Thrombin-stimulated Platelets Research Articles

RXR Ligands Negatively Regulate Thrombosis and Hemostasis.

Platelets have been found to express intracellular nuclear receptors including the retinoid X receptors (RXRα and RXRβ). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2; however, the effects on responses to other platelet agonists and the underlying mechanism have not been fully characterized. The effect of 9-cis-retinoic acid, docosahexaenoic acid and methoprene acid on collagen receptor (glycoprotein VI [GPVI]) agonists and thrombin-stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilization, integrin αIIbβ3 outside-in signaling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses after stimulation by GPVI agonists or thrombin and inhibition of integrin αIIbβ3 outside-in signaling. Treatment with 9-cis-retinoic acid caused inhibition of thrombus formation in vitro and an impairment of thrombosis and hemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and nuclear factor κ-light-chain-enhancer of activated B cell activity. This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of protein kinase A, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo after treatment with RXR ligands.

Modulation of Oxidative Stress in Thrombin-Stimulated Platelets by Almond by-Product

Almond (Al) green hulls are waste materials that are generated annually in the almond harvest and recently gained much attention due to their antioxidant properties. However, paucity information is in literature relevant to the modern pharmacological effects of Al green hulls. The aim of this study was to evaluate anti-platelet activity of Persian Al hull extract. The antioxidant activity of Al hull extract was investigated employing various established in vitro system including total antioxidant activity in linoleic acid emulsion system and DPPH assay. Platelet aggregation was monitored by spectrophotometer and intracellular ROS activity was evaluated by flow cytometry. Chemical composition of extract was determined by gas chromatography-mass spectrometry. In vitro study showed that Al hull extract at 100 μg/ml inhibited thrombin-induced platelet aggregation and protein secretion by 50%, without any cytotoxic effects on platelets. Moreover, based on different free radical trapping assay, it has been revealed that the examined extract had high antioxidant capacity and could suppress ROS generation in thrombin-stimulated platelets. GC-MS analyses revealed that Al hull extract contains a variety of phenolic and benzoic acid compounds dominated by ferulic acid, p-coumaric acid, quercetin, homovanilic acid, catechin and methoxysalsylic acid. Collectively high antioxidant capacity of Al hull extract and also its anti-platelet activity with lack of toxicity, give validity to this agricultural waste material as appropriate candidate for treatment of cardiovascular diseases and thrombosis.

Abstract B62: Role of the blood microenvironment in the upregulation of oncoproteins in circulating pancreatic cancer cells

Abstract Background: Platelets are promoters of cancer cell proliferation at the metastatic niche, however the exact mechanism(s) by which platelets drive cancer cell proliferation remain ill-defined. The oncoprotein c-MYC, which governs neoplastic proliferation, is overexpressed in pancreatic tumors at the metastatic site as compared to primary tumors, with the overexpression of c-MYC indicative of a poor clinical outcome for patients with cancer. At present, the oncogenic pathways mediating c-MYC upregulation in metastatic cancer cells are largely unknown. Our exciting preliminary data demonstrate for the first time that platelets upregulate c-MYC protein levels in PANC-1 pancreatic cancer cells, suggesting a novel prometastatic role for platelets in pancreatic cancer. Aims: To define the mechanisms by which platelets upregulate c-MYC protein levels in PANC-1 pancreatic cancer cells. Methods: We utilized a co-culture platform to examine the effect of resting and thrombin-stimulated human platelets as well as platelet fractions (platelet-derived membrane bound molecules and soluble molecules) on c-MYC expression in PANC-1 cancer cells. Platelet inhibitors, including the GPIIbIIIa blocker Integrilin, were used to study the molecular mechanisms by which platelets upregulate c-MYC expression in cancer cells. Changes in c-MYC oncoprotein were detected by Western Blotting. The physical association between platelets and PANC-1 cancer cells was quantified by immunoblotting with an anti-GPIIb antibody against the platelet-specific integrin GPIIbIIIa. Results: Resting and activated platelets as well as platelet fractions induced an upregulation of c-MYC expression in PANC-1 cancer cells. The effect of platelets on c-MYC expression was largely dependent on direct contact with the cancer cells, as the preincubation of platelets with Integrilin reduced platelet-induced c-MYC expression. Conclusion: We show that the expression of c-MYC in PANC-1 cancer cells can be upregulated by platelets, revealing a novel mechanism of platelet action in the progression of cancer and suggesting that the overexpression of c-MYC at the metastatic niche may be inhibited through the use of platelet inhibitors. We are currently characterizing the molecular underpinnings of platelet-mediated c-MYC regulation with respect to the contribution of platelets to cancer cell proliferation and growth. Citation Format: Annachiara Mitrugno, Katherine Pelz, Rosalie Sears, Owen McCarty. Role of the blood microenvironment in the upregulation of oncoproteins in circulating pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B62.

Platelets Mediate Host Defense against Staphylococcus aureus through Direct Bactericidal Activity and by Enhancing Macrophage Activities.

Platelets are the chief effector cells in hemostasis. However, recent evidence suggests they have multiple roles in host defense against infection. Reports by us and others showed that platelets functionally contribute to protection against Staphylococcus aureus infection. In the current study, the capacity of mouse platelets to participate in host defense against S. aureus infection was determined by assessing two possibilities. First, we determined the ability of platelets to kill S. aureus directly; and, second, we tested the possibility that platelets enhance macrophage phagocytosis and intracellular killing of S. aureus In this study we report evidence in support of both mechanisms. Platelets effectively killed two different strains of S. aureus. A clinical isolate of methicillin-resistant S. aureus was killed by platelets (>40% killing in 2 h) in a thrombin-dependent manner whereas a methicillin-sensitive strain was killed to equal extent but did not require thrombin. Interestingly, thrombin-stimulated platelets also significantly enhanced peritoneal macrophage phagocytosis of both methicillin-resistant S. aureus and methicillin-sensitive S. aureus by >70%, and restricted intracellular growth by >40%. Enhancement of macrophage anti-S. aureus activities is independent of contact with platelets but is mediated through releasable products, namely IL-1β. These data confirm our hypothesis that platelets participate in host defense against S. aureus both through direct killing of S. aureus and enhancing the antimicrobial function of macrophages in protection against S. aureus infection.

Platelet Membranes and Activation Pathway Determine Inhibitory Activity of Anti-Factor VIII C2 Domain Antibodies

Inhibitory antibodies against factor VIII (fVIII) are the major complication of hemophilia A treatment, causing severe bleeding in spite of infused fVIII. Most antibodies are directed against the A2 or the C2 domain of fVIII and the degree of inhibition in clinical fVIII assays does not predict the severity of bleeding risk. We recently discovered that fVIII binds to a macromolecular complex including low molecular weight fibrin and the αIIbβ3integrin on membranes of thrombin-stimulated platelets rather than exposed phosphatidylserine (PS). Because clinical fVIII assays utilize PS-rich vesicles to support fVIII activity we asked whether the degree of inhibition of platelet-dependent activity by anti-C2 domain mAb’s differs from inhibition on PS-rich phospholipid vesicles.Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides for PAR-1 and PAR-4, and either factor XIa (intrinsic pathway) or factor fXa (extrinsic pathway) without standard exogenous activators or vesicles. aPTT assays were performed on the same plasma-preparations using a commercial preparation of activator and phospholipid vesicles. Assays were performed using BBL fibrometers, electromechanical devices. A panel of 12 defined, epitope-mapped mAb’s against the fVIII-C2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies. mAb’s at 10 µg/ml were pre-incubated with fVIII for 60 min in the absence of VWF before mixing the fVIII-antibody mixture with plasma.Results: Platelets from nine donors supported a similar, near log-linear relationship between fVIII concentration and fibrin strand formation spanning at least 10,000-fold (0.0001 - 1 unit/ml). Time to fibrin strand formation was inversely related to platelet concentration over a range of at least 1 x 106 - 2 x 108/ml. Factor XIa and factor Xa concentrations of 20 pM and 0.01 pM, respectively were chosen emulating the intrinsic and extrinsic pathways.Four mAb’s inhibited fVIII activity <10% in the aPTT assay, four others by 50-80%, with the final four in the 90-99% range. fVIII activity in the intrinsic APT had little correlation to inhibition in the aPTT assay with four mAb’s causing less inhibition of the APT and the remaining 8 antibodies having 5-10 fold more inhibition. No mAb caused inhibition that was within 2-fold for the aPTT and APT assays.For the extrinsic APT, triggered by fXa, all antibodies produced greater than 99% inhibition of fVIII activity with 10 antibodies having 2-10 fold greater inhibition than with the intrinsic pathway.Conclusions: These results show that both platelet membranes and the pathway to fVIII activation determine the degree of inhibition by anti-C2 antibodies. The inhibition of platelet-dependent fVIII activity has little correlation to inhibition of the aPTT fVIII activity. Further, anti-C2 antibodies show greater inhibition of platelet-based activity when the fVIII activation pathway is via factor Xa vs. factor XIa. Additional studies will be needed to determine whether residual fVIII activity from the intrinsic APT or extrinsic APT better correlates to bleeding risk and whether the basis for activation pathway-dependent inhibition correlates to different proteolytic cleavage sites for factor VIII. DisclosuresMeeks:Genentech: Membership on an entity’s Board of Directors or advisory committees; Grifols: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Membership on an entity’s Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Shire: Membership on an entity’s Board of Directors or advisory committees. Novakovic:Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity. Gilbert:Baxalta/Shire: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity; Grifols: Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Platelet Integrin αIIbβ3 Inhibitor Rescues Progression of Apoptosis in Human Platelets.

BackgroundApoptosis plays an important role in the physiology of platelet function. We aimed to detect the effect of the platelet integrin αIIbβ3 inhibitor, tirofiban, on apoptotic events, including mitochondrial inner-membrane potential (ΔΨm), phosphatidylserine (PS) exposure on platelet surface, and the generation of reactive oxygen species (ROS), when washed platelets were stimulated with thrombin.Material/MethodsThe study included washed platelets from healthy humans, divided into 4 groups: vehicle, and tirofiban (0.05 μg/ml, 0.25 μg/ml, and 0.5 μg/ml). Platelets were pretreated with vehicle or tirofiban and incubated at 37°C with agitation for 6 h and 24 h. Before thrombin addition, the vehicle group divided into 2 equal groups. Except one vehicle group, the other 4 groups were all stimulated with thrombin (1 U/ml) for 30 min at 37°C. Using flow cytometry, we studied the ΔΨm and PS exposure on platelet surfaces, and the generation of ROS in platelets.ResultsWe observed that at the time of 6 h and 24 h, thrombin-stimulated vehicle platelets induced significant depolarization of ΔΨm, higher PS exposure, and increased ROS production compared with the vehicle group (P<0.01). However, the tirofiban group had significantly more recovery of ΔΨm, PS exposure, and ROS production compared with the thrombin group (P<0.01).ConclusionsThe platelet integrin αIIbβ3 inhibitor, tirofiban, inhibits the depolarization of ΔΨm, PS exposure on platelet surface, and ROS production when stimulated with thrombin. These results suggest that αIIbβ3 inhibitor inhibits the initiation of apoptosis in platelets, showing a potential clinical application of tirofiban as an apoptosis inhibitor.

Short fungal fractions of β-1,3 glucans affect platelet activation.

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains β-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of β-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 μmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbβ3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-β1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.

Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

Platelets mediate host-defense against S. aureus through direct bactericidal activity and by enhancing macrophage activities

Abstract Platelets are chief effector cells in hemostasis. However, recent evidence suggests that platelets have multiple roles in host defense against infection. Recent reports by us and others showed that platelets functionally contribute to protection against Staphylococcus aureus infection. In the current study, the capacity of mouse and human platelets to participate in host defense against S.aureus infection was determined by assessing two possibilities: First, the ability of platelets to directly kill S. aureus; and second, the possibility that platelets enhance macrophage phagocytosis and intracellular killing of S. aureus. Both human and mouse platelets effectively killed S. aureus (&amp;gt;35% and &amp;gt;40% killing in 60 min., respectively) in an activation-dependent (0.1U.ml thrombin) manner. Interestingly, thrombin-stimulated mouse platelets also significantly enhanced peritoneal macrophage phagocytosis of S. aureus by 70% and intracellular killing by 43%. To determine if platelet-enhanced uptake and killing was due to direct interaction or through releasable products, peritoneal macrophages were exposed to releasates collected from resting or thrombin-activated platelets. The data show that releasates collected from thrombin-activated platelets induce similar increases in both uptake and killing of S. aureus by macrophages. These data confirm our hypothesis that platelets participate both directly and indirectly in protection against S. aureus infection.

Relationship between calcium mobilization and platelet α- and δ-granule secretion. A role for TRPC6 in thrombin-evoked δ-granule exocytosis

Changes in cytosolic Ca2+ concentration ([Ca2+]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca2+ from distinct intracellular stores, which, in turn, activates store-operated Ca2+ entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca2+ mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca2+, and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca2+ mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573–586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca2+ mobilization.

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

AbstractThrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites.

The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets.

In blood platelets, stimulation of G protein-coupled receptors (GPCRs) by thrombin triggers the activation of Src family kinases (SFKs), resulting in the tyrosine-phosphorylation of multiple substrates, but the mechanism underlying this process is still poorly understood. In the present study, we show that the time-dependent protein-tyrosine phosphorylation triggered by thrombin in human or murine platelets was totally suppressed only upon concomitant chelation of intracellular Ca(2+) and inhibition of SFKs. Thrombin-induced activation of SFKs was regulated by intracellular Ca(2+) and accordingly the Ca(2+) ionophore A23187 was sufficient to stimulate SFKs. A23187 also triggered the phosphorylation and activation of the Ca(2+)-dependent focal adhesion kinase Pyk2 and Pyk2 activation by thrombin was Ca(2+)-dependent. Stimulation of SFKs by thrombin or A23187 was strongly reduced in platelets from Pyk2 knockout (KO) mice, as was the overall pattern of protein-tyrosine phosphorylation. By immunoprecipitation experiments, we demonstrate that Lyn and Fyn, but not Src, were activated by Pyk2. Inhibition of SFKs by PP2 also reduced the phosphorylation of Pyk2in thrombin or A23187-stimulated platelets. Analysis of KO mice demonstrated that Fyn, but not Lyn, was required for complete Pyk2 phosphorylation by thrombin. Finally, PP2 reduced aggregation of murine platelets to a level comparable to that of Pyk2-deficient platelets, but did not have further effects in the absence of Pyk2. These results indicate that in thrombin-stimulated platelets, stimulation of Pyk2 by intracellular Ca(2+) initiates SFK activation, establishing a positive loop that reinforces the Pyk2/SFK axis and allows the subsequent massive tyrosine phosphorylation of multiple substrates required for platelet aggregation.

Kaempferol inhibits thrombosis and platelet activation.

The objectives of the present study were to investigate whether kaempferol affects pro-coagulant proteinase activity, fibrin clot formation, blood clot and thrombin (or collagen/epinephrine)-stimulated platelet activation, thrombosis, and coagulation in ICR (Imprinting Control Region) mice and SD (Sprague-Dawley) rats. Kaempferol significantly inhibited the enzymatic activities of thrombin and FXa by 68 ± 1.6% and 52 ± 2.4%, respectively. Kaempferol also inhibited fibrin polymer formation in turbidity. Microscopic analysis was performed using a fluorescent conjugate. Kaempferol completely attenuated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and phosphoinositide 3-kinase (PI3K)/PKB (AKT) in thrombin-stimulated platelets and delayed aggregation time (clotting) by 34.6% in an assay of collagen/epinephrine-stimulated platelet activation. Moreover, kaempferol protected against thrombosis development in 3 animal models, including collagen/epinephrine- and thrombin-induced acute thromboembolism models and an FeCl3-induced carotid arterial thrombus model. The ex vivo anticoagulant effect of kaempferol was further confirmed in ICR mice. This study demonstrated that kaempferol may be clinically useful due to its ability to reduce or prevent thrombotic challenge.

Platelet-derived MIF: A novel platelet chemokine with distinct recruitment properties

ObjectiveMacrophage migration inhibitory factor (MIF) is an inflammatory cytokine with chemokine-like functions that plays a role in several inflammatory diseases including atherosclerosis. We recently demonstrated that in addition to macrophages and endothelial cells, platelets are a source of MIF. However, the functional relevance of platelet-derived MIF and differences to other platelet chemokines are unclear. Here, we sought to define the secretion pattern of platelet MIF and to characterize its functional profile in comparison with known atherogenic platelet chemokines. Methods and resultsApplying ELISA, we show that MIF is released from thrombin-stimulated platelets after 2 h, whereas CXCL12 and CXCL4 are secreted within minutes. Applied to platelets, MIF, unlike CXCL12, did not enhance platelet activation as analyzed by platelet aggregation, CD62P exposure and chemokine secretion studies. In contrast, both MIF and CXCL12 attenuated ADP-induced calcium transients in platelets. Transmigration and monocyte flow adhesion assays toward conditioned platelet supernatants together with MIF antibody blockade or supernatants from Mif−/− mice suggested that platelet-derived MIF has a stronger chemotactic activity than CXCL12 at its respective optimal secretion interval, and showed that platelet MIF substantially contributes to monocyte adhesion on endothelial layers. Moreover, MIF was found to delay clot retraction. ConclusionsWe demonstrate that MIF differs from other platelet-derived chemokines by delayed secretion kinetics and by a distinct autocrine/paracrine modulation potential. Importantly, MIF was found to be a major platelet-derived chemotactic recruitment factor with clot-modulating properties and therefore might be relevant in inflammatory diseases such as atherosclerosis.

Clinically relevant HOCl concentrations reduce clot retraction rate via the inhibition of energy production in platelet mitochondria

Using porcine blood, we examined the impact of hypochlorite, product of activated inflammatory cells, on clot retraction (CR), an important step of hemostasis. We found that, in vitro, HOCl is able to reduce CR rate and enlarge final clot size in whole blood (t.c. 100 μM), platelet-rich plasma (PRP) threshold concentration (t.c. 50 μM), and an artificial system (washed platelets and fibrinogen) (t.c. 25 nM). Combination of low HOCl and peroxynitrite concentrations resulted in synergistic inhibition of CR by these stressors. Concentrations of HOCl completely inhibiting CR failed to affect the kinetics of coagulation measured in PRP and in platelet-free plasma. Concentrations of HOCl reducing CR rate in PRP augmented production of lactate, inhibited consumption of oxygen by platelets, and decreased total adenosine triphosphate (ATP) content in PRP-derived clots. In an artificial system, concentrations of HOCl resulting in inhibition of CR (25–100 nM) reduced mitochondrial transmembrane potential and did not affect actin polymerization in thrombin-stimulated platelets. These concentrations of HOCl failed to affect the adhesion of washed platelets to fibrinogen and to evoke sustained calcium signal, thus excluding stressor action on glycoprotein IIb/IIIa receptors. Exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Concentrations of HOCl higher than those affecting CR reduced thromboelastometric variables (maximum clot firmness and α angle). We conclude that low clinically relevant HOCl concentrations may evoke the inhibition of CR via the reduction of platelet contractility resulted from malfunction of platelet mitochondria. At the inflammatory conditions, CR may be the predominant HOCl target.

Ginkgolide B inhibits platelet release by blocking Syk and p38 MAPK phosphorylation in thrombin-stimulated platelets

IntroductionAtherosclerosis is a chronic vascular inflammatory disease. Platelets play a critic role in the initiation of vascular inflammation in atherosclerosis. In the present study, we investigated the effects of ginkgolide B on the inhibition of platelet release and the potential mechanisms. MethodsExperiments were performed in freshly human platelets. Platelet aggregation and ATP release were measured with a Lumi-aggregometer. Thrombin (0.5 U/ml) was used to induce platelet activation. Protein expression and phosphorylation was examined by Western blotting. ResultsThe results showed that ginkgolide B significantly suppressed ATP release by 50.8% in thrombin-activated platelets. Ginkgolide B completely abolished the expression of platelet factor 4 (PF4) and CD40 Ligand (CD40L). Moreover, ginkgolide B fully attenuated the phosphorylation of Syk and p38MAPK. Similarly, R788 (a syk inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited the expression PF4 and CD40L, respectively. Furthermore, the combination of low concentrations of ginkgolide B and R788 or SB203580 has synergistic inhibition on the expression of PF4 and CD40L. Ginkgolide B partially reduced calcium efflux by 52.7% in thrombin-stimulated platelets. ConclusionGinkgolide B potently inhibited the expression of PF4 and CD40L in thrombin-activated platelets. Ginkgolide B partially decreased ATP release and Ca2+ efflux. The mechanism might be associated with the inhibition of Syk and p38 MAPK phosphorylation. These results demonstrated that ginkgolide B might be a promising drug on inhibiting platelet function and reducing inflammation in atherosclerosis.

531Platelet-derived deoxyribose-1-phosphate promotes endothelial cell motility and angiogenesis in vivo via redox stimulation

Redox regulation of angiogenesis is an important and emerging topic in vascular sciences; reactive oxygen species (ROS) modulate vascular endothelial cell (EC) proliferation and migration, but the molecular mechanism underlying this phenomenon remains largely unclear. We have shown micromolar concentrations of the pro-angiogenic metabolite deoxyribose-1-phosphate (dRP) detected by mass spectrometry (MS) and nuclear magnetic resonance (NMR) in the supernatants of collagen- and thrombin-stimulated platelets. The genetic silencing of dRP-generating enzymes uridine phosphorylase (UP) and thymidine phosphorylase (TP) in mouse platelets significantly reduced the levels of dRP in platelet supernatants and impaired the ability of protein-free platelet supernatants to enhance human umbilical vein endothelial cell (HUVEC) motility. Chick chorioallantoic membrane (CAM) vascularisation assays performed with protein-free supernatants suggested that the release of dRP by platelets stimulates angiogenesis in vivo. To understand the pro-angiogenic effect of dRP, we investigated further the molecular mechanism of action of dRP and whether this effect was observed in other nucleoside derivatives deoxyribose-5-phospate (dR5P), ribose-1-phosphate (R1P) and deoxyribose (dR). Using live cell real-time confocal imaging and fluorescence assays, HUVEC treatment with dRP significantly increased ROS generation. Similarly, dR5P and R1P also induced ROS accumulation in HUVECs, whereas dR did not. The protein expression of the redox stress marker heme oxygenase-1 (HO-1) and Integrin β3 were shown to be upregulated by dRP and its phosphorylated derivatives (except dR) by immunoblot experiments. Vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), interleukin 8 (IL-8), and stromal cell-derived factor 1α (SDF-1α) showed a similar effect. In parallel, only dRP and its phosphorylated derivatives increased HUVEC cell motility in scratch-healing assays and capillary tube formation using Matrigel™ assays. The causative link between integrin β3 upregulation and increased HUVEC motility/capillary tube formation was proved using integrin-specific inhibitory antibodies'. Furthermore the angiogenic potential of dRP was confirmed in vivo. In summary, dRP and its phosphorylated derivatives were shown to play a relevant role in the paracrine regulation of EC motility and pro-angiogenic activity by up-regulating integrin β3 in a ROS-dependent manner possibly signaling via VEGFR2. dRP is likely to play a role in the stimulation of postnatal angiogenesis and tissue regeneration.

P66Shc mediates increased platelet activation and aggregation in hypercholesterolemia

Background and hypothesisHypercholesterolemia leads to a prothrombotic phenotype. Platelet hyperactivity associated with hypercholesterolemia has been attributed, in part, to oxidative stress. P66Shc is a well-known determinant of cellular and organismal oxidative stress. However, its role in platelet biology is not known. We hypothesized that p66Shc mediates platelet hyperactivation and hyperaggregation in hypercholesterolemia. Methods and resultsP66Shc was expressed in both human and mouse platelets, as determined by qRT-PCR and immunoblotting. Mouse platelet p66Shc expression was upregulated by hypercholesterolemia induced by high-fat diet feeding. Compared to wild-type mice, high-fat diet-induced p66Shc expression in platelets was suppressed in transgenic mice expressing a short hairpin RNA targeting p66Shc (p66ShcRNAi). High-fat diet feeding of wild-type mice amplified surface P-selectin expression on platelets stimulated by the thrombin receptor agonist protease-activated receptor-4 (PAR4), and increased aggregation of platelets induced by thrombin. These exaggerated platelet responses induced by high-fat diet feeding were significantly blunted in p66ShcRNAi mice. Finally, thrombin-stimulated platelet reactive oxygen species were suppressed in p66ShcRNAi mice. ConclusionsHypercholesterolemia stimulates p66Shc expression in platelets, promoting platelet oxidative stress, hyperreactivity and hyperaggregation via p66Shc.

0382: AMPKalpha1 regulates actin polymerization, lamellipodia formation and clot retraction, in thrombin-stimulated platelets

Platelet activation requires sweeping morphological changes, supported by contraction and remodelling of platelet actin cytoskeleton. In epithelial and endothelial cells, AMP-activated protein kinase (AMPK) controls actin cytoskeleton organization through the phorphorylation of cytoskeletal targets, namely myosin regulatory light chains (MLC), cofilin and the vasodilator-stimulated phosphoprotein (VASP), extending the role of AMPK beyond metabolism. In this study, we hypothesized that AMPK was activated in thrombin-stimulated platelets and played a role in platelet secretion, aggregation and clot retraction, by regulating polymerization and/or organization of actin cytoskeleton through the phosphorylation of MLC, cofilin and VASP. Human platelets expressed exclusively the AMPKalpha1 isoform. In human purified platelets, thrombin led to a transient activation of AMPKalpha1 and to phosphorylation of its bona fide substrate, acetyl coA carboxylase (ACC). Platelets isolated from mice lacking AMPKalpha1 exhibited reduced aggregation and secretion response to thrombin, associated with a defect in ACC, MLC, cofilin and VASP phosphorylation. These changes were associated with an abrogration of thrombin-dependent F-actin formation. Moreover, the percentage of platelets able to form lamellipodia after immobilization on fibrinogen-coated coverslips and stimulation by thrombin, was significantly reduced in the absence of AMPKalpha1, indicating an altered cytoskeleton reorganization during spreading. More importantly, clot retraction was slower and less effective in KO platelets. AMPKalpha1 plays a critical role in platelet function in response to thrombin through the phrophorylation of cytoskeletal targets and the subsequent regulation of cytoskeleton organization-dependent processes.

The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.