Platelet Phosphatidylserine Exposure Research Articles

MARCKS ED Inhibits Fibrin Formation By Blocking Coagulation Protein Complex Assembly on Phosphatidylserine

Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va. The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine. The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme's turnover number. Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex. Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine.Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces. Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay. The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays.Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes. Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05). Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles. In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05). The MARCKS ED probes did not alter platelet surface area coverage. Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment.Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood. The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure. DisclosuresKastelowitz:University of Colorado Boulder: Patents & Royalties. Yin:University of Colorado Boulder: Patents & Royalties.

Desmopressin treatment improves platelet function under flow in patients with postoperative bleeding

Patients undergoing major cardiothoracic surgery are subjected to dilution, owing to massive fluid infusion and blood component transfusion. These patients may experience bleeding perioperatively, and are frequently treated with the endothelium-activating agent desmopressin. To investigate the effect of desmopressin administration on von Willebrand factor (VWF)-dependent coagulant and platelet functions under flow conditions. Blood from 16 patients with postoperative bleeding was obtained before and after desmopressin treatment (0.3μgkg(-1) body weight), and assessed for coagulant properties and platelet function. Furthermore, VWF antigen levels and multimer composition were determined in both samples. Desmopressin treatment did not change thrombin generation in plasma or whole blood thromboelasticity. Also coagulation factor levels (other than factorVIII) and coagulation times were unchanged, suggesting that desmopressin treatment did not have a major effect on the coagulant activity. On the other hand, desmopressin treatment raised the already high plasma levels of VWF from a median of 116IUmL(-1) (interquartile range [IQR]102-154IUmL(-1) ) to a median of 160IUmL(-1) (IQR126-187IUmL(-1) ) (P=0.007), owing to accumulation of the high molecular weight VWF multimers. Furthermore, desmopressin treatment caused an increase in collagen-dependent thrombus formation and platelet phosphatidylserine exposure. Markers of thrombus formation correlated with the plasma levels of VWF. Invitro control experiments confirmed a major contribution of VWF to thrombus formation and procoagulant activity under conditions of blood dilution. Desmopressin treatment of patients with bleeding complications after cardiothoracic surgery induces the release of high molecular weight VWF multimers, which enhance platelet activation and thrombus formation under flow conditions.

Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow.

The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.

Platelets interact with Coxsackieviruses B and have a critical role in the pathogenesis of virus‐induced myocarditis

To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackieviruses B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well-characterized CVB3-induced myocarditis murine model. Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times post-infection. CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals. Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice. Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection.

Staurosporine Induces Platelet Apoptosis Through p38 Mitogen-Activated Protein Kinase Signaling Pathway.

Staurosporine (STS), a microbial alkaloid and potent PKC inhibitor, has become one of the most promising anti-cancer drugs. STS effectively induces apoptosis in many nucleated cells; however, it is still unclear whether STS induces apoptosis in enucleated platelets. Apoptotic events in platelets treated with STS were assessed by flow cytometry or western blotting. STS induced depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and Bak, phosphatidylserine (PS) exposure, release of mitochondrial cytochrome c, and activation of caspase-8 and caspase-9 in human platelets. Furthermore, STS stimulation induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activation significantly reduced ΔΨm depolarization and PS exposure in platelets stimulated with STS. These data indicate that STS induces platelet apoptosis via the p38 MAPK signaling pathway. These findings suggest that platelet apoptosis-related hemorrhage should be noticed in STS and its derivatives in clinical tests.

X-Ray Structure of a Calcium Activated TMEM16 Lipid Scramblase

The TMEM16s or anoctamins constitute a class of eukaryotic membrane proteins that in mammals contain ten members with high sequence conservation. Despite their close relationship these proteins are characterized by a remarkable functional diversity. The family includes the long sought-after Ca2+-activated chloride channels (TMEM16A and B) but also cation channels and lipid scramblases that support the exchange of lipids between the inner and outer leaflets of the bilayer in an ATP-independent manner. As part of the blood coagulation process, TMEM16F triggers the exposure of phosphatidylserine in blood platelets upon activation by Ca2+. TMEM16C, D, G and J were suggested to work as scramblases as well, but with variable characteristics. Although we have by now gained considerable insight into the functional properties of certain family members, their architecture and the relation to mechanisms of action were so far unknown. Here we present the crystal structure of nhTMEM16, a fungal family member that operates as a Ca2+-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that resembles a ‘spiral staircase’ and is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbors a conserved Ca2+-binding site located within the hydrophobic core of the membrane. Ca2+ binding by six residues, five of which carry a negative charge, controls the activation of scrambling in nhTMEM16. A triple mutant of residues in this site shows only weak scrambling activity that is no longer enhanced by Ca2+.The structure thus reveals the general architecture of the family and its mode of Ca2+-activation. It provides insights into potential scrambling mechanisms and will furthermore serve as a framework to unravel the conduction of ions in certain TMEM16 proteins.

Platelet Mechanosensing of Collagen Matrices

Background: Vascular injury causes platelets to initiate hemostasis by first adhering to exposed subendothelial matrix proteins such as collagen. While the biochemical and biological aspects of platelet adhesion via collagen and von willebrand factor are well characterized, if and whether the mechanical properties of the subendothelial matrix affect platelet function is relatively unknown. As purely mechanical cues, such as substrate stiffness, from collagen matrices are sensed and transduced by endothelial cells to alter their physiological processes, platelets may also exhibit similar behavior (Chein S., AM J Physiol Heart Circ Physiol, 2007). In addition, recent reports demonstrate that the stiffness of the subendothelium varies in different disease states, including atherosclerosis and changes associated with aging (Stroka KM & Aranda-Espinoza H, Blood, 2011). Therefore, understanding the effect of subendothelium stiffness on platelet adhesion, spreading, and activation will provide insight into how biomechanical and biochemical factors interact during clot formation in health and disease. To that end, we fabricated polyacrylamide (PA) gels of varying stiffnesses covalently conjugated with collagen to model the biomechanical properties of the subendothelium. Via differential crosslinking, the stiffness of these PA gels can be varied while maintaining constant collagen concentrations on the gel surface, enabling decoupling of mechanical and biochemical cues (Lam, et al, Mol Cancer, 2010; Pathak A & Kumar S, P Natl Acad Sci, 2012). With these systems, we investigated how the stiffness of the underlying collagen matrix affects platelet adhesion, spreading and activation under static and flow conditions and used pharmacological cytoskeletal inhibitors to investigate the underlying mechanotransductive mechanisms.Results and Discussions: Type I collagen was covalently conjugated to the surface of PA gels with varying substrate stiffnesses (0.25 to 100 kilopascals (kPa)); the range of vessel wall stiffness in vivo. The PA gels were then incubated with 5.5 x 106 platelets/ml for 1 hour and stained with a fluorescent membrane dye. While collagen substrate stiffness did not affect platelet adhesion, determined by the number of platelets on each gel, significant differences were observed in platelet spreading area on collagen-conjugated PA gels with stiffnesses >2.5 kPa compared to PA gels of <2.5 kPa (Fig. 1A and 1B). Using PAC-1-FITC and Annexin-V-AF488 to measure platelet integrin αIIbβ3 activation and platelet phosphatidylserine (PS) exposure, respectively, we found that varying the substrate stiffness of collagen matrices did not affect αIIbβ3 activation on adherent platelets but did result in differential levels of PS exposure on adherent platelets, with increased PS exposure on stiffer PA gels (Fig. 1C). In addition, using the myosin light chain kinase (MLCK) inhibitor ML-7 and the Rho kinase inhibitor Y-27632, we observed that platelet exposure to ML-7 eliminated the substrate stiffness-mediated effect on platelet spreading as platelet spreading on PA gels stiffer than 5 kPa was decreased to the levels of that on soft PA gels of 0.5 kPa. Y-27632 exposure did not cause a similar effect, as platelet spreading was increased for all stiffness conditions (Fig. 1D). Finally, under flow conditions using a shear rate of 150 s-1, platelet adhesion in addition to platelet spreading was mediated by substrate stiffness (Fig. 1E); possibly due to weaker collagen attachment on softer substrates that cannot sufficiently resist drag forces.Conclusion: Our results indicate that platelets adhered on collagen mechanosense the stiffness of the underlying subendothelial substrate and transduce these cues into differential levels of adhesion, spreading and activation. While MLCK mediates aspects of this process, further mechanistic studies are currently being conducted. In addition, as shear stress might interact with the observed substrate stiffness-mediated phenomenon, additional experiments under flow at different shear rates are also ongoing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TMEM16F Deficiency Causes a Strain-Dependent Lethality in Mice and Reduced Phosphatidylserine Exposure on Heterozygous As Well As Homozygous Deficient Mouse Platelets

Scott Syndrome is a rare bleeding disorder characterized by a defect in platelet phosphatidylserine (PS) exposure. The syndrome has recently been linked to mutations in TMEM16F, a Ca++-activated ion channel. Tmem16f-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding (Yang et al, Cell 151: 111-122; 2012). We now report analysis of an additional gene targeted Tmem16f allele generated in C57BL/6 ES cells.JM8 ES cell were obtained from EUCOMM, and successful Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f+/gt (+/gt) intercross progeny identified no surviving Tmem16fgt/gt (gt/gt) mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryo’s exhibiting no morphological abnormalities on gross or routine histologic examination. Though complete deficiency of TMEM16F is lethal in the C57BL/6J genetic background between E17.5 and birth, an F2 intercross of +/gt mice outcrossed one generation to 129x1SvJ resulted in gt/gt mice surviving to weaning, though at reduced numbers (6/75 total progeny compared to ~19 expected, p <0.002). Progeny testing of surviving gt/gt mice suggest a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3.Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time.These data suggest the existence of a viability-determining genetic modifier of TMEM16F in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover novel functions for TMEM16F and the regulation of hemostatic function. The observation of a PS exposure and hemostatic phenotype in +/gt mice also suggests the possibility that heterozygous TMEM16F mutations may subtly influence hemostasis or thrombosis in humans. DisclosuresNo relevant conflicts of interest to declare.

Cofilin-1 – Induced Actin Reorganization and Phosphatidylserine Exposure in Platelets

Background: Platelet activation leads to the transbilayer movement of phosphatidylserine (PS) from inner to the outer leaflet of membrane bilayer. Exteriorization of PS promotes platelet procoagulant activity by promoting the assembly of tenase and the prothrombinase complex on platelet membrane. In a previous study, we observed that Rho-associated coiled-coil kinase-1 (ROCK1) deficiency in murine platelets or ROCK inhibition by Y-27632 in human platelets resulted in increase in PS exposure and platelet procoagulant activity. ROCK1 deficient platelets had a marked decrease in phosphorylation of cofilin-1. Cofilin-1 decreases actin filament length by increasing the rate of dissociation of actin monomers and its activity is abolished by phosphorylation. These studies suggested a role for cofilin-1-induced actin reorganization in collagen-induced PS exposure. Cofilin-1 activity is also modulated by its interactions with phosphatidylinositol 4, 5-bisphosphate (PIP2) and a cofilin-1 binding protein, Wdr1, which enhances capacity of cofilin-1 to accelerate depolymerization by capping their barbed ends. Here, we studied the role of cofilin-1 phosphorylation and its interaction with PIP2 and Wdr1 in activation induced PS exposure in platelets.Materials and Methods: We isolated platelet membrane, cytosol and cytoskeleton through differential centrifugation from resting and collagen-stimulated platelets and assessed the relative abundance of cofilin-1 and phosphocofilin-1 by mobility shift in phosphate-affinity polyacrylamide gel electrophoresis. This method allows simultaneous detection of relative proportions of phosphoproteins and its nonphospho counterparts. For cofilin-1 and Wdr-1 distribution, platelets were immobilized on a polylysine-coated cover slip or on a collagen-coated cover slip, fixed, permeabilized and immunostained with appropriate antibody. In addition, PS exposure (FITC lactadherin binding), F-actin (Alexa Fluor 488-phalloidin) and calcium concentration (Fura-2AM fluorescence) were also measured.Results: Immunofluorescence images show that in resting platelets, cofilin-1 is present in distinct patches in the plasma membrane and following activation with collagen, cofilin-1 is redistributed in a discrete granular pattern throughout the cytoplasm. In parallel, we also studied the relative distribution and the phosphorylation status of cofilin-1 in various subcellular fractions of platelets. In resting platelets, cofilin-1 is present in the plasma membrane and in the cytosol but absent in the cytoskeleton. Following activation with collagen, cofilin-1 moves to the cytoskeleton with a concomitant decrease in the plasma membrane fraction. These results suggest that active cofilin-1 is incorporated into reorganizing actin cytoskeleton during platelet activation. In ROCK1-deficient mice, there is increased cofilin-1 in cytoskeletal fraction, which also correlates with increase in PS exposure. Blocking PIP2 hydrolysis by U73122 prevents cofilin-1 translocation and PS exposure. A cell permeable cofilin-1 peptide containing the actin binding site of cofilin-1 (amino acids Trp104 to Met115) at the carboxy terminus (that competitively inhibits cofilin-1 binding to F-actin), blocks cofilin-1 translocation and decreases PS exposure. Platelets from a Wdr1-deficient mice which express less than 2% of wild-type protein in platelets, showed impaired collagen-induced PS exposure despite equivalent increase in intra platelet calcium compared to wild-type platelets.Conclusion: Our results show cofilin-1 trafficking from membranre to cytoskeleton and subsequent actin reorganization precedes PS exposure during platelet activation. In ROCK1-deficient platelets, there is increased cofilin-1 activity due to decreased serine-3 phosphorylation and it is associated with increased cofilin-1 translocation to actin filaments and enhanced PS exposure. In contrast, inhibiting cofilin-1 translocation, either by inhibiting PIP2 hydrolysis or by competitive inhibition by a cell permeable peptide, prevents cofilin-1 trafficking and decreases PS exposure. Furthermore, in Wdr1-deficient mouse platelets, which have diminished cofilin-1 activity, have impaired PS exposure during platelet activation. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Protein Kinase a Induces Platelet Apoptosis

Background:Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelet, or in the development of some platelet-related diseases. However, up to now, little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase A (PKA) is highly expressed and plays central roles in signal transduction in human platelets. The certain level of PKA activity is indispensability for maintaining circulating platelets in resting state. In nucleated cells, PKA stimulated proliferation or promoted cell death via apoptosis in different cell types. However, it is still unclear whether PKA plays a role in platelets apoptosis. The aim of the current study is to investigate the role of PKA in platelets apoptosis.Methods and Results:Apoptotic events were assessed in platelets by flow cytometry, western blotting. PKA inhibitor H89 dose-dependently elucidated depolarization of mitochondrial inner membrane potential (ΔΨm), activation of caspase-3, -9, cleavage of gelsolin, phosphatidylserine (PS) exposure and membrane shrinkage in platelets. Incubation of platelets with PKA catalytic subunit-α (PKACα)-siRNA specific reduced the production PKACα and obviously elucidated platelet apoptosis. Platelet aggregation induced by ADP, botrocetin, or α-thrombin was significantly reduced in platelets treated with H89. However, H89 does not incur P-selectin surface expression and PAC-1 binding in platelets. After intraperitoneal injection of PKA inhibitor (Rp-cAMPS), the circulating platelets were reduced at 8 hour time point and recovered within 24 hours. H89 down-regulated the expression of Bad in platelets, and phosphorylation of Bad at Ser 112 was reduced in platelets. Cyclosporin A and ROS inhibitor (NAC) significantly reduced H89-induced platelet ΔΨm depolarization. However, z-DEVE-fmk, GM6001, MDL28170, forskolin, SB203580, BAPTA had no obvious effect on depolarization of ΔΨm.Conclusions:These data indicate that inhibition of PKA results in ROS-mediated platelet apoptosis. The findings suggest a novel mechanism for PKA in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis. DisclosuresNo relevant conflicts of interest to declare.

Agonist-induced platelet procoagulant activity requires shear and a Rac1-dependent signaling mechanism

AbstractActivated platelets facilitate blood coagulation by exposing phosphatidylserine (PS) and releasing microvesicles (MVs). However, the potent physiological agonists thrombin and collagen poorly induce PS exposure when a single agonist is used. To obtain a greater procoagulant response, thrombin is commonly used in combination with glycoprotein VI agonists. However, even under these conditions, only a percentage of platelets express procoagulant activity. To date, it remains unclear why platelets poorly expose PS even when stimulated with multiple agonists and what the signaling pathways are of soluble agonist-induced platelet procoagulant activity. Here we show that physiological levels of shear present in blood significantly enhance agonist-induced platelet PS exposure and MV release, enabling low doses of a single agonist to induce full-scale platelet procoagulant activity. PS exposed on the platelet surface was immediately released as MVs, revealing a tight coupling between the 2 processes under shear. Using platelet-specific Rac1−/− mice, we discovered that Rac1 plays a common role in mediating the low-dose agonist-induced procoagulant response independent of platelet aggregation, secretion, and the apoptosis pathway. Platelet-specific Rac1 function was not only important for coagulation in vitro but also for fibrin accumulation in vivo following laser-induced arteriolar injury.

Abstract 197: p300-Based Acetylation Signaling Regulates Cytoskeletal Reorganization and Procoagulant Phenotype of Platelets

Introduction: Reversible acetylation signaling catalyzed by acetyltransferases and deacetylases regulates multiple cellular events. We recently showed a role for deacetylation signaling in platelet function via the actions of the deacetylase enzyme HDAC6. Here we investigate the role of acetylation mediated by the p300 class of acetyltransferase proteins in platelet function. Methods: To test the hypothesis that p300 acetylation signaling controls cytoskeletal remodeling and the exposure of phosphatidylserine (PS) in platelets, we used cell biological and hematological methods, including enzymatic assays, microscopy, cell sorting, aggregometry and in vitro assays of thrombus formation. Results: Platelets express high levels of p300 protein. Upon stimulation with the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP), total platelet acetyltransferase enzyme activity increases. Pre-treatment of platelets with pharmacological inhibitors of p300 blocks agonist-induced acetyltransferase activity and prevents the acetylation of p300 substrate proteins. Inhibition of p300 blocked the deacetylation of HDAC6 and tubulin and disrupted the platelet tubulin cytoskeleton. p300 inhibition also increased basal platelet PS exposure via a caspase-dependent apoptotic pathway. On a functional level, p300 inhibition prevented platelet shape change, aggregation and spreading while increasing the procoagulant activity of platelets in whole blood. Conclusions: These results suggest that p300-based acetylation signaling regulates separable aspects of platelet function. p300 plays a role in the regulation of the platelet cytoskeleton as well as PS exposure to effect platelet spreading, aggregation, thrombus formation as well as platelet procoagulant activities. Future studies of the platelet acetylome will aim to identify p300 target substrates and specify roles for p300 in in the context of platelet activation, thrombus formation and pathologies of the cardiovascular system. Acknowledgements: This work is funded by the American Heart Association (13POST13730003 to J.E.A. and 13EIA12630000 to O.J.T.M.) and the National Institutes of Health (R01HL101972 to O.J.T.M.).

Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure in Platelets

BackgroundThe transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform. Methods and ResultsROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an in vivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). ConclusionsThese studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes.

Chloride channels are necessary for full platelet phosphatidylserine exposure and procoagulant activity

Platelets enhance thrombin generation at sites of vascular injury by exposing phosphatidylserine during necrosis-like cell death. Anoctamin 6 (Ano6) is required for Ca2+-dependent phosphatidylserine exposure and is defective in patients with Scott syndrome, a rare bleeding disorder. Ano6 may also form Cl− channels, though the role of Cl− fluxes in platelet procoagulant activity has not been explored. We found that Cl− channel blockers or removal of extracellular Cl− inhibited agonist-induced phosphatidylserine exposure. However, this was not due to direct inhibition of Ca2+-dependent scrambling since Ca2+ ionophore-induced phosphatidylserine exposure was normal. This implies that the role of Ano6 in Ca2+−dependent PS exposure is likely to differ from any putative function of Ano6 as a Cl− channel. Instead, Cl− channel blockade inhibited agonist-induced Ca2+ entry. Importantly, Cl− channel blockers also prevented agonist-induced membrane hyperpolarization, resulting in depolarization. We propose that Cl− entry through Cl− channels is required for this hyperpolarization, maintaining the driving force for Ca2+ entry and triggering full phosphatidylserine exposure. This demonstrates a novel role for Cl− channels in controlling platelet death and procoagulant activity.

Abstract 18994: Shear-Dependent Membrane Scrambling and Microvesiculation and an Important Role for Rac1

Surface exposure of phosphatidylserine (PS) and the release of membrane microvesicles (microparticles) are critically important for blood platelets and leukocytes to promote coagulation and inflammation during vascular injury. The caspase-dependent apoptotic signaling pathway and high intracellular calcium-dependent mechanisms have been implicated in the induction of these events under static conditions. However, it is known that when stimulated with physiological agonists under static conditions only a small percentage of platelets actually respond in a procoagulant manner. Thus, these mechanisms do not fully explain the potent procoagulant response of platelets to physiological stimuli. Here we demonstrate a novel shear stress-dependent mechanism of PS exposure and microvesiculation in platelets stimulated by physiological agonists. We show that exposing agonist-stimulated mouse and human platelets to increasing levels of shear led to greatly increased levels of surfaced exposed PS, shedding of membrane microvesicles, and in particular an increased sensitivity of platelets to display procoagulant activity at low concentrations of physiological agonists. Thus, shear stress is critical for inducing the surface exposure of PS and the shedding of PS-exposed microvesicles in platelets. Moreover, using the Rac1 inhibitor NSC23766 and platelet-specific Rac1 -/- platelets we identified Rac1, a member of the Rho family small GTPases, as being a critical mediator of the platelet procoagulant response to shear. We also show that Rac1 regulates this shear-induced response independent of the known apoptosis signaling pathway, as Rac1 -/- platelets showed no difference in the caspase-dependent pathway of PS exposure induced by ABT737. Importantly, the role of Rac1 in mediating shear-dependent PS exposure and microvesicle release appears to be independent of its known role in stimulating platelet secretion, and represents a common pathway of PS exposure and microvesicle release induced via several different agonists. This novel PS exposure and microvesculation mechanism is important for the function of platelets to promote coagulation.

Arsenic Trioxide Induces Platelet Apoptosis

Background Arsenic trioxide (ATO), a component of Traditional Chinese Medicine, is known as an effective anticancer drug especially in the treatment of acute promyelocytic leukaemia (APL). APL has emerged as the most curable subtype of acute myeloid leukaemia since the widely use of arsenic trioxide-based chemotherapy. However, recent researches showed that thrombocytopenia occurred in 79% of the relapsed or refractory APL patients treated with arsenic trioxide, and part of the APL patients had to be stopped treatment because of catastrophic bleeding, such as intracranial and pulmonary haemorrhage. Thrombocytopenia also occurred in 43% of the myelodysplastic syndrome patients treated with arsenic trioxide. Recently, arsenic trioxide has been proved to have a pro-apoptotic effect on various kinds of nucleated tumour cells or non-tumour cells. The effect of ATO on enucleated platelet, however, still remains unclear. The aim of current study is to investigate whether ATO induces platelet apoptosis. Methods Washed platelets (3 × 108/ml) were incubated with different concentrations of ATO or vehicle at 37°C for 4 hours. Then, mitochondrial inner transmembrane potential (ΔΨm), phosphatidylserine (PS) exposure, P-selection and PAC-1 binding were tested by flow cytometry. In the mean time, the treated platelets were analyzed by western blot for the expression levels of pro-apoptotic protein (Bax), and anti-apoptotic proteins (Bcl-2 and Bcl-XL). Activation of caspase-3 and c-jun NH2-terminal kinase (JNK) was also examined by western blot. To test whether inhibition of JNK can attenuate ATO-induced platelet apoptosis, JNK specific inhibitor dicumarol was used and ΔΨm was analysis by flow cytometry. For platelet aggregation analysis, PRP was incubated with ATO (2 μM) for 1 hr and then subjected to platelet aggregation assay by collagen. To further investigate whether ATO incurs thrombocytopenia in vivo, clinical therapeutic dosage of ATO (or vehicle (0.9% NS) control) was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were counted. Results and Conclusions ATO dose-dependently induces depolarization of mitochondrial inner ΔΨm, up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and PS exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen-induced platelet aggregation. ATO dose-dependently induced JNK activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after 12 and 24 hours of injection. Taken together, the data demonstrate that ATO induces caspase-dependent apoptosis via mitochondria-mediated intrinsic pathway in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also incurs thrombocytopenia in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. Disclosures: No relevant conflicts of interest to declare.

Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure In Platelets

IntroductionPlatelet activation results in a loss of membrane phospholipid asymmetry with concomitant exposure of procoagulant phosphatidylserine (PS) at the outer leaflet of membrane bilayer. PS provides binding sites for the assembly of prothrombinase and the tenase complex on activated platelets. In spite of its central role in thrombosis and hemostasis, the signal transduction mechanisms leading to PS exposure are not well characterized. Previously, we have shown that ROCK inhibitor, Y-27632, induces increased PS exposure following activation of platelets. However, Y-27632 does not distinguish between ROCK-1 and ROCK-2. Here, we studied activation induced PS exposure in ROCK-1 deficient platelets. MethodsPlatelets were isolated from ROCK-1 deficient and control mice. PS expression was measured on a flow cytometer using FITC-lactadherin (5 μg/ml). Platelet aggregation was carried out by standard turbidometric methods. Platelet procoagulant activity was measured in a prothrombinase assay consisting of prothrombin (1μM), factor Va (10 nM), factor Xa (10 pM), calcium (1.5 mM), collagen (10 μg/ml), and platelets (1x106/ml). Thrombin formation was determined by measuring absorbance at 405 nm in a microplate reader Calcium mobilization was measured by incubating ROCK-1 and WT platelets with Fura 2 AM and measuring the Fura 2 fluorescence after collagen treatment. An in vivo light/dye-induced endothelial injury/thrombosis model was used to assess the occlusion in cremasteric venules of mice. Phosphorylation status was measured by Western blot analysis of platelet lysate before and after activation. ResultsCollagen induced significantly more PS exposure in ROCK-1 deficient platelets compared to platelets from wild type littermates. ROCK-1 deficient platelets are more procoagulant as it generates more thrombin compared to control platelets. ROCK-1 deficient mice have a shorter occlusion time in a light/dye induced endothelial injury/thrombosis model in cremasteric venules compared to its littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). Since PS exposure is associated with increase in cytosolic calcium level, we measured calcium concentration in WT and ROCK-1 deficient platelets following collagen stimulation. There was no significant difference in calcium levels in collagen stimulated ROCK-1 and WT platelets. Hence, it is unlikely that the increased PS exposure in ROCK-1 deficient platelets is due to changes in calcium mobilization. Besides calcium, actin cytoskeleton rearrangement also plays a major role in PS exposure. ROCK1 modulates cytoskeleton rearrangement is through LIM kinase-1 (LMK-1). ROCK-1 mediated activation of LIMK-1 catalyses phosphorylation of cofilin-1, an actin severing protein. Phosphorylation inactivates actin severing activity of cofilin-1. Hence, ROCK-1 deficiency should lead to activation of cofilin-1. Indeed, collagen stimulated ROCK-1 deficient platelets showed a major reduction in phosphorylation of cofilin-1. Increased activity of cofilin-1 in ROCK-1 deficient platelets is further supported by the fact that F- actin content is significantly reduced in ROCK-1 deficient platelets. ConclusionWe postulate that cofilin-1-induced F-actin severing and subsequent cytoskeletal rearrangement plays an important role in collagen-induced PS exposure in platelets. Disclosures:No relevant conflicts of interest to declare.

TMEM16F Plays a Vital Role In Platelet Microparticle Generation and Thrombosis

Transmembrane (TMEM)16f is member of the TMEM16 family of ion channels, recently shown to be essential for optimal Ca2+-dependent phospholipid scrambling in platelets, platelet-dependent pro-coagulant activity and thrombosis. However, relatively little is known about the effect of TMEM16f on platelet signaling, functional activity, and microparticle formation. TMEM16f-/- mice were obtained from Andrea Vortkamp and the platelets of these mice were isolated, stimulated with either dual agonist (thrombin + convulxin) or calcium ionophore (A23187), and platelet surface exposure of phosphatidylserine (PS) and microparticle generation were assessed by both flow cytometry and high resolution immunofluorescent confocal microscopy. To measure PS exposure, annexin V binding to platelet surfaces was quantified: In response to thrombin and convulxin, only 10.65% ± 1.35% of TMEM16f-/- platelets exposed PS after dual agonist exposure, compared to 16.1% ± 2.3%PS+ WT platelets when analyzed by high resolution microscopy. When analyzed by flow cytometry, dual agonist exposure of WT platelets yielded 52.4% ± 7.0% of maximal annexin V binding achieved by 10μM A23187; in contrast, there was no significant increase in annexin V binding detected in TMEM16f-/- platelets treated with dual agonist. Responses to calcium ionophore were also reduced in TMEM16f-/- platelets relative to WT (17.3% ± 8.3% PS+ TMEM16f-/- platelets in response to 1μM A23187 compared to 40% ± 1.4% PS+ WT platelets, by high resolution microscopy). Microparticle generation from TMEM16f-/- platelets compared to WT platelets was also evaluated by high resolution immunofluorescent microscopy and flow cytometry. On average, approximately 5.7 ± 0.33 microparticles were generated by untreated WT platelets however thrombin and convulxin treated platelets yielded 10.66 ± 0.92 microparticles per platelet whereas no significant increase in microparticle generation was observed in TMEM16f-/- platelets after dual agonist treatment. These data suggest that TMEM16f is required not only for PS exposure on platelet membranes, but also for the shedding of PS+ microparticles. TMEM16f-/- mice also have a significant defect in stable occlusive thrombus formation following 10% ferric chloride injury of the carotid artery (0/6 TMEM16f-/- mice form stable thrombi compared to 5/6 WT mice forming stable thrombi under the same conditions). TMEM16f-/- platelets had a slight (15%) reduction in total fibrinogen binding stimulated by PAR4 peptide agonist compared to WT, but showed no significant differences in aggregation to PAR4 agonist peptide or ADP compared to WT control platelets, suggesting that the defect in thrombus formation is likely due to PS- or microparticle-dependent procoagulant activity. This hypothesis is supported by preliminary results demonstrating that when microparticles isolated from ionophore-stimulated WT platelets were injected into TMEM16f knockout mice, 3/3 TMEM16f-/- mice formed stable thrombi, compared to 5/6 WT mice injected with vehicle control. In conclusion, TMEM16f-/- mice are deficient in platelet PS exposure, platelet-derived microparticle formation, and injury-induced thrombus formation, and platelet-derived microparticles appear to contribute to the defect in thrombus formation. Disclosures:No relevant conflicts of interest to declare.

A Role for Platelet TRPC Channels in the Ca 2+ Response That Induces Procoagulant Activity

After vascular injury, platelets are rapidly activated by collagen and other agonists, causing them to adhere and aggregate to prevent blood loss. In addition, phosphatidylserine (PS) exposure on the platelet surface accelerates thrombin formation by the coagulation pathway. Thrombin is a potent platelet agonist and converts fibrinogen to fibrin, thereby stabilizing the platelet plug. PS exposure during hemostasis and thrombosis results from a sustained cytosolic Ca(2+) increase; however, the underlying Ca(2+) mobilization pathways have remained unclear. Store-operated Orai1 channels provide substantial, prolonged Ca(2+) influx after inositol trisphosphate-dependent release, and anoctamin 6 (TMEM16F) may operate as a Ca(2+)-activated, Ca(2+)-permeable channel in addition to its scramblase activity that exteriorizes PS. A new study shows that Na(+) entry, resulting from coactivation of the transient receptor potential (TRP) nonselective cation channels TRPC3 and TRPC6, followed by reverse-mode operation of Na(+)/Ca(2+) exchangers, is an important mechanism for the increase in cytosolic Ca(2+) that triggers PS exposure, particularly during combined thrombin and collagen stimulation.

The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, βPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.