Platelet Phosphatidylserine Research Articles

Adrenaline May Contribute to Prothrombotic Condition via Augmentation of Platelet Procoagulant Response, Enhancement of Fibrin Formation, and Attenuation of Fibrinolysis.

Background: Adrenaline is believed to play a role in thrombosis and hemostasis. The complex effect of its clinically relevant concentrations on thrombus formation, coagulation and fibrinolysis in human blood has never been specifically studied.Methods: Confocal microscopy was used to study thrombus formation under flow, exposure of phosphatidylserine (PS) in adhered platelets, to evaluate clots density, and to measure kinetics of fibrin formation and external fibrinolysis under flow. Flow cytometry was utilized to assess PS exposure in non-adhered platelets. Kinetics of clot formation and internal fibrinolysis was evaluated by thromboelastometry. Platelet aggregation was measured by optical aggremometry. Kinetics of clot retraction was assessed by using digital camera.Results: We found that adrenaline (1–10 nM) is able to enhance platelet activation evoked by subthreshold collagen (150 ng/ml), resulting in augmentation of platelet aggregation, thrombus formation under arterial flow conditions, platelet PS exposure, and formation of platelet-fibrin clots. The development of platelet procoagulant response evoked by adrenaline + low collagen was associated with the formation of denser platelet-fibrin clots and the decrease in rate of fibrinolysis despite whether lysis was initiated inside (internal fibrinolysis) or outside the clot (external fibrinolysis). The above phenomena were abolished by the α2-adrenergic receptor antagonist, rauwolscine. Adrenaline-collagen synergism, expressed as PS exposure, was significantly reduced by cyclooxygenase inhibitor (acetylsalicic acid), GPIIb/IIIa receptor blocker (tirofiban), and P2Y12 receptor antagonist (PSB 0739).Conclusion: Clinically relevant concentrations of adrenaline may significantly augment responses of human platelets in the presence of subthreshold concentrations of collagen, which should be considered during therapies involving adrenaline infusion. Routinely used antiplatelet drugs may reduce the prothrombotic state evoked by adrenaline-collagen synergism.

Aquaporins in platelet function

Structurally, aquaporins (AQPs) are small channel proteins with monomers of ~ 30 kDa that are assembled as tetramers to form pores on cell membranes. Aquaporins mediate the conduction of water but at times also small solutes including glycerol across cell membranes and along osmotic gradients. Thirteen isoforms of AQPs have been reported in mammalian cells, and several of these are likely expressed in platelets. Osmotic swelling mediated by AQP1 sustains the calcium entry required for platelet phosphatidylserine exposure and microvesiculation, through calcium permeable stretch-activated or mechanosensitive cation channels. Notably, deletion of AQP1 diminishes platelet procoagulant membrane dynamics in vitro and arterial thrombosis in vivo, independent of platelet granule secretion and without affecting hemostasis. Water entry into platelets promotes procoagulant activity, and AQPs may also be critical for the initiation and progression of venous thrombosis. Platelet AQPs may therefore represent valuable targets for future development of a new class of antithrombotics, namely, anti-procoagulant antithrombotics, that are mechanistically distinct from current antithrombotics. However, the structure of AQPs does not make for easy targeting of these channels, hence they remain elusive drug targets. Nevertheless, thrombosis data in animal models provide compelling reasons to continue the pursuit of AQP-targeted antithrombotics. In this review, we discuss the role of aquaporins in platelet secretion, aggregation and procoagulation, the challenge of drugging AQPs, and the prospects of targeting AQPs for arterial and venous antithrombosis.

Pediatric immune thrombocytopenia: apoptotic markers may help in predicting the disease course.

In all, 15-30% of pediatric immune thrombocytopenia (ITP) patients will remain chronically thrombocytopenic at 1 year post diagnosis. All attempts to classify patients at diagnosis have proven unsuccessful. We hypothesized that a different pathophysiology is responsible for non-chronic versus chronic pediatric ITP. We aimed to examine differences in the apoptotic markers' presentation at diagnosis between non-chronic and chronic patients. Blood samples were collected from 42 pediatric patients with newly diagnosed ITP prior to initiation of treatment. We incubated patients' sera with control platelets and compared the results among three research groups: healthy controls, chronic ITP, and non-chronic ITP patients. We measured apoptotic markers phosphatidylserine (PS) exposure and mitochondrial inner membrane potential (ΔΨm) by flow cytometry and the level of human apoptotic proteins by Human Apoptosis Array. We found increased platelet PS exposure and decreased ΔΨm in response to all ITP patients' sera compared to control subjects. Human Apoptotic Array revealed an increased expression of five apoptotic proteins: BIM, CD40, IGFBP2, P21, and SMAC, following sera incubation of non-chronic pediatric ITP patients, compared to chronic patients' sera, at diagnosis. Our data contribute to knowledge on apoptosis markers that may aid in predicting the prognosis of children with ITP. The key message of our article is that children with chronic ITP have a different apoptotic profile compared to non-chronic ITP. Addition to existing literature: This is the first study comparing apoptotic markers between children with chronic ITP to non-chronic ITP. Our findings indicate that, in the future, apoptotic markers may help to classify ITP patients into non-chronic versus chronic ones, at diagnosis.

HAuCl4, Putative General Aquaporins Blocker, Reduces Platelet Spreading, Filopodia Formation, Procoagulant Response, and Thrombus Formation Under Flow.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated.Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo.Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg).Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

Ethaninidothioic acid (R5421) is not a selective inhibitor of platelet phospholipid scramblase activity.

Background and PurposeEthaninidothioic acid (R5421) has been used as a scramblase inhibitor to determine the role of phospholipid scrambling across a range of systems including platelet procoagulant activity. The selectivity of R5421 has not been thoroughly studied. Here, we characterised the effects of R5421 on platelet function and its suitability for use as a scramblase inhibitor.Experimental ApproachHuman platelet activation was measured following pretreatment with R5421 and stimulation with a range of agonists. Phosphatidylserine exposure was measured using annexin V binding. Integrin αIIbβ3 activation and α‐granule release were measured by flow cytometry. Cytosolic Ca2+ signals were measured using Cal520 fluorescence. An in silico ligand‐based screen identified 16 compounds which were tested in these assays.Key ResultsR5421 inhibited A23187‐induced phosphatidylserine exposure in a time‐ and temperature‐dependent manner. R5421 inhibited Ca2+ signalling from the PAR1, PAR4 and glycoprotein VI receptors as well as platelet αIIbβ3 integrin activation and α‐granule release. R5421 is therefore not a selective inhibitor of platelet scramblase activity. An in silico screen identified the pesticide thiodicarb as similar to R5421. It also inhibited platelet phosphatidylserine exposure, Ca2+ signalling from the PAR1 and glycoprotein VI, αIIbβ3 activation and α‐granule release. Thiodicarb additionally disrupted Ca2+ homeostasis in unstimulated platelets.Conclusion and ImplicationsR5421 is not a selective inhibitor of platelet scramblase activity. We have identified the pesticide thiodicarb, which had similar effects on platelet function to R5421 as well as additional disruption of Ca2+ signalling which may underlie some of thiodicarb's toxicity.

Procoagulant Phosphatidylserine-Exposing Platelets in vitro and in vivo.

The physiological heterogeneity of platelets leads to diverse responses and the formation of discrete subpopulations upon platelet stimulation. Procoagulant platelets are an example of such subpopulations, a key characteristic of which is exposure either of the anionic aminophospholipid phosphatidylserine (PS) or of tissue factor on the activated platelet surface. This review focuses on the former, in which PS exposure on a subpopulation of platelets facilitates assembly of the intrinsic tenase and prothrombinase complexes, thereby accelerating thrombin generation on the activated platelet surface, contributing importantly to the hemostatic process. Mechanisms involved in platelet PS exposure, and accompanying events, induced by physiologically relevant agonists are considered then contrasted with PS exposure resulting from intrinsic pathway-mediated apoptosis in platelets. Pathologies of PS exposure, both inherited and acquired, are described. A consideration of platelet PS exposure as an antithrombotic target concludes the review.

2-Aminoethoxydiphenylborate (2-APB) inhibits release of phosphatidylserine-exposing extracellular vesicles from platelets

Activated, procoagulant platelets shed phosphatidylserine (PS)-exposing extracellular vesicles (EVs) from their surface in a Ca2+- and calpain-dependent manner. These PS-exposing EVs are prothrombotic and proinflammatory and are found at elevated levels in many cardiovascular and metabolic diseases. How PS-exposing EVs are shed is not fully understood. A clearer understanding of this process may aid the development of drugs to selectively block their release. In this study we report that 2-aminoethoxydiphenylborate (2-APB) significantly inhibits the release of PS-exposing EVs from platelets stimulated with the Ca2+ ionophore, A23187, or the pore-forming toxin, streptolysin-O. Two analogues of 2-APB, diphenylboronic anhydride (DPBA) and 3-(diphenylphosphino)-1-propylamine (DP3A), inhibited PS-exposing EV release with similar potency. Although 2-APB and DPBA weakly inhibited platelet PS exposure and calpain activity, this was not seen with DP3A despite inhibiting PS-exposing EV release. These data suggest that there is a further target of 2-APB, independent of cytosolic Ca2+ signalling, PS exposure and calpain activity, that is required for PS-exposing EV release. DP3A is likely to inhibit the same target, without these other effects. Identifying the target of 2-APB, DPBA and DP3A may provide a new way to inhibit PS-exposing EV release from activated platelets and inhibit their contribution to thrombosis and inflammation.

Comparison of putative BH3 mimetics AT-101, HA14-1, sabutoclax and TW-37 with ABT-737 in platelets

Platelet lifespan is regulated by intrinsic apoptosis. Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL. Here, we investigated several small molecules that are reported to act as BH3 mimetics and compared their effects to the well-established BH3 mimetic, ABT-737. Platelet phosphatidylserine (PS) exposure was determined by flow cytometry. Changes in cytosolic Ca2+ signaling were detected using Cal-520. Plasma membrane integrity was determined by calcein leakage. The roles of caspases and calpain in these processes were determined using Q-VD-OPh and calpeptin, respectively. As previously reported, ABT-737 triggered PS exposure in a caspase-dependent manner and calcein loss in a caspase and calpain-dependent manner. In contrast, AT-101 and sabutoclax triggered PS exposure independently of caspases. HA14-1 also triggered PS exposure in a caspase-independent but calpain-dependent manner. There were also significant differences in the pattern and protease-dependency of cytosolic Ca2+ signaling in response to these drugs compared to ABT-737. Since there are clear differences between the action of ABT-737 and the other putative BH3 mimetics investigated here, AT-101, HA14-1 and sabutoclax cannot be considered as acting as BH3 mimetics in platelets. Furthermore, the platelet death caused by these drugs is likely to be distinct from apoptosis.

PF801 DASATINIB ATTENUATES THROMBIN GENERATION AND CLOT RETRACTION OF CONVULXIN ACTIVATED HUMAN PLATELETS

Background:Tyrosine kinase inhibitors (TKI) are a very effective group of drugs that considerably prolong survival of patients with chronic myeloid leukemia. Several lines of evidence suggest that dasatinib an inhibitor of BCR‐ABL may induce bleeding due to its effect on platelets. Previously it was shown that dasatinib induced impaired platelet adhesion, aggregation and secretion. In our in vitro studies we investigated the effect of dasatinib on the procoagulant activity and on clot retraction of human platelets.Aims:We hypothesized that dasatinib treatment may impair various platelet functions in activated platelets.Methods:Platelet‐rich plasmas (PRPs) were prepared from citrate‐anticoagulated blood samples of healthy volunteers using low speed centrifugation. Subsequently PRPs were incubated with or without dasatinib, then platelets were activated by a GPVI agonist, the snake venom convulxin. Phosphatidylserine (PS) expression of activated platelets was examined by flow cytometry. The thrombin generation test (TGT) of PRPs was performed in the presence of 1pM Tissue factor (PRP reagent) by a fluorimetric assay. Fluorescence was detected by a Fluoroskan Ascent fluorimeter (Thermo Fischer Scientific), and the thrombin generation curves were analyzed by the Thrombinoscope software (Thrombinoscope BV). The activated form of GPIIb/IIIa was detected by measuring PAC1 expression by flow cytometry. Clot retraction of platelets was elicited by high calcium concentration in PRP and results were expressed as retracted percent of the original volume and was followed up to 1 hour. Sarcoma Family Kinases (SFK) play role in platelet activation, thus their phosphorylation levels were examined from dasatinib pretreated platelet lysates by Western‐blot.Results:Convulxin resulted in a dose‐dependent increase of platelet PS expression peaking at 51% when 12.5 ng/mL convulxin was used. The lower end of the therapeutic range of dasatinib (10 nM) showed no effect on the rate of this activation marker while the high therapeutic dasatinib concentration (100 nM) considerably prevented this activating effect. PS expressed on the platelet surface may serve as an anchor for the multiprotein complexes of the coagulation cascade. Thus, we investigated the effect of dasatinib on thrombin generation in PRP and found that 100 nM dasatinib attenuated thrombin formation as it reduced the peak thrombin values from 322 ± 31 nM thrombin to 204 ± 62 nM. Similarly, PAC1 expression on convulxin activated platelets was significantly prevented by preincubation with 100 nM dasatinib (8% versus 81%). Clot retraction was 80% in non activated samples and this was decreased to 53% upon convulxin activation that was completely prevented by dasatinib preincubation. We found that dasatinib pretreatment downregulated the phosphorylation levels of Fyn, Lyn and Src kinases in a concentration dependent manner.Summary/Conclusion:Dasatinib is an effective TKI that alters platelet reactions to activation via the collagen receptor GPVI. This effect may be mediated via its effect on phosphorylation status of SFK and can contribute to bleeding symptoms observed during dasatinib treatment.

Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

Platelet Dysfunction and Cellular Microparticles May be Involved in the Hipercoagulable State Observed in Obstructive Sleep Apnea Syndrome

Introduction: Obstructive sleep apnea syndrome (OSA) is a common disorder characterized by repetitive episodes of nocturnal breathing cessation due to upper airway collapse that cause intermittent hypoxia. Because of this, there is an increase in oxidative stress, endothelial damage and mortality associated with the incidence of thrombotic events. The evaluation of platelet function by flow cytometry (FCM) and hemostatic capacity through global hemostasis tests have been successfully used in the study of the prothrombotic state associated to numerous diseases. We anticipate that these techniques may help to clarify the mechanism involved in the prothrombotic state of OSA.Materials and Methods: We included 16 OSA patients diagnosed in the Pneumology Unit of the University Hospital La Paz and 59 healthy controls recruited in the Donor Unit of the same Hospital.Platelet surface receptors were analysed by FCM (FACScan, BD Biosciences) with specific monoclonal antibodies (mAbs) against CD41/αIIb and CD61/β3, CD42a and CD42b. Platelet activation was assessed by FCM through PAC1-FITC binding (mAbs that recognizes activated conformation of fibrinogen receptor), P-selectin and CD63 exposure in baseline condition and after stimulation with 100 µM TRAP (agonist of the PAR-1 receptor) and 10 µM ADP. Platelet apoptosis was analysed by FCM through FITC-annexin V (BD Pharmingen) binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions and by determination of activity of caspases -3/7, -8, and -9 (BD Millipore).The overall hemostatic capacity was determined by the evaluation of thrombin generation using the Calibrated Automatic Thrombinography (CAT) with fresh platelet-poor plasma (obtained after double centrifugation of whole blood at 2500g, 20 minutes and 24ºC) and by the assessment of kinetics of clot formation using Rotational Thromboelastometry (ROTEM® Gamma, Tem Innovations GmbH, Spain) using whole blood and activation by recalcification (naTEM® test, Tem Innovations GmbH, Spain).The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Statistical significance was established at p <0.05.Results: No significant differences in age or sex were found between groups. Patients presented anxiety-depressive disorder (15%), ischemic cardiomyopathy (8%), dyslipidemias (38%), arterial hypertension (69%) and lower minimum oxygen pressures during sleep (mean±SD; patients: 76±8 % vs controls: 89±1 %; p< 0.001) indicating hypoxia. In basal conditions, an increase of CD42a subunit of the von Willebrand factor receptor was observed in patients with OSA but no differences were found in the exposure of fibrinogen receptor either at basal condition or after activation with TRAP or ADP (Table 1 and Figure 1). Platelet PS exposure before and after TRAP stimulation was higher in the patient group (Figure 1) but no differences between groups were observed in caspase activities (Table 2). Clotting time (CT), maximum clot firmness (MCF) and MP-associated thrombin generation were higher in patients with OSA (Figure 2).Conclusion: Mechanisms related to the mobilization of P-selectin to platelet surface and with the maintenance of phospholipids distribution in platelet membrane might be altered in OSA. The increment of P-selectin exposure may favor the interaction between white cells and platelets through PSGL-1 ligand from lymphocytes which in turn may produce endothelial damage explaining part of OSA's pathophysiology. A lower CT and higher MCF reflected a hypercoagulable state in patients, perhaps related to their higher MP-associated thrombin generation and, at least in part, due to the enhanced platelet-PS exposure. Our results also suggest that both ROTEM and CAT are useful tools for characterizing prothrombotic states in OSA patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). DisclosuresÁlvarez-Roman:Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding; Sobi: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

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.

Microfluidic flow-based platforms for induction and analysis of dynamic shear-mediated platelet activation-Initial validation versus the standardized hemodynamic shearing device.

A microfluidic flow-based platform (μFP), able to stimulate platelets via exposure of shear stress patterns pertinent to cardiovascular devices and prostheses, was compared to the Hemodynamic Shearing Device (HSD)-a state-of-the-art bench-top system for exposure of platelets to defined levels and patterns of shear. Platelets were exposed to time-varying shear stress patterns in the two systems; in detail, platelets were recirculated in the μFP or stimulated in the HSD to replicate comparable exposure time. Shear-mediated platelet activation was evaluated via (i) the platelet activity state assay, allowing the measurement of platelet-mediated thrombin generation and associated prothrombotic tendencies, (ii) scanning electron microscopy to evaluate morphological changes of sheared platelets, and (iii) flow cytometry for the determination of platelet phosphatidylserine exposure as a marker of shear activation. The results revealed good matching and comparability between the two systems, with similar trends of platelet activation, formation of microaggregates, and analogous trends of activation marker exposure for both the HSD and microfluidic-stimulated samples. These findings support future translation of the microfluidic platform as a Point-of-Care facsimile system for the diagnosis of thrombotic risk in patients implanted with cardiovascular devices.

Platelet procoagulant phenotype is modulated by a p38-MK2 axis that regulates RTN4/Nogo proximal to the endoplasmic reticulum: utility of pathway analysis.

Upon encountering physiological cues associated with damaged or inflamed endothelium, blood platelets set forth intracellular responses to ultimately support hemostatic plug formation and vascular repair. To gain insights into the molecular events underlying platelet function, we used a combination of interactome, pathway analysis, and other systems biology tools to analyze associations among proteins functionally modified by reversible phosphorylation upon platelet activation. While an interaction analysis mapped out a relative organization of intracellular mediators in platelet signaling, pathway analysis revealed directional signaling relations around protein kinase C (PKC) isoforms and mitogen-activated protein kinases (MAPKs) associated with platelet cytoskeletal dynamics, inflammatory responses, and hemostatic function. Pathway and causality analysis further suggested that platelets activate a specific p38-MK2 axis to phosphorylate RTN4 (reticulon-4, also known as Nogo), a Bcl-xl sequestration protein and critical regulator of endoplasmic reticulum (ER) physiology. In vitro, we find that platelets drive a p38-MK2-RTN4-Bcl-xl pathway associated with the regulation of the ER and platelet phosphatidylserine exposure. Together, our results support the use of pathway tools in the analysis of omics data sets as a means to help generate novel, mechanistic, and testable hypotheses for platelet studies while uncovering RTN4 as a putative regulator of platelet cell physiological responses.

Characteristics of thrombin generation in a fulminant porcine sepsis model

IntroductionThe activation of blood coagulation has been demonstrated in most cases of sepsis, however previous studies in humans could not detect hypercoagulability with global hemostasis assays. In a fulminant porcine sepsis model we analysed coagulation screening tests and thrombin generation to evaluate hemostatic alterations. Materials and methodsLive Escherichia coli bacteria were inoculated to female pigs and prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen were measured by coagulometry. Platelet counts, platelet aggregates and platelet phosphatidyl serine (PS) expression were studied, furthermore in in vitro experiments the PS-inducing ability of septic and control plasmas was investigated by flow cytometry. Thrombin generation was carried out by the Ascent Fluoroscan reader and results were evaluated by the Thrombinoscope software. ResultsClotting assays showed a large variability, but no systematic changes during the 4-hour observation period. Platelet count significantly decreased and the number of platelet aggregates increased already by 2h compared to baseline values and to control animals. Although the increase in platelet PS expression was non-significant in the septic group, the septic plasma elicited PS expression on normal human red blood cells. Thrombin generation became significantly faster, but the quantity of formed thrombin demonstrated both hypo- and hypercoagulability depending on the setting of the assay. ConclusionsEnhanced thrombin generation without activators and the PS-inducing capacity of septic plasma are signs of hemostatic activation during fulminant sepsis while the decreased amount of generated thrombin upon tissue factor and phospholipid induced activation demonstrates attenuated thrombin forming ability.

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.

Abstract 351: Plasma Phospholipid Transfer Protein Promotes Platelet Aggregation

Hypothesis: Phospholipid transfer protein (PLTP) has a direct effect on platelet aggregation, since PLTP knockout mice have longer bleeding time. Methods and Results: Platelets from humans or mice were prepared as were mouse platelet-rich plasma and human recombinant PLTP (rPLTP). In mice, we assessed ADP- and collagen-induced platelet aggregation, phosphatidylserine (PS) externalization, and photothrombosis-induced cerebral infarction. We found that human platelets produce PLTP. Platelet aggregation increased upon PLTP overexpression whereas it decreased with PLTP deficiency in a gene dose-dependent manner. Human rPLTP increased mouse or human platelet aggregation in a dose-dependent manner. PS externalization provides a water/lipid surface for the interaction of coagulation factors, which accelerates thrombosis. Compared with wild type controls, platelets from PLTP transgenic mice had significantly greater amounts of PS on the exterior surface of the plasma membrane, whereas platelets from PLTP-deficient mice had significantly less on the surface, thus influencing fibrinogen binding. Moreover, rPLTP together with ADP significantly increased PS exposure on the plasma membrane of PLTP-deficient platelets, thereby increasing fibrinogen binding. Importantly, PLTP overexpression significantly accelerated the incidence of photothrombosis-induced infarction, whereas PLTP deficiency reduced the incidence. Conclusions: PLTP promotes PS externalization at the plasma membrane of platelets and accelerates ADP- or collagen-induced platelet aggregation. Thus, PLTP is involved in hypercoagulation. Therefore, PLTP inhibition could be a novel approach for countering thrombosis.

Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation.To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs.In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibition of SiPP formation in the absence of VWF, in VWF with deletion of the GPIbα-binding A1 domain, and in the presence of antibodies blocking the interaction of GPIb-IX and VWF. Calcium-dependent processes play an essential role in agonist-initiated PSer externalization. However, extracellular Ca2+ is not required for SiPP formation. Inhibition of Ca2+ entry, removal of extracellular Ca2+ and chelation of intracellular Ca2+, all had no effect on SiPP formation. Caspase activation mediates PSer externalization in the apoptotic platelet, but neither pharmacologic inhibition of caspases nor calpain inhibitors prevented SiPP formation. In contrast to the negligible roles of calcium- and caspase mediated events, SiPP formation is abrogated both in the absence of the mPTP potentiator cyclophilin D and in cyclosporine A-treated platelets. Furthermore, SiPP formation was closely correlated with mPTP formation as measured by loss of mitochondrial transmembrane potential. Together, these results identify a new mechanistic pathway of SiPP formation distinct from previously described initiating mechanisms for PSer externalization. Targeting of this distinct pathway may provide a novel pharmacologic strategy for the treatment of high shear hematopathologies, like arterial thrombosis. DisclosuresNo relevant conflicts of interest to declare.

14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function.

The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function.