Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

Role of calcium and calpain in complement-induced vesiculation of the platelet plasma membrane and in the exposure of the platelet factor Va receptor

The role of calcium and intracellular calpains in the expression of platelet prothrombinase activity was investigated. Incubation of gel-filtered platelets with complement proteins C5b-9 resulted in alpha-granule and dense granule secretion and exposure of membrane binding sites for coagulation factors Va and Xa. This was accompanied by the release of microparticles from the cell surface that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa and the alpha-granule membrane protein GMP-140. Generation of these membrane microparticles was dependent on the presence of extracellular calcium and was accompanied by proteolytic degradation of the cytoskeletal proteins, actin binding protein (ABP), talin, and myosin heavy chain. Microparticle formation was also detected when unstirred platelets were activated by thrombin plus collagen, although proteolysis of ABP, talin, or myosin was not observed. Preincorporation of the calpain inhibitor leupeptin into the platelet cytosol completely blocked C5b-9-induced proteolysis of ABP, talin, and myosin. However, inhibition of this calpain-mediated proteolysis had no effect on platelet secretion, the generation of microparticles, the exposure of membrane sites for factors Va and Xa, or the expression of prothrombinase activity. Furthermore, the microparticles that formed in the presence of leupeptin contained intact ABP, talin, and myosin heavy chain. Prior depletion of ATP with metabolic inhibitors eliminated all platelet responses to thrombin plus collagen, but did not affect C5b-9-induced microparticle formation or exposure of binding sites for factor Va on the microparticles. These data indicate that the formation of microparticles and the expression of platelet prothrombinase activity in response to C5b-9 are dependent upon an influx of calcium into the platelet cytosol, but do not require metabolic energy or calpain-mediated proteolysis of cytoskeletal proteins.     

Fluoride-dependent calcium-induced platelet procoagulant activity shows that calpain is involved in increased phospholipid transbilayer movement

Treatment of platelets with fluoride (10 mM) was found to result in a transient increase in Ca2+-permeability of the platelet plasma membrane. This phenomenon was used to provide supplementary evidence for the suggestions made earlier (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143; Verhallen et al. (1987) Biochim. Biophys. Acta 903, 206), that cytoskeletal disrupture by calpain is involved in the process leading to transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. This was achieved by relating both calpain activity and exposure of phosphatidylserine with platelet procoagulant activity. It was found that only upon addition of extracellular Ca2+ to fluoride-treated platelets, procoagulant activity, expressed as prothrombinase activity, and calpain activity, estimated from protein patterns after gel electrophoresis, were generated. Both Ca2+-inducible prothrombinase activity and calpain activity followed an identical time-course during incubation with fluoride: after a time-lag of about 10 min they sharply increased towards a peak level. Upon further incubation with fluoride, both activities decreased towards a final plateau, still above basal level. The presence of leupeptin during incubation with fluoride was found to inhibit Ca2+-inducible calpain activity and prothrombinase activity in an identical way. Ca2+-inducible exposure of phosphatidylserine, as determined with extracellular phospholipase A2, showed a similar pattern as Ca2+-inducible calpain activity and prothrombinase activity. From the strict parallelism between prothrombinase activity, calpain activity and exposure of phosphatidylserine, it is concluded that calpain plays an important role in the activation-dependent transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. It is suggested that degradation of the platelet membrane-skeleton by calpain disturbs the structural organization of the lipid bilayer of the platelet plasma membrane leading to enhanced transbilayer movement of phospholipids and appearance of phosphatidylserine at the platelet outer surface.     

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca²⁺ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca²⁺/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca²⁺ titration of intact platelets using A23187 and Ca²⁺/HEDTA buffers, revealed half-maximal response at about 15 μM free Ca²⁺ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

The platelet cytoskeleton contains elements of the prothrombinase complex

Triton-insoluble cytoskeletons prepared from thrombin-activated platelets were found to potentiate the activation of prothrombin (prothrombinase activity). Cytoskeletons prepared from red cells or lymphoblasts contained no prothrombinase activity. The platelet prothrombinase activity was dependent on cytoskeletal-associated Factor Va, and exogenously added Factor Xa and prothrombin. Cytoskeletons contained 38% of the total platelet prothrombinase activity. Both platelets and cytoskeletons displayed half-maximal activities at similar prothrombin concentrations. The role of lipids in the cytoskeletal prothrombinase activity was investigated. Cytoskeletons were found to contain 3.8% of the total platelet phospholipids, consisting of the following lipids expressed as percentage of total present in platelets: 6.0% sphingomyelin, 3.8% phosphatidylcholine, 2.9% phosphatidyl-ethanolamine, 4.4% phosphatidylinositol, and 2.2% phosphatidylserine. The cytoskeletal prothrombinase activity and the lipid phosphorus content of cytoskeletons decreased after treatment of cytoskeletons with various doses of phospholipase C. Incubation of cytoskeletons with the highest concentrations tested (10 micrograms/ml) resulted in a 72% loss of phosphatidylserine and 84% loss of cytoskeletal prothrombinase activity. Cytoskeletal prothrombinase activity destroyed by phospholipase C treatment could be restored to control levels by treatment of hydrolyzed cytoskeletons with total cytoskeletal lipid or mixtures of phosphatidylserine/phosphatidylcholine (25:75% by weight). These results suggest that the cytoskeletal prothrombinase complex in addition to containing Factor Va, as has been previously shown (15), contains a lipid cofactor activity consisting in part of phosphatidylserine.     

Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.     

The involvement of cytoskeleton in the regulation of transbilayer movement of phospholipids in human blood platelets

Activation of human platelets by different activators resulted in a different extent of degradation of the cytoskeletal proteins actin-binding protein and myosin, as well as of the non-cytoskeletal protein P235. The highest extent of proteolysis was observed with Ca-ionophore A23187 and decreased on going from A23187 greater than collagen plus thrombin greater than collagen greater than thrombin = ADP. The same order of potency has been found previously ((1983) Biochim. Biophys. Acta 736, 57-66) for the ability of platelet activators to induce exposure of aminophospholipids in the outer leaflet of the platelet plasma membrane, and to stimulate platelets to become procoagulant. Degradation of cytoskeletal proteins as a result of platelet stimulation by collagen plus thrombin was prevented in the presence of dibutyryl cAMP or EDTA but not in the presence of aspirin. This also runs in parallel with platelet procoagulant activity. Moreover, platelets from a patient with a partial deficiency in platelet procoagulant activity revealed a diminished extent of degradation of cytoskeletal proteins upon platelet stimulation with collagen plus thrombin. It is concluded that alterations in cytoskeletal organization upon platelet stimulation may lead to alterations in the orientation of (amino)phospholipids in the plasma membrane, and may therefore play a regulatory role in the expression of platelet procoagulant activity.     

Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A- Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.     

Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity

Activation of human platelets by complement proteins C5b-9 is accompanied by the release of small plasma membrane vesicles (microparticles) that are highly enriched in binding sites for coagulation factor Va and exhibit prothrombinase activity. We have now examined whether assembly of the prothrombinase enzyme complex (factors VaXa) is directly linked to the process of microparticle formation. Gel-filtered platelets were incubated without stirring with various agonists at 37 degrees C, and the functional expression of cell surface receptors on platelets and on shed microparticles was analyzed using specific monoclonal antibodies and fluorescence-gated flow cytometry. In addition to the C5b-9 proteins, thrombin, collagen, and the calcium ionophore A23187 were each found to induce formation of platelet microparticles that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa. These microparticles were enriched in binding sites for factor Va, and their formation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Little or no microparticle release or prothrombinase activity were observed when platelets were stimulated with epinephrine and ADP, despite exposure of platelet fibrinogen receptors by these agonists. When platelets were exposed to thrombin plus collagen, the shed microparticles contained activated GP IIb-IIIa complexes that bound fibrinogen. By contrast, GP IIb-IIIa incorporated into C5b-9 induced microparticles did not express fibrinogen receptor function. Platelets from a patient with an isolated defect in inducible procoagulant activity (Scott syndrome) were found to be markedly impaired in their capacity to generate microparticles in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors. Taken together, these data indicate that the exposure of the platelet factor Va receptor is directly coupled to plasma membrane vesiculation and that this event can be dissociated from other activation-dependent platelet responses. Since a catalytic membrane surface is required for optimal thrombin generation, platelet microparticle formation may play a role in the normal hemostatic response to vascular injury.     

Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin.Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.     

Interaction between phosphatidylserine and the isolated cytoskeleton of human blood platelets

Binding experiments were performed to demonstrate a direct interaction between cytoskeletons from human blood platelets and phosphatidylserine. A centrifugation technique using radiolabeled phosphatidylserine-vesicles and Triton X-100 insoluble residues from unstimulated human platelets was used to assess the binding. Interaction between cytoskeleton and phospholipid is demonstrated to be specific for phosphatidylserine. No binding was observed for phosphatidylcholine. The binding of phosphatidylserine was saturable and dependent on the concentration of cytoskeleton used. The interaction between phosphatidylserine and the cytoskeleton appeared to be completely reversible. The existence of a reversible and specific interaction between phosphatidylserine and the cytoskeleton of unstimulated platelets would suggest a role for the cytoskeleton in the maintenance of the asymmetric distribution of this lipid in the plasma membrane. We have previously shown (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143-148) that in activated platelets a strong correlation exists between degradation of platelet cytoskeletal proteins by the endogenous calcium-dependent proteinase (calpain) and exposure of phosphatidylserine at their outer surface. Nevertheless, hydrolysis of the isolated cytoskeleton by calpain did not result in a change in the parameters of the binding between phosphatidylserine and cytoskeleton. Also, sulfhydryl oxidation of the cytoskeleton by diamide did not affect its binding properties for phosphatidylserine, in spite of the fact that diamide treatment of platelets results in exposure of phosphatidylserine at the outer surface. Exposition of phosphatidylserine upon activation of platelets cannot be directly ascribed to a change in affinity or number of binding sites of the modified cytoskeleton as measured in model systems. However, it cannot be excluded that topological rearrangements of the cytoskeleton as occur within the cell during platelet activation lead to a decreased contact between cytoskeleton and lipid, irrespective of the binding parameters.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase

Influx of calcium in platelets and red cells produces formation of vesicles shed from the plasma membrane. The time course of the shedding process closely correlates with the ability of both cells to stimulate prothrombinase activity when used as a source of phospholipid in the prothrombinase assay. This reflects increased surface exposure of phosphatidylserine, presumably resulting from a loss in membrane asymmetry. Evidence is presented that the shed vesicles have a random phospholipid distribution, while the remnant cells show a progressive loss of membrane phospholipid asymmetry when more shedding occurs. Removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not of that of the shed vesicles. This is consistent with reactivation of aminophospholipid translocase activity, being first inhibited by intracellular calcium and subsequently reactivated upon calcium removal. Involvement of aminophospholipid translocase is further supported by the observation that reversibility of procoagulant activity is also dependent on metabolic ATP and reduced sulfhydryl groups. The finding that this reversibility process is not apparent in shed vesicles may be ascribed to the absence of translocase or to a lack of ATP. These data support and extend the suggestion made by Sims et al. [1989) J. Biol. Chem. 264, 17049-17057) that membrane fusion, which is required for shedding to occur, produces transient flip-flop sites for membrane phospholipids. Furthermore, the present results indicate that scrambling of membrane phospholipids can only occur provided that aminophospholipid translocase is inactive.     

Platelet storage under in vitro condition is associated with calcium-dependent apoptosis-like lesions and novel reorganization in platelet cytoskeleton

Platelets are cleared from circulation after a life span of 8-10 days. The molecular mechanisms underlying platelet senescence remain poorly characterized. Here we report that, progressive functional impairment in the platelets incubated in vitro in a plasma-free isotonic medium for up to 24 h at 37 degrees C is associated with release of cytochrome c from platelet mitochondria and cleavage of procaspase-9, but without evidence of caspase-3 activation. Concomitantly, there was proteolysis of survival proteins like focal adhesion kinase, Src, gelsolin, and specific cytoskeleton-associated peptides, in a manner regulated by extracellular calcium and calpain activity. Cytoskeleton played a critical role as evidenced from the association of these proteins and their degradation products, as well as procaspase-3 and the actin regulatory small GTPase, CDC42Hs, with the cytoskeleton of the stored platelets. The cytoskeletal enrichment with specific proteins was not associated with increase in the content of F-actin and was cytochalasin-resistant, thus signifying a novel mechanism of interaction of the translocating proteins with the pre-existing cytoskeleton. There was progressive exposure of phosphatidylserine on the outer leaflet of platelet membrane and specific electron microscopic changes suggestive of apoptotic lesions. Based on these observations we discuss the caspase-independent but calpain-mediated signaling events in the stored platelets resembling the features of apoptosis in the nucleated cells.     

Rearrangement of aminophospholipids in bilayers from sheep platelet plasma membranes and platelet liposomes by increasing their cholesterol levels

Phospholipid orientation in platelet plasma membranes and other blood cells, such as erythrocytes, appears to be rather similar. The negatively charged phospholipids are almost exclusively located on the inner leaflet of the bilayer. No phosphatidylserine is present on the outer membrane bilayer. The results of the present study, using a specific reagent for amino groups, trinitrobenzenesulfanilic acid, showed that in sheep platelet plasma membranes enriched with free exogenous cholesterol, an alteration in the aminophospholipid topology occurs, with a portion of phosphatidylserine moving from the inner to the outer side. A progressive appearance of aminophospholipids in the outer membrane bilayer was also observed in artificial vesicles prepared with total lipids from sheep platelets supplemented with increased amounts of free cholesterol.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.     

Regulation of platelet factor Va-dependent thrombin generation by activated protein C at the surface of collagen-adherent platelets

Recent studies have indicated that factor Va bound to activated platelets is partially protected from inactivation by activated protein C (APC). To explore whether this sustained factor Va activity could maintain ongoing thrombin generation, the kinetics of platelet factor Va-dependent prothrombinase activity and its inhibition by APC were studied. In an attempt to mimic physiologically relevant conditions, platelets were adhered to collagen type I-coated discs. These discs were then spun in solutions containing prothrombin and factor Xa either in the absence or presence of APC. The experiments were performed in the absence of platelet-derived microparticles, with thrombin generation and inhibition confined to the surface of the adherent platelets. APC completely inactivated platelet-associated prothrombinase activity with an overall second order rate constant of 3.3 x 10(6) m(-)1 s(-)1, which was independent of the prothrombin concentration over a wide range around the apparent K(m) for prothrombin. Kinetic studies on prothrombinase assembled at a planar phospholipid membrane composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine revealed a similar second order rate constant of inhibition (2.5 x 10(6) m(-1) s(-1)). Collectively, these data demonstrate that ongoing platelet factor Va-dependent thrombin generation at the surface of collagen-adherent platelets is effectively inhibited by APC. No differences were observed between the kinetics of APC inactivation of plasma-derived factor Va or platelet factor Va as part of the prothrombinase associated with, respectively, a planar membrane of synthetic phospholipids or collagen-adherent platelets.     

Effects of diamide and iodoacetamide on the expression of the glycoprotein IIb/IIIa complex on blood platelets

We have examined the effects of two agents that alter platelet thiol-disulphide status on platelet aggregation and on the ability of platelets to bind a monoclonal antibody (M148) that is directed toward an epitope on the glycoprotein IIb/IIIa complex. The immediate effect of both diamide and iodoacetamide is to enhance aggregation but after further incubation diamide, but not iodoacetamide, inhibits platelet aggregation. Incubation of platelets with diamide, but not iodoacetamide, is accompanied by a marked increase in the amount of M148 that binds to platelets. This is presumably a reflection of an altered distribution of glycoproteins on the platelet surface. It is known that diamide, but not iodoacetamide, leads to polymerisation of cytoskeletal proteins in platelets. Thus evidence is provided that agents that interact with the cytoskeleton inhibit platelet behaviour via an effect on surface glycoproteins.     

Effect of the thiol group inhibitor monobromobimane and other inhibitors on the composition of the platelet cytoskeletal core and its association with glycoprotein IIIa

SDS-polyacrylamide gel electrophoresis was used to study the effects of the thiol inhibitor monobromobimane (MB), EDTA, and prostaglandin E1 (PGE1) on the formation and composition of the platelet cytoskeletal core (Triton-insoluble residue) and its association with glycoprotein (GP) IIIa. Stimulation or aggregation of platelets in response to ADP or thrombin increased the amount of Triton-insoluble myosin. Aggregation resulted in incorporation of [125I]GP IIIa and a new band at about 210 kDa into the cytoskeletal core. EDTA and PGE1 caused little disaggregation of platelets that were aggregated in PRP with ADP and that had secreted the contents of their granules. In contrast to EDTA, PGE1 decreased the amount of Triton-insoluble residue and its association with GP IIIa. MB added after ADP-induced aggregation caused an increase in the amount of cytoskeletal core despite marked disaggregation and a substantial decrease in core-associated GP IIIa. With aspirin-treated platelets that had not secreted, EDTA, PGE1, and MB all caused disaggregation and loss of cytoskeletal GP IIIa. MB diminished, but did not reverse, thrombin-induced aggregation of washed platelets and arrested GP IIIa incorporation into the cytoskeletal core. Concanavalin A (Con A) cross-links glycoproteins on a single platelet and induces incorporation of GP IIIa into the Triton-insoluble residue in the absence of platelet aggregation. This induction was not inhibited by MB, although this reagent, as well as aspirin, inhibited Con A-induced secretion. Since GP IIIa incorporation caused by ADP-induced aggregation differs from that caused by Con A in its susceptibility to MB, it seems unlikely that thiol groups are directly involved in the association of GP IIIa with the cytoskeletal core.