Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.     

Differential effects of spermine on aggregation, inositol phosphate formation and protein phosphorylation in human platelets in response to thrombin, arachidonic acid and lysophosphatidic acid

Platelet aggregation stimulated by thrombin, arachidonic acid or lysophosphatidic acid is associated with rapid phosphorylation of two platelet proteins, myosin light chain and a 47 kDa protein. The polyamine, spermine, inhibited platelet aggregation stimulated by all three agents. Spermine inhibited thrombin-stimulated phosphorylation of myosin light chain and the 47 kDa proteins as well as thrombin-induced production of the inositol phosphates and phosphatidic acid. In contrast, spermine did not inhibit phosphorylation of either protein or the formation of inositol phosphates and phosphatidic acid in response to arachidonic acid or lysophosphatidic acid. Although spermine has been demonstrated to inhibit both phosphatidylinositol-specific phospholipase C and calcium-dependent protein kinases in cell free systems, these results suggest that, in the intact platelet, spermine does not directly inhibit these enzymes. Inhibition of aggregation stimulated by arachidonic acid and lysophosphatidic acid is secondary to interference with platelet-platelet interaction but not with platelet activation. In contrast, spermine inhibits thrombin-induced platelet activation. This thrombin-specific inhibition may be related to interference with the binding of thrombin to its receptor or to its catalytic substrate on the cell surface.     

The behavior of platelets at foreign surfaces

Many conditions affect the interaction of platelets with foreign surfaces, including the type of surface, modifications of the surface, conditions of blood flow, the adsorbed layer of plasma proteins, changes in this protein layer with time, and the animal species in which experiments are done. Platelets probably never adhere directly to a foreign surface in vivo, because upon exposure of the surface to blood, plasma proteins, principally fibrinogen, are adsorbed almost immediately. When platelets adhere to such a surface and spread on it, they are activated in much the same way as when they are exposed to a strong aggregating and release-inducing agent, but in contrast to aggregation caused by some agonists, adhesion is not dependent on the formation of TXA2 or the release of ADP. It does appear to depend on external Ca2+. Much less is known about the initial adhesion reaction than about platelet aggregation (thrombus formation) on the adherent platelets, although the morphological changes resulting from adhesion have been described. It is surmised that the metabolic and cytoskeletal changes upon adhesion are similar to those that are involved in the response of platelets to other activating agents. The consequences of adhesion include the formation of thrombi and thromboemboli, thrombocytopenia, reduced platelet survival, reduced platelet function in response to hemostatic stimuli, and the appearance in the circulation of products released or formed by activated platelets. Many efforts are being made to develop surfaces and to set up conditions that will minimize platelet adhesion, but it has not yet been possible to find a foreign surface that has and can maintain the nonthrombogenic characteristics of the normal endothelium.     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Proteomic identification of pleckstrin‐associated proteins in platelets: Possible interactions with actin

Pleckstrin (plek)‐null platelets from a knockout mouse have been shown to be defective in granule secretion, aggregation and actin polymerization. However, the mechanism of plek signaling is currently unknown. Therefore, we sought to identify plek‐binding proteins in platelets by using GST pulldown assays and immunoprecipitation to isolate proteins from extracts of protein kinase C‐activated or inhibited human platelets. Co‐purified plek‐binding proteins were resolved by SDS‐PAGE and identified via nanospray quadruple TOF MS. Identified proteins may be involved in various cellular processes including cytoskeletal reorganization (moesin, radixin and α‐actinin) and signal transduction (serum deprivation response protein, 17 β‐hydroxysteroid dehydrogenase 4 and factor XIIIA). Both platelet aggregation and/or secretion require actin polymerization. However, studies have shown no direct association between plek and actin. Based on our findings we propose indirect associations between plek and actin through 17 β‐hydroxysteroid dehydrogenase 4, α‐actinin, moesin, radixin and factor XIIIA, which in turn suggest new roles for plek in platelet biology.     

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.     

Thrombin-reactive polypeptides of platelets may regulate inhibition of thrombin by antithrombin

The central enzyme involved in blood coagulation and activation of platelets is the serine proteinase thrombin. The principal inhibitor of this proteinase in plasma is antithrombin. The mechanism of regulation of the thrombin-antithrombin reaction remains unknown. Two polypeptides of 74 and 55 kDa present on the platelet surface and in plasma are known to specifically enhance the activity of thrombin on different substrates. This study was undertaken to assess the effects of these platelet proteins on thrombin-antithrombin interaction. Direct measurements of residual thrombin activity in mixtures of thrombin and antithrombin, in the presence or absence of the platelet proteins, were made utilizing a specific chromogenic substrate. Under these conditions, when 60% of thrombin activity was inhibited by antithrombin in controls, 100% of enzyme activity was retained in the presence of the platelet proteins. When heparin was used in these assays, the rate of inhibition of thrombin by antithrombin was much more rapid and 62% of thrombin activity remained after 1 min. Under these conditions, the platelet proteins continued to protect thrombin from inactivation with 98% activity remaining at 1 min and 85% activity at 5 min. In contrast, the inhibition of trypsin by antithrombin was not affected by the platelet proteins. Additional studies in platelet aggregation showed that the platelet polypeptides have two effects on thrombin: (i) protection of the enzyme inhibition by antithrombin and (ii) stabilization of thrombin from loss of activity due to aging. The results suggest a novel role for the platelet proteins in hemostasis - regulation of the inhibition of thrombin by antithrombin.     

The perturbation of thrombin binding to human platelets by trypsin and neuraminidase

The initial step in the interaction of thrombin with human platelets in binding of the enzyme to the platelet surface. The effects of digestion of isolated platelets with trypsin and neuraminidase on aggregation, release of serotonin and binding of thrombin have been examined.Trypsin is a powerful inducer of platelet aggregation as well as the release reaction. The aggregation effect of trypsin may be blocked with disodium ehtylenediaminetatraacetate (EDTA). Further, in the presence of EDTA, trypsin-induced release of [¹⁴C]serotonin is 15–20% lower compared to controls and the initial lag period is prolonged. Conditions were developed under which trypsin did neither aggregate nor release serotonin from platelets. Even under these conditions, trypsin caused a profound loss in the thrombin binding capacity of platelets. Thus, the trypsin-induced fall in the thrombin binding capacity and the platelet response are dissociated. This loss in the thrombin binding by trypsin is due to a lower number of binding sites available on the platelet surface and is not due to an altered affinity.Neuraminidase did not induce platelet aggregation or the release reaction. The ability of platelets to bind thrombin was also unimpaired by prior digestion with neuraminidase. Thus, the sialic acid at the platelet surface is not essential in the function of thrombin recognition by the receptor. This moiety may nontheless be a constituent of a glycoprotein which might act as the thrombin receptor.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

Proteolytic alterations of factor Va bound to platelets

The coagulation protein Factor Va forms the receptor for the serine protease Factor Xa at the platelet surface. This membrane-bound complex of Factor Va and Factor Xa plus calcium constitutes the enzymatic complex prothrombinase, which effects the conversion of prothrombin to the clotting enzyme, thrombin. Studies were undertaken to investigate the proteolytic events accompanying the inactivation of platelet-bound Factor Va by activated protein C as well as the ability of Factor Xa to protect Factor Va from activated protein C inactivation. During the course of these studies, observations were made which indicated that Factor Va was also cleaved by both a platelet-associated protease, as well as Factor Xa. When Factor Va was incubated with washed platelets, electrophoresis and autoradiography of solubilized platelet pellets indicated that three Factor Va peptides were associated with the platelet: component D (Mr = 94,000), component E (Mr = 74,000), and a 90,000-dalton peptide (component D') which appeared with time as the result of a platelet-associated protease cleavage of component D. The Factor Va peptides bound to platelets were proteolytically inactivated by activated protein C, resulting in five peptide products, all of which remained associated with the platelet-membrane surface. Factor Va was protected from activated protein C proteolysis by complex formation with Factor Xa or active site-blocked Factor Xa. However, active Factor Xa cleaved platelet-bound Factor Va to peptide products which also remained associated with the platelet. Whereas activated protein C rapidly cleaved components D and D' with secondary cleavages occurring in component E, Factor Xa rapidly cleaved component E with secondary cleavages occurring in components D and D'. The Factor Xa-cleaved Factor Va is catalytically functional. To determine whether cleavage was necessary for function, prothrombin conversion reaction mixtures were monitored for thrombin formation and Factor Va cleavage with time in a defined phospholipid vesicle model system. The results indicated that Factor Xa cleavage of Factor Va is not essential for Factor Va activity but may promote its ability to function in the prothrombinase complex.     

Specific binding of blood coagulation factor XIIIa to thrombin-stimulated platelets

We studied the binding of 125I-platelet and plasma Factor XIII (125I-Factor XIII) to human platelets. When 125I-Factor XIII was incubated with gel-filtered platelets, calcium chloride (5 mM) and thrombin (1 unit/ml) at 37 degrees C, saturable binding was observed. Half-maximal binding occurred at 1 min. Binding was inhibited 93% by a 100-fold molar excess of unlabeled ligand but not by other purified proteins. Greater than 87% of platelet-bound radioactivity migrated as thrombin-cleaved a-chains (a'-chains) in sodium dodecyl sulfate-polyacrylamide gels indicating that Factor XIIIa but not Factor XIII binds to platelets. 125I-Factor XIIIa does not bind to unstimulated platelets. When platelet secretion was blocked, binding was markedly inhibited. 125I-Factor XIIIa bound minimally to platelets stimulated with agonists other than thrombin. Thus, binding is dependent on platelet activation, as well as modification of platelets by thrombin. 125I-Factor XIIIa bound to gamma-thrombin-stimulated platelets, at concentrations which did not clot fibrinogen. Therefore, Factor XIIIa is not bound to fibrin associated with platelets. Binding was only partially reversible. Approximately 12,000 molecules of Factor XIIIa were bound per platelet. 125I-Factor XIIIa bound normally to platelets from patients with severe Glanzmann's thrombasthenia indicating that 125I-Factor XIIIa does not bind to platelet glycoproteins IIb or IIIa, or platelet-bound fibrinogen. Chymotrypsin treatment of platelets inhibited 125I-Factor XIIIa binding by 78% without inhibiting secretion. Methylamine and putrescine, Factor XIIIa substrates, and N-ethylmaleimide, an active site inhibitor, did not inhibit binding. Factor XIIIa bound to platelets was enzymatically active and catalyzed [3H]putrescine incorporation into platelet proteins. The specific binding of Factor XIIIa to platelets suggests it may play a role in physiologic reactions involving platelets.     

Effect of cerebrocrast on the function of human platelets and release of the arachidonic acid from plasma membrane

Diabetes mellitus (DM) is accompanied by several cardiovascular complications such as coronary artery disease, atherosclerosis, hypertension, cerebral and myocardial infarction, etc. DM induces the alteration of platelet functions including activation, hyperaggregation, adhesiveness, and formation of thrombi. Release of AA from phospholipids of the PM, synthesis of TxA(2),PGE(2), activity of PLA(2), and PLC are increased in the platelets of the DM patients. Stimulation of PLA(2) activity and accumulation of bioactive metabolites such as AA, its oxygenated derivatives, prostaglandins and PAF can evoke glucose production, also. In this study we explored the effect of the 1,4-dihydropyridine compound cerebrocrast at a low concentration (10(-6)-10(-8)M) on the level of intracellular calcium in unstimulated human platelets and those stimulated with thrombin as well as release of [(3)H] AA from phospholipids of platelet PM. Cerebrocrast at a concentration of 10(-6) M decreased the basal level of intracellular calcium concentration (platelets were loaded with Fura-2) in unstimulated as well as in thrombin stimulated platelets. Cerebrocrast at concentrations of 10(-6), 10(-7), 10(-8) M inhibited release of [(3)H] AA from phospholipids of platelet PM. We conclude that blockade of human platelet activation with cerebrocrast can prevent aggregation, adhesion and formation of thrombi. The inhibition of [(3)H] AA release from phospholipids of platelet PM can prevent formation of eicosanoids such as TxA(2), PGG(2), and PGH(2) plus AA oxygenated derivatives. These effects of cerebrocrast are very significant in the treatment of DM-evoked cardiovascular complications.     

Biophysical insight into the heparin‐peptide interaction and its modulation by a small molecule

The heparin‐protein interaction plays a vital role in numerous physiological and pathological processes. Not only is the binding mechanism of these interactions poorly understood, studies concerning their therapeutic targeting are also limited. Here, we have studied the interaction of the heparin interacting peptide (HIP) from Tat (which plays important role in HIV infections) with heparin. Isothermal titration calorimetry binding exhibits distinct biphasic isotherm with two different affinities in the HIP‐heparin complex formation. Overall, the binding was mainly driven by the nonionic interactions with a small contribution from ionic interactions. The stoichiometric analysis suggested that the minimal site for a single HIP molecule is a chain of 4 to 5 saccharide molecules, also supported by docking studies. The investigation was also focused on exploiting the possibility of using a small molecule as an inhibitor of the HIP‐heparin complex. Quinacrine, because of its ability to mimic the HIP interactions with heparin, was shown to successfully modulate the HIP‐heparin interactions. This result demonstrates the feasibility of inhibiting the disease relevant heparin‐protein interactions by a small molecule, which could be an effective strategy for the development of future therapeutic agents.     

Adaptor proteins of blood platelets

Adaptor proteins play a pivotal role in the regulation of signal transduction events elicited after the engagement of cell surface receptors. Platelets exhibit a number of integral membrane receptors capable of initiating a cellular response. These include collagen receptors, von Willebrand factor receptors, the fibrinogen receptor, and a number of G-protein coupled receptors, such as those for thrombin and ADP. The primary function of platelet receptors is the translation of externally applied signals into appropriate responses leading to platelet activation being a prerequisite for normal hemostasis. Multitude of signalling pathways described in platelets is based on the interaction of compounds of many different categories, such as transmembrane receptors, protein kinases, protein phoshatases, G-proteins, transmembrane and cytosolic adaptor proteins, phosphoinositides, cyclic AMP or GMP. Adaptor proteins lack intrinsic effector function, but contain distinct molecular domains, which mediate protein-protein and protein-lipid interactions. These molecules thus serve as a scaffolding, around which effectors and their substrates are assembled into three-dimensional signaling complexes. Adaptor proteins integrate receptor-mediated signals at intracellular levels and couple signaling receptors to cytosolic signaling pathways. While the function of adaptor proteins is well established in immune cells, the knowledge about their role in platelet activation is still at the onset Over the last decade numerous adaptor proteins have been identified in platelets and shown to be involved in accurate assembly of intracellular signaling complexes. Collagen-induced platelet intracellular signaling through GPVI resembles the functional response of B- and T-cell antigen receptors and is the best described in the literature. This review focuses on the structure and functional role of the most extensively studied adaptor proteins during platelet activation induced by physiological agonists.     

Unequal distribution of membrane components between pseudopodia and cell bodies of platelets

Platelet pseudopodia were compared to platelet cell bodies with respect to their lipid composition, fatty acid distribution and protein composition. The methodology for producing pseudopodial preparations of platelets stimulated with thrombin, ADP or calcium ionophore was established. The separation of pseudopodia and cell bodies was verified by electron microscopic examination of the respective platelet components. Lipid analyses demonstrated a preponderance of lysophospholipids and sphingomyelin in pseudopodial preparations and a large increase in mono-, di- and tri-ene fatty acids as compared to cell bodies. Changes were also evident in the protein composition evaluated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis and by [32P]ATP labeling of exofacial membrane proteins. A protein of approximately 68 kDa which reacted strongly with antibody to PlA1, was prominantly displayed in platelet pseudopodia. Thus, our studies demonstrate a heterogeneous distribution of lipids and proteins in a mammalian membrane system which may have important implications for the functional behavior of the cell.     

The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.     

Protein S is required for bovine platelets to support activated protein C binding and activity

Gel-filtered platelets accelerate activated protein C inactivation of factor Va in a reaction that requires the presence of protein S. With protein S present, specific activated protein C binding to the platelet surface is observed (Kd = 11 +/- 3 nM, 203 +/- 20 sites/platelet). The concentration dependence of the activated protein C-mediated factor Va inactivation is in close agreement with the binding. The observed binding is specific since protein C does not compete with activated protein C. Platelet-bound activated protein C is approximately 8000 times more active than the solution-phase enzyme. Platelet activation with thrombin results in formation of a site capable of accelerating factor Va inactivation by activated protein C in the absence of added protein S. This cell surface site is blocked by the addition of affinity purified antibodies to protein S. We conclude that protein S is required for activated protein C binding to the platelet surface and subsequent rapid factor Va inactivation. Platelet activation leads to the expression of either protein S or an antigenically related protein which can substitute for exogenously added protein S.     

Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of SNAP-23 in activated human platelets

Phosphorylation of SNARE proteins may provide a critical link between cell activation and secretory processes. Platelets contain all three members of the SNAP-23/25/29 gene family, but by comparison to brain tissue, SNAP-23 is the most highly enriched of these proteins in platelets. SNAP-23 function is required for exocytosis from platelet alpha, dense, and lysosomal granules. SNAP-23 was phosphorylated largely on serine residues in platelets activated with thrombin. Phosphorylation kinetics paralleled or preceded granule secretion. Inhibition studies suggested that SNAP-23 phosphorylation proceeds largely through a protein kinase C (PKC) mechanism and purified PKC directly phosphorylated recombinant (r-) SNAP-23 (up to 0.3 mol of phosphate/mol of protein). Five major tryptic phosphopeptides were identified in cellular SNAP-23 isolated from activated platelets; three phosphopeptides co-migrated with those identified in PKC-phosphorylated r-SNAP-23. In contrast, only one major phosphopeptide was identified when SNAP-23, engaged in a ternary SNARE complex, was phosphorylated by PKC. Ion trap mass spectrometry revealed that platelet SNAP-23 was phosphorylated at Ser23/Thr24 and Ser161, after cell activation by thrombin; these sites were also identified in PKC-phosphorylated r-SNAP-23. SNAP-23 mutants that mimic phosphorylation at Ser23/Thr24 inhibited syntaxin 4 interactions, whereas a phosphorylation mutant of Ser161 had only minor effects. Taken together these studies show that SNAP-23 is phosphorylated in platelets during cell activation through a PKC-related mechanism at two or more sites with kinetics that parallel or precede granule secretion. Because mutants that mimic SNAP-23 phosphorylation affect syntaxin 4 interactions, we hypothesize that SNAP-23 phosphorylation may be important for modulating SNARE-complex interactions during membrane trafficking and fusion.