Platelet Procoagulant Function Research Articles

Procoagulant platelet activation promotes venous thrombosis

AbstractPlatelets are key players in cardiovascular disease, and platelet aggregation represents a central pharmacologic target, particularly in secondary prevention. However, inhibition of adenosine diphosphate and thromboxane signaling has low efficacy in preventing venous thromboembolism, necessitating the inhibition of the plasmatic coagulation cascade in this disease entity. Anticoagulation carries a significantly higher risk of bleeding complications, highlighting the need of alternative therapeutic approaches. We hypothesized that procoagulant activation (PA) of platelets promotes venous thrombus formation and that targeting PA could alleviate venous thrombosis. Here, we found elevated levels of procoagulant platelets in the circulation and in thrombi of patients with deep vein thrombosis (DVT) and pulmonary embolism, and in mice developing DVT following inferior vena cava stenosis. Furthermore, we detected PA of recruited platelets within murine venous thrombi and human pulmonary emboli. Mice with platelet-specific deficiency in central pathways of PA—cyclophilin D and transmembrane protein 16F—were more resistant toward low flow–induced venous thrombosis. Finally, we found that a clinically approved carbonic anhydrase inhibitor, methazolamide, reduced platelet procoagulant activity and alleviated murine thrombus formation without affecting trauma-associated hemostasis. These findings identify an essential role of platelet procoagulant function in venous thrombosis and delineate novel pharmacologic strategies targeting platelets in the prevention of venous thromboembolism.

AGK (Acylglycerol Kinase) Potentiates Arterial Thrombosis by Affecting Talin-1 and αIIbβ3-Mediated Bidirectional Signaling Pathway.

AGK (acylglycerol kinase) was first identified as a mitochondrial transmembrane protein that exhibits a lipid kinase function. Recent studies have established that AGK promotes cancer growth and metastasis, enhances glycolytic metabolism and function fitness of CD8+ T cells, or regulates megakaryocyte differentiation. However, the role of AGK in platelet activation and arterial thrombosis remains to be elaborated. We performed hematologic analysis using automated hematology analyzer and investigated platelets morphology by transmission electron microscope. We explored the role of AGK in platelet activation and arterial thrombosis utilizing transgenic mice, platelet functional experiments in vitro, and thrombosis models in vivo. We revealed the regulation effect of AGK on Talin-1 by coimmunoprecipitation, mass spectrometry, immunofluorescence, and Western blot. We tested the role of AGK on lipid synthesis of phosphatidic acid/lysophosphatidic acid and thrombin generation by specific Elisa kits. In this study, we found that AGK depletion or AGK mutation had no effect on the platelet average volumes, the platelet microstructures, or the expression levels of the major platelet membrane receptors. However, AGK deficiency or AGK mutation conspicuously decreased multiple aspects of platelet activation, including agonists-induced platelet aggregation, granules secretion, JON/A binding, spreading on Fg (fibrinogen), and clot retraction. AGK deficiency or AGK mutation also obviously delayed arterial thrombus formation but had no effect on tail bleeding time and platelet procoagulant function. Mechanistic investigation revealed that AGK may promote Talin-1Ser425 phosphorylation and affect the αIIbβ3-mediated bidirectional signaling pathway. However, AGK does not affect lipid synthesis of lysophosphatidic acid/lysophosphatidic acid in platelets. AGK, through its kinase activity, potentiates platelet activation and arterial thrombosis by promoting Talin-1 Ser425 phosphorylation and affecting the αIIbβ3-mediated bidirectional signaling pathway.

Update on platelet procoagulant mechanisms in health and in bleeding disorders.

Platelet procoagulant mechanisms are emerging to be complex and important to achieving haemostasis. The mechanisms include the release of procoagulant molecules from platelet storage granules, and strong agonist-induced expression of procoagulant phospholipids on the outer platelet membrane for tenase and prothrombinase assembly. The release of dense granule polyphosphate is important to platelet procoagulant function as it promotes the activation of factors XII, XI and V, inhibits tissue factor pathway inhibitor and fibrinolysis, and strengthens fibrin clots. Platelet procoagulant function also involves the release of partially activated factor V from platelets. Scott syndrome has provided important insights on the mechanisms that regulate procoagulant phospholipids expression on the external platelet membrane, which require strong agonist stimulation that increase cystolic calcium levels, mitochondrial calcium uptake, the loss of flippase function and activation of the transmembrane scramblase protein anoctamin 6. There have been advances in the methods used to directly and indirectly assess platelet procoagulant function in health and disease. Assessments of thrombin generation with platelet rich plasma samples has provided new insights on how platelet procoagulant function is altered in inherited platelet disorders, and how platelets influence the bleeding phenotype of a number of severe coagulation factor deficiencies. Several therapies, including desmopressin and recombinant factor VIIa, improve thrombin generation by platelets. There is growing interest in targeting platelet procoagulant function for therapeutic benefit. This review highlights recent advances in our understanding of platelet-dependent procoagulant mechanisms in health and in bleeding disorders.

Determination of fibrin clot growth and spatial thrombin propagation in the presence of different types of phospholipid surfaces

In this work, we present a new method—Thrombodynamics-4D—for the assessment of both plasma and platelet contributions to clotting. Thrombodynamics-4D potentially allows for the determination of plasma or platelet disorders and the effects of various drugs on plasma clotting or on platelet procoagulant function. In this assay, clot formation in platelet-rich plasma or platelet-free plasma supplemented with phospholipids is activated with tissue factor immobilized on a surface. Spatial fibrin clot growth and thrombin concentration dynamics are registered by measuring light scattering of the fibrin clot and fluorescence of the product formed by cleavage of the synthetic fluorogenic substrate by thrombin, respectively. Here, we describe the preanalytical requirements, measurement methodology and calculation principles of assay parameters. Preanalytical and analytical variability and reference ranges of the assay are given. Additionally, we show some clinical examples, which determine the effect of anticoagulants, measure clotting dysfunction in patients with platelet or coagulation disorders and evaluate the effect of surgery.

Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

CD36 and ERK5 link dyslipidemia to apoptotic-like platelet procoagulant function.

Metabolic diseases, including dyslipidemia, diabetes mellitus, and chronic inflammation are risk factors for clinically significant thrombotic events. Thrombosis in these settings is multifaceted with coordinated mechanisms between platelet activation and the hemostatic pathways. This review focuses on recent advances in platelet procoagulant and apoptotic signaling with emphasis on the pathophysiologic mechanisms induced by platelet CD36 in dyslipidemia, and the key unaddressed questions relating to the field. CD36 promotes platelet activation and increases the risk for thrombosis through signaling events. These include generation of reactive oxygen species, activation of redox-sensitive MAP kinase ERK5, and promotion of a pro-thrombotic phenotype. CD36 promotes phosphatidylserine externalization leading to a procoagulant function downstream from MAP kinase ERK5 that is separate from a pro-aggregatory function. Phosphatidylserine externalization requires maladaptive caspase activation, promotes assembly of the factor tenase and prothrombinase complex, and promotes fibrin formation. It is distinct from the canonical pathways mediating platelet procoagulant function by strong physiologic stimuli or by the platelet apoptotic-like Bak/Bax-mediated pathway for cellular clearance. Understanding CD36 signaling in the context of dyslipidemia, or other metabolic diseases will identify important and novel signaling hubs that could be potential therapeutic targets for intervention without impacting hemostasis.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation

AbstractIt is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β3−/− platelets or by integrin antagonists. The impaired MV release and PS exposure in β3−/− platelets were rescued by expression of wild-type β3 but not a Gα13 binding–deficient β3 mutant (E733EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα13-integrin interaction, suggesting that Gα13-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β3 integrins serve as a shear sensor activating the Gα13-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα13-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets.

Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo. Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus.

Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood

Essentials The platelet thrombin receptor, PAR4, is an emerging anti-thrombotic drug target. We examined the anti-platelet & anti-thrombotic effects of PAR4 inhibition in human blood. PAR4 inhibition impaired platelet procoagulant activity in isolated cells and during thrombosis. Our study shows PAR4 is required for platelet procoagulant function & thrombosis in human blood. Background Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via protease-activated receptor (PAR)1 and PAR4. PAR1 has higher affinity for thrombin, and the first PAR1 antagonist, vorapaxar, was recently approved for use as an antiplatelet agent. However, vorapaxar is contraindicated in a significant number of patients, owing to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. Objectives To determine the specific antiplatelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. Methods and Results We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4, and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium level and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin αII b β3 activation, α-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (fluorescence resonance energy transfer-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. Conclusions These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood, and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.

Regulation of Platelet Procoagulant Activity

Platelets play prominent and unique roles in the kinetics of the coagulation process. Modulated by the flow conditions, they control coagulation in various ways: (1) by integrin activation and ensuing clot retraction; (2) via exposure of the procoagulant phospholipid phosphatidylserine (PS), which causes binding of a specific set of coagulation factors at the platelet surface with as a result tenase activation and thrombin generation; (3) by initiating fibrin fiber formation at the platelet surface, a process specifically catalyzed by coated platelets. Recent evidence points to fibrin as an active structural network, which provides binding sides for plasma factors and platelets and, thereby, provides an important feedback mechanism for the stimulation of platelet activation. This overview will focus on novel insights into the signaling and activation processes in platelets that regulate these distinct procoagulant functions. One key mechanism is the activation and secondary closure of integrin alphaIIb-beta3. Another key mechanism concerns the calcium-dependent exposure of PS, which is accompanied by swelling of the platelets. Together, these processes control not only platelet adhesion, but also the accessibility for coagulation factors. Major distortions in these procoagulant functions are seen in platelets from a patient with the Scott syndrome, a rare bleeding disorder associated with mutations in the calcium-dependent ion channel protein anoctamin-6 (gene ANO6, previously TMEM16A). These platelets are deficient in calcium-induced PS exposure, swelling, integrin inactivation and in the formation of thrombin and fibrin at the platelet surface. These dysfunctions are precisely phenocopied in mice lacking anoctamin-6 expression. Advanced platelet proteomics analysis indicated that the deficiency in anoctamin-6 was accompanied by decreased calpain-dependent cleavage of a whole spectrum of intracellular proteins, and an increased phosphorylation state of many signaling and cytoskeletal proteins. However, calcium signaling in the Scott platelets was unchanged. In contrast, reports on patients with a gain in platelet procoagulant function, the Stormorken syndrome, indicate a link with genetic mutation in the calcium signaling protein STIM1. The possible consequences of these novel insights for our understanding of haemorrhagic and thrombotic deseases will be discussed. DisclosuresNo relevant conflicts of interest to declare.

Vessel wall BAMBI contributes to hemostasis and thrombus stability

AbstractBone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the transforming growth factor-β superfamily, and is highly expressed in platelets and endothelial cells. We previously demonstrated its positive role in thrombus formation using a zebrafish thrombosis model. In the present study, we used Bambi-deficient mice and radiation chimeras to evaluate the function of this receptor in the regulation of both hemostasis and thrombosis. We show that Bambi−/− and Bambi+/− mice exhibit mildly prolonged bleeding times compared with Bambi+/+ littermates. In addition, using 2 in vivo thrombosis models in mesenterium or cremaster muscle arterioles, we demonstrate that Bambi-deficient mice form unstable thrombi compared with Bambi+/+ mice. No defects in thrombin generation in Bambi−/− mouse plasma could be detected ex vivo. Moreover, the absence of BAMBI had no effect on platelet counts, platelet activation, aggregation, or platelet procoagulant function. Similar to Bambi−/− mice, Bambi−/− transplanted with Bambi+/+ bone marrow formed unstable thrombi in the laser-induced thrombosis model that receded more rapidly than thrombi that formed in Bambi+/+ mice receiving Bambi−/− bone marrow transplants. Taken together, these results provide strong evidence for an important role of endothelium rather than platelet BAMBI as a positive regulator of both thrombus formation and stability.

Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation

Several studies have implicated the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in inhibition of normal platelet function, suggesting a role for platelets in EPA- and DHA-mediated cardioprotection. However, it is unclear whether the cardioprotective mechanisms arise from alterations to platelet-platelet, platelet-matrix, or platelet-coagulation factor interactions. Our previous results led us to hypothesize that EPA and DHA alter the ability of platelets to catalyze the generation of thrombin. We tested this hypothesis by exogenously modifying platelet membranes with EPA and DHA, which resulted in compositional changes analogous to increased dietary EPA and DHA intake. Platelets treated with EPA and DHA showed reductions in the rate of thrombin generation and exposure of platelet phosphatidylserine. In addition, treatment of platelets with EPA and DHA decreased thrombus formation and altered the processing of thrombin precursor proteins. Furthermore, treatment of whole blood with EPA and DHA resulted in increased occlusion time and a sharply reduced accumulation of fibrin under flow conditions. These results demonstrate that EPA and DHA inhibit, but do not eliminate, the ability of platelets to catalyze thrombin generation in vitro. The ability of EPA and DHA to reduce the procoagulant function of platelets provides a possible mechanism behind the cardioprotective phenotype in individuals consuming high levels of EPA and DHA.

Inherited platelet disorders and oral health

Platelets play a key role in thrombosis and hemostasis. Accumulation of platelets at the site of vascular injury is the first step in the formation of hemostatic plugs, which play a pivotal role in preventing blood loss after injury. Platelet adhesion at sites of injury results in spreading, secretion, recruitment of additional platelets, and formation of platelet aggregates. Inherited platelet disorders are rare causes of bleeding syndromes, ranging from mild bruising to severe hemorrhage. The defects can reflect deficiency or dysfunction of platelet surface glycoproteins, granule contents, cytoskeletal proteins, platelet pro-coagulant function, and signaling pathways. For instance, Bernard-Soulier syndrome and Glanzmann thrombasthenia are attributed to deficiencies of glycoprotein Ib/IX/V and GPIIb/IIIa, respectively, and are rare but severe platelet disorders. Inherited defects that impair platelet secretion and/or signal transduction are among the most common forms of mild platelet disorders and include gray platelet syndrome, Hermansky-Pudlak syndrome, and Chediak-Higashi syndrome. When necessary, desmopressin, antifibrinolytic agents, and transfusion of platelets remain the most common treatment of inherited platelet disorders. Alternative therapies such as recombinant activated factor VII are also available for a limited number of situations. In this review, we will discuss the management of patients with inherited platelet disorders in various clinical situations related to dental cares, including surgical intervention.

Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome.

Like all eukaryotic cells, platelets maintain plasma membrane phospholipid asymmetry in normal blood circulation via lipid transporters, which control transbilayer movement. Upon platelet activation, the asymmetric orientation of membrane phospholipids is rapidly disrupted, resulting in a calcium-dependent exposure of the anionic phospholipid, phosphatidylserine (PS), at the outer platelet surface. This newly-exposed PS surface is a major component of normal hemostasis because it supports platelet procoagulant function. Binding of blood clotting enzyme complexes to this negatively-charged membrane surface allows a dramatic increase in the rate of conversion of zymogens to active serine proteases, which in turn produce a burst of thrombin leading to the formation of a fibrin clot and further platelet activation. Cells have the capacity to catalyze transbilayer phospholipid exchange via ATP-requiring translocase enzymes (flippases and floppases), which control unidirectional phospholipid transport against a concentration gradient. They also use an energy-independent, calcium-dependent scramblase activity to govern the bidirectional exchange of phospholipids between the two leaflets of the bilayer; this activity is essential for PS exposure during platelet activation. Scramblase activity, biochemically characterized in the 1980s, is deficient in patients with Scott syndrome, a rare inherited bleeding disorder with defective platelet procoagulant activity. Despite considerable efforts, the platelet scramblase protein remained elusive for years but a significant advance has recently been made with the identification of TMEM16F, a membrane protein essential for calcium-dependent PS exposure whose loss of function mutations are found in Scott syndrome. This review recalls historical aspects of platelet membrane asymmetry characterization, summarizes the mechanisms and roles of PS exposure following platelet activation and discusses the recent identification of TMEM16F and its significance in the scrambling process.

Procoagulant platelets: are they necrotic?

AbstractApoptosis and necrosis represent distinct cell death processes that regulate mammalian development, physiology and disease. Apoptosis characteristically leads to the silent destruction and removal of cells in the absence of an inflammatory response. In contrast, necrotic cell death can induce physiologic inflammatory responses linked to tissue defense and repair. Although anucleate, platelets undergo programmed cell death, with apoptosis playing an important role in clearing effete platelets from the circulation. While it has long been recognized that procoagulant platelets exhibit characteristic features of dying cells, recent studies have demonstrated that platelet procoagulant function can occur independent of apoptosis. A growing body of evidence suggest that the biochemical, morphologic and functional changes underlying agonist-induced platelet procoagulant function are broadly consistent with cell necrosis, raising the possibility that distinct death pathways regulate platelet function and survival. In this article, we will discuss the mechanisms underlying apoptotic and necrotic cell death pathways and examine the evidence linking these pathways to the platelet procoagulant response. We will also discuss the potential contribution of these pathways to the platelet storage lesion and propose a simplified nomenclature to describe procoagulant platelets.

A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbα N-terminal extracellular domain

AbstractActivated platelets become procoagulant and efficiently promote the conversion of prothrombin to thrombin. A role of the GPIb-V-IX complex has long been postulated in view of the decreased prothrombin consumption in Bernard-Soulier patients. We evaluated the impact of GPIb-V-IX deficiency and the requirement for the GPIbα extracellular domain. In GPIbβ−/− mice, thrombin generation was profoundly decreased in tissue factor– or collagen-related peptide (CRP)–activated platelet-rich plasma and in washed platelets supplemented with normal plasma or with FVa, FXa, and prothrombin. Phosphatidylserine (PS) exposure was similarly decreased in response to thrombin, CRP, or CRP + PAR4 peptide despite a normal platelet phospholipid composition. The hypothesis that these defects originate from lack of the GPIbα N-terminal domain was evaluated after its removal from normal mouse and human platelets with Nk protease or O-sialoglycoprotein endopeptidase. Unexpectedly, the treated platelets exhibited normal thrombin generation and PS exposure, indicating that GPIb-V-IX regulates procoagulant activity independently of its GPIbα-binding region. These results suggested a more general structuring role through intracellular cytoskeleton-anchoring portions regulating responses leading to PS exposure. This hypothesis was supported by the decreased calcium mobilization observed in GPIbβ−/− platelets in response to several agonists, some acting independently of GPIb, in contrast to the normal calcium responses in Nk protease–treated platelets.

Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

AbstractProcoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak−/−Bax−/− or caspase inhibitor–treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.

Novel methodology for assessment of prophylactic platelet transfusion therapy by measuring increased thrombus formation and thrombin generation

Currently, patients developing severe thrombocytopenia during chemotherapy treatment are prophylactically transfused with platelets. We developed two platelet function tests to report the improved haemostasis in the transfused patients, which were capable of detecting aberrant responsiveness of the platelets after transfusion. First, in a whole-blood flow test, platelet adhesion and thrombus formation were determined under high-shear flow conditions. Second, the procoagulant function of platelets was assayed in platelet-rich plasma by measurement of thrombin generation. Experimental conditions were established, where flow-induced adhesion and thrombin generation test parameters increased semi-linearly with the platelet concentration, and informed on the activation properties of platelets. The transfusion effects were evaluated for 38 thrombocytopenic patients, who were transfused with platelets stored in plasma or in synthetic medium (platelet additive solution II). In most but not all patients, transfusion resulted in increased adhesion and thrombus formation, as well as in improved platelet-dependent coagulation. Taken together, the increase in platelet count after transfusion explained 57% of the overall improvement in platelet function. In acute graft-versus-host disease, thrombus formation was normal, while platelet-dependent coagulation was higher than expected. We conclude that assessment of flow-induced adhesion and thrombin generation in acquired thrombocytopenia adequately determines the improved haemostatic activity by transfused platelets.

Serotoninergic Mechanisms Play a Major Role on Platelet-Mediated Thrombogenicity: Studies under Flow Conditions and Effects of Inhibitory Strategies.

Serotoninergic mechanisms are reciprocally implicated in depression and cardiovascular disorders. Serotonin (5-HT) may facilitate the development of a subpopulation of platelets with increased procoagulant activity. We have investigated the involvement of serotoninergic mechanisms in adhesive, cohesive and procoagulant function of platelets. Furthermore, we evaluated the antithrombotic properties of selective serotonin reuptake inhibitors (SSRI). For these purposes we used a series of experimental strategies including standard aggregometry, flow cytometry, perfusion techniques and determination of coagulation parameters. Serotonin concentrations ranging 0.5–5 μM were used in these studies. Citalopram, a selective serotonin reuptake inhibitor (SSRI), was used at concentrations equivalent to those reached in clinical practice.Aggregation studies indicate that 5-HT is a weak agonist for platelets in comparison with standard activating agents. Only the highest concentrations of 5-HT tested (5 μM) caused minimal and reversible platelet aggregation. Despite this modest effect on aggregation, 5-HT potentiated the aggregation induced by low concentrations of ADP (0.5 μM) in PRP samples (16.3±5.9 % vs. 28.1±5.5 %). Potentiation of aggegation was more evident when PRP was obtained from blood anticoagulated with LMWH (p<0.05), where some thrombin could be generated. Citalopram at 0.6 μM caused statistical reductions in % of maximal aggregation induced by ADP (2 or 0.5 μM) and COL (2.5 μg/ml) (p<0.05). Platelet aggregation was maximally inhibited with citalopram at concentrations exceeding clinical doses (6 μM). Flow cytometry studies revealed a mild increase of P-selectin expression in washed platelets in the first minute after activation with 5-HT. In isolated platelets, 5-HT produced irreversible aggregation when studies were performed in the presence of human-TF enriched microvesicles. A significant increase in Annexin V binding and presence of factor Va was detected by flow cytometry in the latter studies. Further studies performed in perfusion models using human blood flowing though chambers exposing a surface rich in collagen and TF demonstrated that presence of 5-HT in the perfusates increased thrombin generation (p<0.05) as demonstrated by elevations of F1+2 levels. Presence of citalopram in the perfusates significantly inhibited the formation of large platelet aggregates on the perfused thrombogenic surfaces (20.2±3.4 % vs. 11.5±1.7 %; p<0.05) and prevented elevations of F1+2 levels observed in previous studies. Our studies confirm that 5-HT is a weak agonist for platelets, though it possesses a marked ability to enhance the activation induced by other agonists. Moreover, 5-HT showed a powerful capacity to potentiate procoagulant responses of platelets under different experimental conditions and to enhance the thrombogenesis on damaged vascular surfaces. The fact that SSRI inhibited the responses elicited by 5-HT in the previous situations further confirm the implications of serotoninergic mechanisms in platelet activation. Our data reinforce the opinion that serotoninergic mechanisms play an important role in platelet-mediated thrombogenicity, suggesting that modulation of 5-HT mediated responses may offer a new potential target for the development of more powerful antithrombotic drugs.

SHIP1 and Lyn Kinase Negatively Regulate Integrin αIIbβ3 Signaling in Platelets

Integrin alpha(IIb)beta(3) plays a critical role in platelet function, promoting a broad range of functional responses including platelet adhesion, spreading, aggregation, clot retraction, and platelet procoagulant function. Signaling events operating downstream of this receptor (outside-in signaling) are important for these responses; however the mechanisms negatively regulating integrin alpha(IIb)beta(3) signaling remain ill-defined. We demonstrate here a major role for the Src homology 2 domain-containing inositol 5-phosphatase (SHIP1) and Src family kinase, Lyn, in this process. Our studies on murine SHIP1 knockout platelets have defined a major role for this enzyme in regulating integrin alpha(IIb)beta(3)-dependent phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) accumulation, necessary for a cytosolic calcium response and platelet spreading. SHIP1 phosphorylation and PtdIns(3,4,5)P(3) metabolism is partially regulated through Lyn kinase, resulting in an enhanced calcium flux and spreading response in Lyn-deficient mouse platelets. Analysis of platelet adhesion dynamics under physiological blood flow conditions revealed an important role for SHIP1 in regulating platelet adhesion on fibrinogen. Specifically, SHIP1-dependent PtdIns(3,4,5)P(3) metabolism down-regulates the stability of integrin alpha(IIb)beta(3)-fibrinogen adhesive bonds, leading to a decrease in the proportion of platelets forming shear-resistant adhesion contacts. These studies define a major role for SHIP1 and Lyn as negative regulators of integrin alpha(IIb)beta(3) adhesive and signaling function.