL ELECTROPHORETIC STUDIES OF PLATELETS FROM DOGS AFFECTED WITH BASSET HOUND HEREDITARY THROMBOPATHY: A THROMBASTHENIA-LIKE AGGREGATION DEFECT Wayne R. Patterson, Thomas J. Kunicki*, and Thomas G. Bell Department of Pathology, Animal Health Diagnostic Laboratory, Michigan State University, East Lansing, Michigan 48824, USA, and *The Blood Center of Southeastern Wisconsin, 1701 W. Wisconsin Avenue, Milwaukee, Wisconsin 53226, USA ABSTRACT Because of a thrombasthenia-like platelet aggregation defect, platelets from dogs affectedv with Basset Hound Hereditary Thrombopathy were compared to normal control dog platelets by three different techniques in order to assess platelet membrane glycoprotein content. Crossed immunoelectrophoresis (CIE), two-dimensional nonreduced- reduced electrophoresis (NR-R), and O'Farrell two-dimen- sional electrophoresis were used for the assays. CIE and NR-R gels detected no differences between affected Basset Bound and control dog platelets. Gels run by the O'Farrell technique detected no differences in glycoprotein/protein content, however, there appear to be several constituents missing from BHT affected dog platelet samples. The missing components appear to be either lipids or sialoglycoproteins as they were detectable by silver staining but not by Coomassie Blue staining. INTRODUCTION Previous studies on platelets from a group of closely related Basset Hounds revealed a thrombasthenia-like intrinsic Key Words: Platelets, platelet membranes, platelet glyco- proteins, thrombasthenia-like. l9 20 platelet aggregation defect which was named Basset Hound Hereditary Thombopathy (BHT)(l,2), and has been shown to be inherited as an autosomal recessive trait (2). Other studies on BHT affected dogs revealed normal intrinsic and extrinsic coagulation mechanisms, variably prolonged template bleeding times, normal platelet count and morphology. normal clot retraction, and defective platelet aggregation responses to adenosine diphosphate (ADP) (2). More recently, studies on release of storage pool adenosine triphosphate (ATP) detected normal quantities of ATP being released in response to ADP and thrombin, but in response to ADP (and not thrombin), the release occurred so rapidly that it was complete in about one-sixth of the time required for release to occur from normal control dog platelets (3). Several plasma and platelet constituents have been shown to be required for normal platelet aggregation. Amoung these are divalent calcium ions (4,5), fibrinogen (6,7), and platelet membrane glycoproteins IIb and IIIa (8,9). It has been shown by various methods that the calcium dependent glycoprotein IIb-IIIa (GP IIb-Illa) complex is the receptor for fibrinogen on the platelet membrane (10-12). In Glanzmann's Thrombasthenia, a disease in which several platelet membrane glycoproteins are reduced or absent, platelet aggregation and fibrinogen binding are severely affected, being either totally absent or greatly reduced (13,14). Because of the evidence that the complex of glycoproteins IIb and IIIa in platelet membranes mediates platelet aggregation and that an absence or reduction of the complex results in defective platelet aggregation, platelets from dogs affected with BHT were compared to normal control dog platelets as to their platelet protein and glycoprotein composition. Two- dimensional nonreduced-reduced electrophoresis (NR-R), crossed immuno— electrophoresis (CIE), and two-dimensional O'Farrell sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) techniques were used to determine the presense of platelet membrane abnormalities. HAIEBIALS.AND_METHQD§ All reagents, buffers, and chemicals used were of the highest purity. SDS-PAGE and staining reagents were obtained from Bio-Rad (Richmond, California). GEL-BOND, GEL-BOND PAS, and agarose were obtained from FMC Corporation (Rockland, Maine), electrophoresis equipment used was a Bio-Rad PROTEAN II system (Bio-Rad, Richmond, Calif.) and an LKB Multfphor system (LKB, Stockholm, Sweden). AP-2, a monoclonal antibody to the GP IIb-IIIa complex was acquired from Dr. Thomas Kunicki (Milwaukee, Wisconsin), and an anti-human platelet antibody was obtained from Accurate Chemical & Scientific Corporation (Westbury, NY). 21 Blood was collected by jugular venipuncture and platelet rich plasma prepared from BHT and normal control dogs as previously described (2). Platelet aggregation was performed on a LUMI-AGGREGOMETER (Chronolog, Haverstown, PA) as described previously (2). Washed platelets for electrophoresis procedures were prepared as described by Kristopeit and Kunicki (15) and Nurden, et a1 (16). After the final wash, the platelets were resuspended in a volume of Tris-glygine buffer (15) sufficient to give a platelet count of 5-6 X 10 platelets/ml. Platelets for nonreduced-reduced two-dimensional electrophoresis (NR-R) were solubilized with SDS in the presence of lOmM N-ethyl maleamide (l6). Platelets for O'Farrell two - dimensional electrophoresis were solubilized with SDS in the presence of 2-mercaptoethanol acc rding to Anderson et al (17). The specimens were incsbated at 100 C for 5 minutes, aliquoted, and stored at -80 C until assayed. Platelets for crossed immunoelectrophoresis were radiolabeled and solubilized according to Phillips, et a1 (18). ElectrOphoresis of the solubilized platelet samples for NR-R gels was performed according to Nurden, et a1 (16) with modifica- tions. Tubes for first dimension gels were 3.0mm (ID) X 125mm and 200 ug samples were electrophoresed for 4-5 hours at 100 volts until the brom0phenol blue front was about 1/2 cm from the bottom of the tube. The gels were removed from the tubes and placed in 15 ml reducing buffer (16), and placed on a rocker for one hour, after which they were annealed to the second dimension slab gels with 1% agarose.E1ectrophoresis by the O'Farrell method uses isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. First dimension O'Farrell gels and buffers were prepared according to O'Farrell (19) using an ampholyte pH range of 3-10 (Pharmolytes, Sigma Chemical Company, St. Louis, MO) and 2.0mm (ID) X 125mm tubes. The samples were electrophoresed for 16 hours at 400 volts and then one additional hour at 600 volts. The gels were then placed in equilibration buffer (2% SDS, 5% 2-ME, 10% glycerol, SOmM Tris) with rocking for two hours after which they were annealed to second dimension gels with It agarose. Slab gels for second dimension electrophoresis were 16 x 16 cm square and 1.5 mm thick and were cast using GEL-BOND PAG as a backing. Buffer for second dimension electrophoresis was 0.05 M Tris, 0.384 M glycine, and 0.1% w/v SDS for both NR-R and O'Farrell gels. Second dimension electrophoresis was performed at 40 volts for 16 hours. Staining of the gels was with Coomassie Blue R (CBR) for NR-R gels and silver - coomassie blue double staining according to Dzandu, et a1 (20) for O'Farrell gels. Crossed immunoelectrophoresis was performed according to Kunicki, et al (10). Two antibodies were used: AP-2 which has been characterized as specific for the GP IIb-IIIa complex by Pidard, et a1 (21), and an anti-human platelet antibody. Autoradiographs were performed using Kodak XRP-l ofilm (Kodak, Rochester, NY) and were exposed for 36 hours at -80 C. 22 RESULTS Platelet aggregation. Aggregation tggcings of BHT and control dog platelets in response to 1 X 10 M ADP are shown in figure 1. Control dog platelets displayed a normal response to ADP, however, BHT dog platelets responded only by changing shape and no aggregation was noted. Increasing the concentration of ADP had no effect on BHT platelets (data not shown). Further discussion of the platelet aggregation abnormalities in BHT can be found in previous articles (2,3). NORMAL AF FECTED 3.2m. 320.129.. x 1’4 3T4 '“________—- FIG 1 Normal and BHT platelet aggregation tracings in response to 1.0 X 10 M ADP. The BHT (Affected) platelet aggrega- tion response shows only a decrease in percent transmission indicative of platelet shape change. Crossed immunoelectrophoresis. Figure 2 demonstrates precipitin arcs detected when solubilized platelets were electro- phoresed and autoradiographed using both AP-2 and an anti-human platelet antibody. The major precipitin arc seen in the gels is represented by the glycoprotein IIb-IIIa (GP IIb-IIIa) complex. Using AP-2, only GP IIb-IIIa is stained and autoradiographed(10). Dog platelets showed little antigen cross-reactivity with the anti- human platelet antibody with some variability in the number of precipitin arcs that are detected by CBR staining. However, the major platelet membrane glycoprotein antigen, the GP IIb-Illa complex, is precipitated in all samples tested and appears normal in charge and quantity using both AP-2 and an anti-human platelet antibody. Nonreduced-reduced two-dimensional gels. Figure 3 shows CBR FIG 2 Crossed immunoelectrophoresis gels of radiolabeled, Triton X-100 solubilized platelets stained with Coomassie Blue R (A,B,C,D) and corresponding autoradiographs (E,F,G,H). Normal dog (A,E) and BHT platelets (B,F) using an anti-human platelet antibody. Normal dog (C,G) and BHT platelets (D,H) using AP-2, a monoclonal antibody specific for the GP IIb— IIIa complex. The gels show no differences between normal and BHT platelets. Normal FIG 3 Nonreduced-reduced two dimensional electrophoresis gels of SDS solubilized normal and BHT platelets stained with Coomassie Blue R. Several components have been labeled; ThrombOSpondin (Tsp), fibrinogen (Fbg), actin (Ac), and glycoproteins IIb, 111a, and Ib (IIb, 111a, and lb). The gels revealed no differences between normal and BHT platelets by this technique. DI 40 Normal n . 4 FIG 4 O'Farrell two-dimensional gels of SDS solubilized normal and BHT platelets stained by a Coomassie Blue R - silver double staining technique. The gels revealed no differences in the glycoprotein/protein content (Coomassie Blue staining spots) however, they did show apparent differences in that there are several missing components in the BHT sample (surrounded by arrows). These components stained yellow in the normal sample which indicates that they are possibly lipid or sialoglycoprotein in nature. stained NR-R gels from normal and BHT affected dogs. Again, GP's IIb and IIIa are present in normal quantities as are glycoprotein Ib, platelet fibrinogen, thrombospondin, and other membrane and intracellular constituents. The NR—R gels showed complete homology with human specimens as NR—R gels of solubilized human platelets were indistinguishable from those of the dog samples (personal observation). O'Farrell two—dimensional gels. Representative gels from normal control and BHT dog platelets are.shown in Figure 4. The gels were stained by a silver—CBR double staining technique (20). There are no apparent differences between between normal and BHT platelets with respect to protein/glycoprotein content (Coomassie Blue R staining spots). The area of the gels surrounded by arrows shows several spots missing in the BHT sample when compared to the normal control sample. with the double staining technique these spots were yellow—brown in color and according to Dzandu, et a1 (20) they are probably lipids. All the CBR stained spots appeared identical in number and location when comparing normal 25 staining intensity which would indicate differences in protein concentration between samples. Every attempt was made to use lOOug protein samples, however, minor errors in protein measurement or pipetting cannot be ruled out. The missing spots in the BHT sample do not appear to be due to variations in concentration since they are completely absent rather than less intensely stained. Also, the silver staining technique is several times more sensitive than CBR staining. 2152285193 It is now well recognized that some major events in the hemostatic process are mediated by platelet membrane glycoproteins. By studying platelets from patients with Bernard- Soulier Syndrome, glycoprotein Ib has been shown to be responsible for platlet adherence to subendothelium , since Bernard—Soulier platelets are deficient in glycoprotein Ib and do not adhere to exposed subendothelium (13,22). However, these platelets did show normal aggregation responses to ADP and collagen (22). Another inherited bleeding disorder, Glanzmann's Thrombasthenia, shows absent platelet aggregation responses to ADP, thrombin, and collagen, and is deficient in platelet membrane glycoproteins IIb and IIIa (14). Subsequent to these reports, the GP IIb-IIIa complex has been shown to be the calcium dependent platelet fibrinogen receptor which is necessary for normal platelet aggregation (8-12). BHT resembles Glanzmann's Thrombasthenia in that there is an absence of platelet aggregation in reponse to various aggregating agents, but it is dissimilar in that there is normal clot retraction in BHT (2). Investigation of the BET platelet membrane glycoproteins was indicated, however, due to the absence of platlet agrregation response. The presence of an apparently normal GP IIb-IIIa complex as demonstrated _by CIE and NR-R gels rules out the possibility of BHT being an animal homologue of thrombasthenia, but, the possibility remains that the GP IIb-111a complex may be functionally abnormal. O'Farrell two- dimensional electrophoretic gels could theoretically demonstrate abnorm- alities in platelet composition if they resulted in changes in isoelectric point and/or molecular weight. Over a pH range large enough to encompass the majority of platelet components, however, the abnormalities would have to be gross enough to cause a relatively large change in the isoelectric point or molecular weight. Therefore, minor abnormalities could go undetected in this procedure. The comparison of BHT and normal control dog solubilized platelets by the O'Farrell technique did not reveal differences in glycoprotein and/or protein content, as was also evidenced by CIE and NR-R gels. However, O'Farrell gels did reveal some apparent lipid abnormalities, with several spots missing from the BHT sample. Further studies of BHT platelet membrane lipid, phospholipid, and glycolipid contents by more analytical techniques will be required to establish if an actual t." u: teci gre. PIO‘ ackl 26 lipid abnormality exists in BHT platelet membranes. Lipid differences, if present, could affect platelet membrane reorganization or fluidity or membrane receptor movement after stimulation which is essential to normal platelet function. Membrane reorganization may be essential for exposure of fibrinogen and other membrane receptors, and impr0per reorganization could adversely affect agonist induced platelet aggregation. Studies of agonist induced fibrinogen binding to BHT platelets is imperative in order to establish if GP IIb-IIIa, shown by electrophoretic techniques to be quantitatively normal, is functionally normal in BHT and is capable of binding fibrinogen. Fibrinogen binding studies are currently in progress. AQKNQHLEDGEHENIE This work was supported by NIH grant HL31753-02. The expert technical assistance of Sue Kristopeit and Randy Piotrowicz is greatly appreciated. The expertise of Jan Grace who always provided excellent care of the Basset Hounds is also gratefully acknowledged. ~ BEEEBENQES 1. JOHNSTONE, I., AND LOTZ, E. An inherited platelet function defect in Basset Hounds. Qan_¥et, 2Q, 211-215, 1979. 2. BELL, T.G., LEADER, R.W., OLSON, F.H., PADGETT, G.A., PENNER, J.A., AND PATTERSON, W.R. Basset Hound Hereditary Thrombopathy: An autosomally recessively inherited platelet dysfunction. an: Medicine, (Ryder/Byrd), Springer-Verlag, 335-344, 1984. 3. PATTERSON, W.R., PADGETT, G.A., AND BELL, T.G. Abnormal release of storage pool adenine nucleotides from platelets of dogs affected with Basset Hound Hereditary Thrombopathy. Thrgmb Res, 31, 61-71, 1985. 4. DETWILER, T.G., CHARO, I.F., AND FEINMAN, R.D. Evidence that calcium regulates platelet function. Ih;gmh_ng§,‘gn, 207-211, 1978. 5. GERRARD, J.M., PETERSON, D.A., AND WHITE, J.G. Calcium mobil- ization. In:zlat.els.ts_in_nielnsx_and_2athnlnul J.L. Gordon (86) Amsterdam - Elsevier/North Holland Biomedical Press, 407-436, 1981. 6. CROSS, M.J. Effect of fibrinogen on the aggregation of platelets by adenosine diphosphate. Thrgmhg§_niath_nagmgrzhp 12: 524-527, 1964. a}! PI (a :3 e a.“ 9 a Punl Old f ”Hart; 1 which 1 Pt Fruit . a l (lurks: 7e halal l‘uhbsl I Cy ~\.u‘ 27 7.NIEWIAROWSKI, S., KORNECKI, E., BUDZYNSKI, A.Z., MORINELLI, T.A., AND TUSZYNSKI, G.P. Fibrinogen interactions with platelet receptors. Ann_NX_A&afi_§gi, 438, 536-554, 1983. 8. JENNINGS, L.K., AND PHILLIPS, D.R. Purification of glycoprot- eins IIb and IIIa from human platelet plasma membranes and char- acterization of a calcium-dependent glycoprotein IIb-III complex. .J_Ein_Chfim, 251, 10458- -10466, 1982. 9. LEGRAND, C., DUBERNARD, V., AND CAEN, J.P. Platelet aggreg- ation: Its relation with ADP-induced fibrinogen binding to platelets and ADP-related membrane enzyme activities. Eur_1 Eimhem 112. 465-471, 1984. 10. KUNICKI, T 1., PIDARD, D., ROSA, J. P., AND NURDEN, A.T. The formation of Ca -dependent complexes of platelet membrane glyco- proteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis. Bleed, 58, 268-278, 1981. " 11. NACHMAN, R. L., AND LEUNG, L. L. K. Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen. W 5.9.. 263- -269. 1983. 12. BENNETT, J.S., HOXIE, J.A., LEITMAN, S.F., VILAIRE, G., AND CINES, D.B. Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. 2;gg_Natl_A&nd_£si_fl£A, 8Q. 2417-2421, 1983. 13. GEORGE, J.N., NURDEN, A.T., AND PHILLIPS, D.R. Molecular defects in interactions of platelets with the vessel wall. N_Engl J_H£fi4 111: 1084-1098, 1984. 14. NURDEN, A.T., DIDRY, D., KIEFFER, N., AND NCEVER, R.P. Resid- ual amounts of glycoprotein IIb and IIIa may be present in the platelets of most patients with Glanzmann's Thrombasthenia. Blond 3g, 1021-1024, 1985. 15. KRISTOPEIT, S.M., AND KUNICKI, T.J. Quantitation of platelet membrane glycoproteins in Glanzmann's Thrombasthenia and the Bernard-Soulier Syndrome by electroimmunoassay. Ihrgmb__£gfi..1§, 133-142, 1984. 16. NURDEN, A.T., DUPUIS, D., KUNICKI, T.J., AND CAEN, J.P. Analysis of the glycoprotein and protein composition of Bernard- Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ‘1_£11n_1n2g§t, 51, 1431-1440, 1981. 17. ANDERSON, L., AND ANDERSON, N. G. High resolution two-dimen- sional electrophoresis of human plasma proteins. 2L29_Nat1_A§afi smi_flSA, 14, 5421- -5425, 1977. 28 18. PHILLIPS, D.R., AND POH AGIN, P. Platelet plasma membrane glycoproteins. Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis. 1_Biel_ghem, 252, 2121-2126, 1977. 19. O'FARRELL, P.H. High resolution two-dimensional electrophor- esis of proteins. 1_Biel_§hem, 250, 4007-4021, 1975. 20. DZANDU, J.K., DEH, M.E., BARRETT, D.L., AND WISE, G.E. Detec- tion of erythrocyte membrane proteins, sialoproteins, and lipids in the same polyacrylamide gel using a double staining technique. I .81, 1733-1737; 1984. 21. PIDARD, D., MONTGOMERY, R.R., BENNETT, J.S., AND KUNICKI, T.J. Interaction of AP-2, a monclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets. 1_Biel_§h£m. 255. 12582-12586, 1983. 22. CAEN, J.P., AND LEVY-TOLEDANO, S. Interaction between platelets and von Willebrand factor provides a new scheme for primary hemostasis. Nature, 214, 159-160, 1973. CHAPTER 4 PIBRINOGEN BINDING TO PLATELETS PROM DOGS WITH BASSET HOUND HEREDITARY THROHBOPATHY: A THRONBASTHENIA-LIKE AGGREGATION DEFECT Wayne R. Patterson, Douglas W. Estry, Kenneth A. Schwartz, and Thomas G. Bell The Departments of Pathology, Medical Technology, and Medicine, Michigan State University, East Lansing, Michigan 48824 This work Supported by NIH grant HL31753-02 Running Title: BHT Fibrinogen Binding Address all Correspondence to: Wayne Patterson Dept. of Pathology A522 East Pee Hall Michigan State University East Lansing, Michigan 48824 29 30 53518521 Platelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) display a thrombasthenia-like aggrega- tion defect but have been shown to have normal amounts of platelet membrane glycoproteins IIb and IIIa (GP IIb-IIIa). The presence of normal quantities of GP IIb-IIIa, however, did not rule out the possibility of a functionally abnormal glycoprotein complex which would be unable to bind radio- labeled fibrinogen. Therefore, fibrinogen binding in BHT platelets was evaluated. Fibrinogen preparations from BHT and normal control dogs, as well as a human fibrinogen preparation were used. Platelets from BHT and normal dogs were activated with 1 x 10-5M ADP in the presence of 1251- 1abeled fibrinogen and the surface bound radioactivity was quantitated. For all fibrinogen preparations, the amount of fibrinogen bound by BHT platelets was not significantly different than that bound by normal dog platelets. BHT platelets bound 23,972 1, 3612 and normal dog platelets bound 23,033 1 3971 molecules of fibrinogen per platelet. The quantitatively normal GP IIb- IIIa complex binds fibrinogen in normal amounts and does not seem to be the abnormality responsible for the aggregation defect in BHT platelets. The results show that the binding of fibrinogen is normal and that other factors, such as receptor mobility may help to explain the abnormal aggregation of BHT platelets. 31 INIBQDHQIIQN Central to the normal hemostatic process is the aggregation of platelets at the injury site. It has long been recognized that certain plasma factors, especially calcium and fibrinogen, are required for platelet aggregation to occur (1,2). Afibrinogenemic individuals have prolonged bleeding times (3), and platelets from individuals with Glanzmann's Thrombasthenia do not aggregate (4) or adsorb fibrinogen (5,6) which suggests that during clot formation fibrinogen and platelets are involved in specific interactions. Studies have shown that ADP induces the exposure of specific and saturable fibrinogen receptors on the platelet surface and that fibrinogen binding and platelet aggregability are closely correlated (7,8), with some studies indicating that the dimeric fibrinogen molecule may actually form a bridge between receptors on adjacent platelets (9). Platelets from Glanzmann's thrombasthenia patients are characterized by a severely reduced fibrinogen binding capacity, as well as an inability to aggregate in response to ADP (6.9). One of the major defects in this disorder is the absence or reduction of platelet membrane glycoproteins IIb and IIIa (GP IIb- IIIa)(10-12). Evidence has accumulated that the calcium dependent GP IIb-IIIa complex is the platelet membrane receptor for fibrinogen and is responsible for platelet- platelet cohesion in ADP 32 antibodies specific for the GP IIb- IIIa complex, have been shown to cause inhibi- tion of both fibrinogen binding and platelet aggregation (18,19). Basset Hound Hereditary Thrombopathy (BHT) is described as a thrombasthenia-like aggregation defect (20,21) with an abnormally rapid but quantitatively normal release of storage pool adenine nucleotides (22). In view of the thrombasthenia-like aggregation defect, the platelet membrane glycoprotein content was investigated and found to be normal (23). The study of radiolabeled fibrinogen binding to ADP activated BHT platelets was undertaken to ascertain if the quantitatively normal GP IIb-IIIa complex was functionally normal with respect to its fibrinogen binding capability. 33 HEIEQDS Platelets. Platelets were isolated and washed according to the method of Kunicki et a1.(24). Briefly, six volumes of whole blood were drawn into one volume of acid-citrate-dextrose (ACD, NIH formula A) by non-traumatic jugular puncture as described by Bell et a1. (21). Platelet rich plasma (PRP) was prepared by centrifugation at 1470 x g for 1.5 minutes. The centrifugation step was repeated two times and provided approximately 10 m1 of PRP from 24 m1 of blood. A11 manipulations were performed at room temperature. During washing, apyrase (2 U/ml) and prostaglandin E1 (20 nmol/L)(Sigma Chemical Company, St. Louis, M0.) were used in the wash buffer to prevent inadvertent platelet activation. Platelets were resuspended to a concentration of 1 x 109 platelets/m1. Prepared platelets maintained their ability to bind fibrinogen for at least four hours. EIBRINQGEN. Plasma fibrinogen was purified from BHT affected and normal control dogs according to Takeda (25). Human fibrinogen was obtained from Rabi Vitrum, Stockholm, Sweden. Purity of the fibrinogen preparations was checked by SDS- Polyacrylamide Gel Electrophoresis (SDS-PAGE). Clottability was determined to be approximately 958 before radioiodination and >90% after radioiodination. Fibrinogen 12S preparations were radioiodinated with carrier free I (Amersham, Arlington Heights, IL) using lacto- 34 peroxidase-conjugated beads (Enzymobeads, BioRad Laboratories, Richmond, CA) according to the manufacturer's instructions. Unbound 125 I was removed from the fibrinogen reaction mixture by gel filtration through Sephadex 6-50. The specific activity of the fibrinogen preparations was determined to be approximately 100 cpm/ng. Labeled fibrinogen was used within one week and stored at 4°C. EIBBINQ§£N_BINDIN§. Binding assays were performed as described by Kunicki, et. a1. (24). Non-specific binding was determined and was subtracted to give specific binding. BHT and normal dog platelets were reacted in triplicate with each of the three fibrinogen preparations, i.e. BHT, normal, and human fibrinogen. 2LATELET__A§GBE§ATTQN. Blood was collected and PRP prepared according to Bell et a1. (21). Platelet aggregation studies were performed using a Lumi-Aggrego- meter (Chronolog Corp, Haverstown, PA). PRP was adjusted as necessary to a final platelet count of 300 x 103/ul with platelet poor plasma. ADP, to a final concentration of 1x10'5, was added to 0.45 ml PRP with mixing at 900 rpm and at 37°C. The chart recorder was run at 2.5 cm/min. STATISTICS. The Students t Test was used to compare BHT platelets to normal dog platelets as to the number of fibrinogen molecules bound per platelet. Significance is specified as p < .05. 35 BESQLIS Wm. As seen in figure 1. the aggregation response of BHT platelets, when compared to platelets from normal dogs, is totally absent except for a decrease in percent transmission corresponding to platelet shape change. Increasing the concentration of ADP to 5 2x10' M had no effect on the aggregation response as has been previously reported (22). EihLinegen___Binding. Three different purified fibrinogen preparations were used to exclude the possibility that there might be a fibrinogen abnormality that would affect binding to stimulated platelets. As seen in table 1, the mean values of number of molecules of fibrinogen bound per ADP activated platelet for each fibrinogen preparation, and the overall means for all preparations are not significantly different between BHT and normal dog platelets. The overall values, 23,972 1 3612 molecules per platelet for BHT and 23,033 1,3971 molecules per platelet for normal control dogs show that BHT platelets do in fact bind fibrinogen. Fibrinogen binding values for both test groups are comparable to those obtained by other investigators for human platelets (26,27). It is also interesting to note that human fibrinogen will bind to dog platelets with no apparent species specific differences in total binding. It appears that a quantitatively normal GP IIb-IIIa complex, as reported previously (23), is also functionally normal in 36 its fibrinogen binding capacity. Competitive inhibition of 125 I-fibrinogen binding to ADP activated platelets by unlabeled fibrinogen is illustrated in figure 2. The total amount of fibrinogen in the reaction mixture was constant with only the percentage of labeled fibrinogen being varied. The linear relationship of the number of 125 I-fibrinogen molecules bound versus the percentage labeled fibrinogen in the reaction mixture established that the binding of fibrinogen to ADP activated dog platelets is specific as has been previously reported by Marguerie et al. for human platelets (28). 37 DISQHEEIQN Fibrinogen is a cofactor in ADP- induced platelet aggregation (1-7), and its binding to specific receptors on the platelet membrane mediates platelet- platelet cohesion and aggregation (4,6,9,29). A four phase sequence for fibrinogen binding as proposed by Marguerie and Plow (29) involves platelet activation, induction of the fibrinogen receptor, reversible fibrinogen binding, and fibrinogen-platelet complex stabilization. Many agonists cause platelet activation, some by seemingly different pathways, but each seems to lead to exposure of fibrinogen receptors and fibrinogen binding. The mechanics of fibrinogen receptor exposure on platelets is somewhat of a controversy at present. Shattil et a1.(30) reported that receptor exposure involves a conformational change in GP IIb-IIIa which exposes the fibrinogen binding site, while Coller (31) presents evidence for a membrane microenviron- mental change which exposes the receptor. Studies with platelets from patients with Bernard- Soulier and Glanzmann's thrombasthenia show that the calcium dependent membrane GP IIb-IIIa complex is the major platelet receptor for fibrinogen (4,6,9). Platelet activation by ADP is attributed to an agonist induced increase in cytoplasmic calcium ion concentration (32), with exposure of fibrinogen receptors being part of the overall response. These responses can be blocked by raising 38 the c-AMP levels in platelets (33,34). BHT is characterized by an absence of a platelet aggregation response with an abnormally rapid release of normal quantities of storage pool adenine nucleotides (22). The aggregation tracings are similar to those observed with Glanzmann's thrombasthenic platelets (21,22). Investigation of the platelet membrane GP IIb-IIIa complex in BHT platelets revealed apparently normal quantities and complexing of the glycoproteins, in contrast to Glanzmann's platelets which show absent or reduced amounts of GPIIb- IIIa (9). Two obvious possibilities for the aggregation defect were that the GP IIb-IIIa complex was present in the membrane in normal amounts, but was unavailable to fibrinogen because of other defects which prevented its exposure after ADP stimulation or, that the receptor was unable to bind fibrinogen once it was exposed. Either of these alternatives would evidence themselves as absent or reduced fibrinogen binding. However, in this report we show that the number of fibrinogen molecules bound per platelet by BHT platelets is identical to that bound by normal dog platelets. Therefore, fibrinogen receptor exposure and binding must be normal in BHT. This suggests that fibrinogen binding is necessary for platelet aggregation but is not sufficient in and of itself for normal aggregation. This phenomenon was reported previously by Peerschke and Zucker (34), who observed that fixed ADP stimulated platelets bound fibrinogen but failed to 39 aggregate when shaken. Receptor movement within the membrane may also be essential for normal platelet aggregation, and simply binding fibrinogen is not enough to support aggregation. Fibrinogen receptor redistribution in platelets after activation has been reported by Loftus and Albrecht (35) and Estry et a1. (36), who used fibrinogen-labeled colloidal-gold to directly visualize fibrinogen receptors on platelet membranes. Their experiments showed no fibrinogen binding in platelets immediately after contact which was followed by fibrinogen binding and redistribution with localization over the granulomere. Receptor movement would seem to be part of the normal course of events in platelet aggregation and abnormalities in membrane fluidity may have an antagonistic effect on platelet aggregation. It is interesting to note that an apparent lipid defect was observed in BHT platelets by two-dimensional O'Earrell electrophoretic techniques (23), but more analytical techniques are required to fully establish if a lipid defect is actually present. Another possible explanation for the defect may be an abnormal membrane glycoprotein-cytoskeletal protein interaction. Kalomiris and Coller (37) provide evidence to suspect that protein sulhydryl and disulfide groups may contribute to various platelet functions, especially those in which membrane related events influence cytoplasmic events and vice versa. Since BHT platelets have normal amounts of GP IIb-IIIa and, as we show, normal fibrinogen binding, the 40 possibility of abnormal membrane fluidity and membrane protein-cytoskeletal protein interactions needs to be investigated. 41 ACKNQHLEDQHENIS The authors wish to thank Jan Grace for her excellent professional care of the animals used in this project, and Maggie Hoffman for her expert preparation of graphics. We are also grateful to Stoneybluff Basset Hounds for their ongoing support in all facets of study of this bleeding disorder by supplying heterozygote and normal dogs as needed. FIG'I 42 LEGENDS Eignre_l. Aggregation tracings of BHT and normal dog platelets in response to 1 x 10.5 M ADP. The only response noted with BHT platelets was a decrease in percent transmission indicative of shape change. of 1251- labeled Eigure__2. Competitive inhibition fibrinogen binding. The total amount of fibrinogen was constant with only the percentage of 125I- fibrinogen being varied. Unlabeled fibrinogen was able to compete with labeled fibrinogen for binding to receptors, after ADP stimulation, directly proportional to the amount of unlabeled fibrinogen present, which indicates specific binding to membrane receptors. 4r Fibrinogen Molecules Bound x 103 N U'l l N O l G l 5 I U) l 2'5 52) 7'5 .60 96 125 I Labeled Fibrinogen F|G2 45 COMPARATIVE FIBRINOGEN BINDING (MOLECULES/PLATELET) IN BASSET BOUND HEREDITARY THROMBOPATHY Normal Dog BHT Platelets Platelets Type Fibrinogen 22 c 37°C 22°C 37°C BHT (dog) 20939 7385 21748 6461 13269 1507 13970 12028 Normal (dog) 23217 7893 25315 7283 12998 1704 1841 .1288 Human 24942 7384 25408 8197 15596 12327 14134 12211 Mean Binding 23033 7554 23972 7573 All types of 13971 11267 13612 411848 Fibrinogen Table 1 Number of molecules of 1251- labeled fibrinogen bound to washed platelets from BHT and normal control dogs. 5M, final Platelets were stimulated with ADP (1 x 10- concentation) at 22°C and 37°C. Purified fibrinogen preparations of indicated types were used. Both homologous and heterologous results were averaged. values are expressed as mean 1 SD, (Three affected Basset Hound and three normal dog samples were run in triplicate). 46 BEEEBENQES 1. Cross MJ. Effect of Fibrinogen on the Aggregation of Platelets by Adenosine Diphosphate. Thrombos Diathes Haemorrh 12:521, 1964 2. McLean JR, Maxwell RE, and Herther D. Fibrinogen and Adenosine Diphosphate-Induced Aggregation of Platelets. Nature (London) 202:605, 1964 3. Weiss HJ, and Rogers J. Fibrinogen and Platelets in the Primary Arrest of Bleeding. N Engl J Med 285:369, 1971 4. Caen JP. Glanzmann Thrombasthenia. Clin Heamatol 1:383, 1972 5. Bang NU, Heidenreich R0, and Trygstad CW. Plasma Protein Requirements for Human Platelet Aggregation. Ann NY Acad Sci 201:280, 1972 6. Kornecki E, Niewiarowski S, Morinelli TA, and Kloczewiak M. Effects of Chymotrypsin and Adenosine Diphosphate on the Exposure of Fibrinogen Receptors on Normal Human and Glanzmann's Thrombasthenia Platelets. J Biol Chem 256:5696, 1981 47 7. Mustard JF, Packham MA, Kinlough-Rathbone RL, Perry DW, and Regoeczi E. Fibrinogen and ADP-Induced Aggregation. Blood 52:453. 1978 8. Peerschke EI, Zucker MB, Grant RA, Egan JJ, and Johnson MM. Correlation Between Fibrinogen Binding to Human Platelets and Platelet Aggregability. Blood 55:841, 1980 9. Lee H, Nurden AT, Thomaidis A, and Caen JP. Relationship Between Fibrinogen Binding and the Platelet Glycoprotein Deficiencies in Glanzmann's Thrombasthenia Type I and Type II. Br J Haematol 48:47, 1981 10. Phillips DR, and Poh Agin P. Platelet Membrane Defects in Glanzmann's Thrombasthenia. J Clin Invest 60:535, 1977 11. Nurden AT, and Caen JP. The Different Glycoprotein Abnormalities in Thrombasthenic and Bernard-Soulier Platelets. Semin Hematol 28:253. 1974 12. Hagen I, Nurden AT, Hjerrum OJ, Solum NO, and Caen JP. Immunochemical Evidence for Protein Abnormalities in Platelets from Patients with Glanzmann's Thrombasthenia and Bernard-Soulier Syndrome. J Clin Invest 65:722, 1980 48 13. Bennett JS, and Vilaire G. Exposure of Platelet Fibrinogen Receptors by ADP and Epinephrine. J Clin Invest 64:1393, 1979 14. Mustard JF, Rinlough-Rathbone RL, Packham MA, Perry DW, Harfenist EJ, and Pai KRM. Comparison of Fibrinogen Association With Normal and Thrombasthenic Platelets on Exposure to ADP or Chymotrypsin. Blood 54:987. 1979 15. Nachman RL, and Leung LLK. Complex Formation of Platelet Membrane Glycoproteins IIb and IIIa with Fibrinogen. J Clin Invest 69:263. 1982 16. Gogstad GO, Brosstad P, Krutnes MB, Hagen I, and Solum NO. Fibrinogen-Binding Properties of the Human Platelet Glycoprotein IIb-IIIa Complex: A Study Using Crossed-Radioimmunoelectrophoresis. Blood 60:663. 1982 17. Bennett JS, Vilaire G, and Cines DB. Identifi- cation of the Fibrinogen Receptor on Human Platelets by Photoaffinity Labeling. J Biol Chem 257:8049, 1982 18. McEver RP, Bennett EM, and Martin MN. Identifica- tion of Two Structurally and Functionally Distinct Sites on Human Platelet Membrane Glycoprotein IIb-IIIa Using Mono- clonal Antibodies. J Biol Chem 258:5269, 1983 49 19. Pidard D, Montgomery RR, Bennett JS, and Kunicki, TJ. Interaction of AP-2, A Monoclonal Antibody Specific for the Human Platelet Glycoprotein IIb-IIIa Complex, With Intact Platelets. J Biol Chem 258:12582, 1983 20. Johnstone I, and Lotz E. An Inherited Platelet Function Defect in Basset Hounds. Can Vet 20:211. 1979 21. Bell TG, Leader RW, Olson PM, Padgett GA. Penner JA, and Patterson WR. Basset Hound Hereditary Thrombopathy: An Autosomally Recessively Inherited Platelet Dysfunction. In Ryder/Byrd (eds): One Medicine. Berlin, Springer-Verlag, 1984. p335 22. Patterson WR. Padgett GA, and Bell TG. Abnormal Release of Storage Pool Adenine Nucleotides From Platelets of Dogs Affected With Basset Hound Hereditary Thrombopathy. ‘Thromb Res 37:61, 1985 23. Patterson WR. Padgett GA, and Bell TG. Platelet Aggregation, Adenine Nucleotide Release, and Membrane Glycoproteins in Dogs Affected With Basset Hound Hereditary Thrombopathy. Thrombos Hemostas 54(1):188, 1985 (abstr) 24. Kunicki TJ, Newman PJ, Amrani DL, and Mosesson MW. Human Platelet Fibrinogen: Purification and Hemostatic Properties. Blood 66(4):808, 1985 50 25. Takeda Y. Studies of the Metabolism and Distri- 125 bution of Fibrinogen in Healthy Men With Autologous I- Labeled Fibrinogen. J Clin Invest 45:103. 1966 26. Peerschke EIB. Ca+2 Mobilization and Fibrinogen Binding of Platelets Refractory to Adenosine DiphOSphate Stimulation. J Lab Clin Med 106(2):111. 1985 27. Legrand C. Dubernard v, and Nurden AT. Character- istics of Collagen-Induced Fibrinogen Binding to Human Platelets. Biochim Biophys Acta 812:802. 1985 28. Marguerie GA, Plow EP, and Edington TS. Human Platelets Possess an Inducible and Saturable Receptor Specific for Fibrinogen. J Biol Chem 254:5357, 1979 29. Marguerie GA, and Plow SF. The Fibrinogen-Depend- ent Pathway of Platelet Aggregation. Ann NY Acad Sci 408:556. 1983 30. Shattil SJ, Hoxie JA, Cunningham MC, and Brass LP. Expression of the Platelet Fibrinogen Receptor Involves a Conformational Change in the Membrane Glycoprotein IIb-IIIa Complex. Thrombos Hemostas 54(1):50, 1985 51 31. Coller BS. Evidence That “Exposure" of the Platelet GP IIb-IIIa Complex Receptor Involves a Micro- environmental Change. Thrombos Hemostas 54(1):50. 1985 32. Massini P. The Role of Calcium in the Stimulation of Platelets. in DCB Mills and PI Pareti (eds). Platelets and Thrombosis. New York. Academic Press, 1977, p33 33.Mills DCB. Platelet Aggregation and the Adenylate Cyclase System. in DCB Mills and PI Pareti (eds). Platelets and Thrombosis. New York. Academic Press, 1977. p63 34. Peerschke EI, and Zucker MB. Fibrinogen Receptor Exposure and Aggregation of Human Blood Platelets Produced by ADP and Chilling. Blood 57:663. 1981 35. Loftus JC, and Albrecht RM. Redistribution of the Fibrinogen Receptor of Human Platelets After Surface Activation. J Cell Biol 99:822. 1984 36. Estry DW, Oesterle JR, Mattson JC. Patterson WR. and Bell TG. Analysis of ADP-Induced Fibrinogen Receptors in Dogs With Basset Hound Hereditary Thrombopathia. Blood 66(5).Supl 1. Nov 1985, In Press 52 37. Kalomiris EL and Coller BS. Thio-Specific Probes Indicate That the B-chain of Platelet Glycoprotein Ib is a Tramsmembrane Protein with a Reactive Endofacial Sulfhydryl Group. Biochemistry 24:5430, 1985 CHAPTER 5 Normal hemostasis is accomplished through a complex set of reactions that involve platelets, the vascular wall, and the coagulation factors. Central to the hemostatic process is the role of the platelet which includes its ability to change shape. adhere to subendothelial tissue, release storage pool granule contents, and to aggregate into viable and stable platelet plugs. For some platelet stimuli. there must be a functional receptor on the platelet membrane to which the agonist binds and the stimulus is biochemically transmitted to the platelet interior. Microtubule mediated concentration and release of platelet granules follows, and finally. surface fibrinogen receptors are exposed and interact with fibrinogen thereby mediating aggregation. It has been shown that the platelet membrane fibrinogen receptor is a calcium-dependent heterodimer complex of glycoproteins IIb and IIIa (GP IIb-IIIa). Dysfunction of any of the above series of events can cause platelet aggregation abnormalities. A colony of Basset Hounds with a platelet aggregation defect was identified and various studies have been performed to attempt to pinpoint the exact defect. The results of these studies were reported in the preceding chapters. 53 54 As seen in chapter 1, platelets from dogs with Basset Hound Hereditary Thrombopathy are capable of releasing their storage pool granule contents in normal quantities. however. they do so much more rapidly than normal dog platelets. This seemingly unsynchronized release may have deleterious effects on the overall aggregation reaction and should be investigated further. In chapter 2, the platelet membrane and total platelet protein and glycoprotein content were shown to be normal, with an apparent defect in lipid content which will require more analytical techniques to fully establish. Finally, in chapter 3, it was shown that platelets from BHT affected dogs were able to bind fibrinogen in normal amounts when compared to normal dog platelets. To summarize, it was shown that BHT platelets release normal amounts of storage pool contents, they contain normal amounts of the GP IIb-IIIa complex, and they bind normal amounts of radiolabeled fibrinogen. However, they still fail to aggregate. Possibilities that would explain the defect in light of the presented data would include: 1) the rapid release of storage pool contents is responsible for unsynchronizing the biochemical events necessary for a normal platelet aggregatory response. Perhaps if storage pool granule contents are released too early in the complex chain of events, they simply might not be in the right place at the right time: 2) the presence of an abnormal intracellular calcium content or flux after stimulation. Calcium 55 sequestration. movement, and resequestration are necessary events for proper muscle cell function and are also essential to normal platelet function. Abnormal sequestration or movement of calcium within BHT platelets would adversely affect several biochemical mechanisms which include microtubule reorganization, myosin phosphorylation, and stimulation of protein kinase C and phospholipase C. Since some of these calcium dependent events are directly related to secretion and aggregation, investigating this alternative could provide data which would better explain platelet responses after stimulation: and 3) abnormal membrane fluidity or lipid-protein interactions caused by abnormal lipid content which would adversely affect receptor mobility. Receptor mobility appears to be part of the normal course of events after stimulation. Painter et a1. (1985) proposed that a small population of GP IIb-IIIa does not bind fibrinogen, but rather is associated with the platelet cytoskeleton. After stimulation and fibrinogen binding. the GP IIb-IIIa that does bind fibrinogen moves to become closely associated with the nonfibrinogen binding GP IIb-IIIa population. The final association presumably provides a bridge between the cytoskeletons of adjacent platelets. Another alternative related to abnormal lipid content would be that when some lipids are present in cell membranes the membranes are more rigid. and their presence may affect cell membrane charge. If this were the case perhaps an abnormally charged or rigid membrane would 56 disallow a conformational change in the GP IIb-IIIa complex after stimulation and fibrinogen binding. However this may not be the case in view of normal clot retraction in BHT. Yet another possible explanation for the platelet defect, which was previously discussed. may be an abnormal membrane glycoprotein-cytoskeletal interaction which is mediated by sulfhydryl and/or disulfide groups. 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