RhoA and Rac1 Gtpases Differentially Regulate Agonist-Receptor Mediated ROS Generation in Platelets

Abstract Agonist induced generation of reactive oxygen species (ROS) including superoxide anion (O-2) and hydrogen peroxide (H2O2) enhance platelet aggregation and hence the risk of thrombosis. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Previous studies have shown that small GTPases Rac1 and RhoA are involved in NOX activation. Rac GTPase activates NOX by directly binding to NOX as well as by interacting with p67phox to promote its binding to NOX (Physiol Rev 87: 245–313, 2007), whereas RhoA triggers ROS generation via the ROCK/p38MAPK cascade mediated phosphorylation of p47phox, a critical component of the NOX complex, (Exp Mol Med 37:575-87, 2005). To date, however, the roles of Rac1 and RhoA in platelet ROS production remain unclear. This study was conducted to define the contributions of Rac1- and RhoA- signaling to ROS generation and platelet function. ROS generation was quantified by flow cytometry in dcf-da (10 µM) loaded washed platelets. Thrombin has been shown to generate ROS in human platelets (Blood 106: 2757-2760, 2005). In this study we confirmed that platelets stimulated with thrombin generate ROS in a time- and a concentration- dependent manner. Addition of thrombin to human platelets pre-treated with NSC23766, a Rac-specific inhibitor, or murine platelets with Rac1 gene deletion, produced significantly less ROS than the matching control samples. Further, Phox-I, a pharmacologic inhibitor of Rac-p67phox interaction (Chem Biol 19: 228-24, 2012), potently suppressed thrombin induced ROS production, indicating that a Rac1-p67phox signaling axis is involved in thrombin mediated ROS production. Separately, treatment of washed human platelets with a RhoA specific inhibitor, Rhosin (Chem Biol 19:699-710, 2012) resulted in: (a) inhibition of the U46619, a stable analog of TXA2, induced activation of RhoA, but not that of Rac1or Cdc42; (b) U46619 induced phosphorylation p38MAPK and p47phox; and (c) U46619 or thrombin induced ROS generation. We further investigated the role of RhoA/ROCK/p38MAPK in ROS production by using platelets from RhoA-/- mice, Y27632 (a ROCK inhibitor) and SB203580 (a p38MAPK inhibitor). RhoA-/- platelets or human platelets treated with Y27632 or SB203580 exhibited significantly diminished ROS generation in response to thrombin. Next, we investigated the physiological effects of Rhosin on platelet activation. A pre-incubation of washed human platelets with Rhosin inhibited U46619 or thrombin induced platelet shape change, release of P-selectin, secretion of ATP and aggregation. The anti-platelet effects of Rhosin were reversible as washing of platelets after incubation with Rhosin abolished the inhibitory effect of Rhosin on platelet aggregation. These results suggest that (a) RhoA signaling, through ROCK/MAPK/p47phox activation, leads to ROS generation and platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC, and (b) Rac1 and RhoA differentially regulate platelet ROS generation by directly binding to NOX, promoting binding of p67phox to NOX and by phosphorylation of p47phox, respectively. Disclosures No relevant conflicts of interest to declare.

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Specific Pharmacologic Targeting of Rho GTPases Rac1, Cdc42 and RhoA Reveals Their Differential and Critical Roles In Regulation of Platelet Activation

Blood ◽

10.1182/blood.v116.21.2019.2019 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2019-2019 ◽

Cited By ~ 1

Author(s):

Huzoor Akbar ◽

Xun Shang ◽

Rehana Perveen ◽

Kevin Funk ◽

Mark Berryman ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Rho Gtpase ◽

Conflicts Of Interest ◽

Small Gtpases ◽

Signaling Cascades ◽

Dependent Manner ◽

Exchange Reactions ◽

Concentration Dependent Manner ◽

Induced Aggregation

Abstract Abstract 2019 Rac1, Cdc42 and RhoA, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton and aggregation in platelets. Although they have been implicated in regulation of platelet activation, the unique and redundant roles of each of the Rho GTPase in various signaling cascades and the resulting functional outcomes have yet to be clearly defined. In this study we compared their roles in several aspects of platelet activation by utilizing three small molecule inhibitors, NSC23766, CASIN, and GO4, that specifically suppress endogenous Rac1, Cdc42, and RhoA activities, respectively. These novel pharmacological inhibitors are active in direct binding to their specific GTPase substrates, i.e. NSC23766 to Rac1, CASIN to Cdc42, and G04 to RhoA, and in interfering with the GTP loading exchange reactions of each Rho GTPase catalyzed by respective guanine nucleotide exchange factors at 5–50 uM concentration range. First, effector-domain pull down assays confirmed that treatment of platelets with NSC23766 (30 uM), CASIN (10 uM) or GO4 (30-50 uM) specifically blocked collagen induced Rac1-GTP, Cdc42-GTP, and RhoA-GTP formations, respectively. Incubation of platelets with NSC23766 (30 uM) or CASIN (10 uM) effectively inhibited collagen-induced phosphorylation of the Rac/Cdc42 effector, PAK1. Addition of GO4 (30 uM) to platelets prior to stimulation with thrombin blocked RhoA/ROCK mediated phosphorylation of myosin light chain (MLC). Second, incubation of aspirin treated platelets containing apyrase (3 U/ml) with CASIN (10 uM), but not NSC23766 (30 uM) or GO4 (30 uM), inhibited filopodia formation on immobilized fibrinogen or collagen-related peptide (CRP), a GPVI agonist. On the other hand, treatment of platelets with CASIN (10 uM) or GO4 (30 uM), but not with NSC23766 (30 uM), inhibited spreading of platelets on immobilized fibrinogen in the presence of aspirin and apyrase. Third, NSC23766 (3-30 uM), CASIN (3-10 uM), and GO4 (5-50 uM) all inhibited secretion from platelet granules and secretion-dependent aggregation induced by threshold concentration of ADP, collagen, CRP, or thrombin in a concentration-dependent manner. However, while CASIN (10 uM) or GO4 (30 uM) completely blocked collagen or CRP induced aggregation in aspirin treated platelets containing apyrase, NSC23766 (30 uM) showed no effect. Fourth, while pre-incubation of platelets with 5 uM CASIN or 10 uM G04 alone only partially (15%) inhibited CRP induced platelet aggregation in aspirin treated samples, CASIN at 10 uM or a combination of 5 uM CASIN and 5 uM G04 were able to inhibit platelet aggregation by 90%. Fifth, GO4 (30 uM) but not CASIN (10 uM) inhibited thrombin stimulated phosphorylation of p38-MAPK (137%) in aspirin treated platelets in the presence of apyrase. Addition of GO4 (30 uM) or CASIN (10 uM) to aspirin treated platelets containing apyrase inhibited CRP induced phosphorylation of ERK1/2 by 94% and 53% respectively, However, in the absence of aspirin and apyrase GO4 (30 uM), but not CASIN (10 uM), completely inhibited CRP induced phosphorylation of ERK1/2. Finally, although both GO4 (30 uM) and CASIN (10 uM) completely inhibited CRP induced phosphorylation of MLC in aspirin treated platelets containing apyrase, GO4 (30 uM) maximally (94%) while CASIN (10 uM) partially (36%) inhibited phosphorylation of MLC in the absence of aspirin and apyrase. Taken together, these data suggest that: (a) Cdc42 is involved in integrin alphaIIbbeta3 and GPVI mediated filopodia formation, RhoA is involved in regulation of integrin alphaIIbbeta3 induced platelet spreading, whereas Rac1 is critical in secondary mediators (ADP/TXA2) mediated lamellipodia formation; (b) Cdc42 and RhoA regulate platelet aggregation in parallel pathways, possibly by affecting the RhoA/ROCK-MAPK-dependent and -independent phosphorylation of MLC; and (c) the crosstalk among Cdc42, Rac1 and RhoA plays an important role in signaling cascades involved in platelet activation. Disclosures: No relevant conflicts of interest to declare.

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Fucoidan Is a Novel Platelet Agonist for CLEC-2 Receptor

Blood ◽

10.1182/blood.v120.21.94.94 ◽

2012 ◽

Vol 120 (21) ◽

pp. 94-94

Author(s):

Bhanukanth Manne ◽

Todd M Getz ◽

Craig Hughes ◽

Carol T Dangelmaier ◽

Steve P Watson ◽

...

Keyword(s):

Platelet Aggregation ◽

Animal Models ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Human Platelets ◽

Lag Phase ◽

Dependent Manner ◽

Wild Type ◽

Concentration Dependent Manner

Abstract Abstract 94 Fucoidan, a sulphated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor (TFPI) (Prasad et al., Blood 111:672, 2008). The decrease in bleeding times in the hemophilia animal models by in vivo administration of fucoidan suggests the beneficial effect of fucoidan as a novel treatment. Furthermore, in vitro studies using platelet poor plasma from hemophilia animal models and human patients has shown that fucoidan inhibits TFPI thereby contributing to an increase in the extrinsic coagulation pathway activity. The effect of fucoidan on platelets however has not been studied. As it is known that the platelet count remains unaffected in hemophilia A patients and bleeding times are primarily measured to assess normal platelet function, we hypothesize that the decrease in bleeding times in the hemophilia animal models may be due to platelet activation by fucoidan. In this study, we demonstrate for the first time that fucoidan induces platelet activation in a concentration dependent manner. Fucoidan-induced platelet activation is completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, and in the absence of Syk and PLC-g2. Furthermore, fucoidan-induced platelet activation has a lag phase, which is reminiscent of platelet activation by collagen and by CLEC-2 receptor agonists. Platelet activation by fucoidan however was only slightly inhibited in FcRg-chain null mice indicating that fucoidan is not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in CLEC-2 deficient mouse platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data shows fucoidan as a novel CLEC-2 receptor agonist that activates platelets through an SFK-dependent signaling pathway. Further, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. . / B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. . / C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. Disclosures: No relevant conflicts of interest to declare.

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GRK2 Regulates ADP Signaling in Platelets Via P2Y 1 and P2Y 12

Blood ◽

10.1182/blood-2021-148615 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 578-578

Author(s):

Xuefei Zhao ◽

Matthew Cooper ◽

Yanki Yanman ◽

Aiden Baltz ◽

James Michael ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Heart Diseases ◽

Specific Inhibitor ◽

Human Platelets ◽

Fine Tuning ◽

Regulatory Role ◽

Platelet Accumulation

Abstract Abstract Venous thromboembolism (VTE), heart attack, and stroke are all diseases in which platelets play a role, through inappropriate platelet activation and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which are targeted by many antiplatelet drugs. Along with thrombin and TxA 2, ADP has long been recognized for its important role in hemostasis and thrombosis. It activates platelets via GPCRs, P2Y 1 and P2Y 12. However, little is known about the negative feedback mechanisms governing P2Y receptor-mediated platelet activation and thrombus formation. Here, we provide the first evidence that GPCR kinase 2 (GRK2) serves this regulatory role during platelet activation and thrombus formation by using a platelet-specific GRK2 deletion mouse model and a GRK2-specific inhibitor in human platelets. Deletion of GRK2 in mouse platelets causes increased platelet accumulation following laser-induced injury in cremaster muscle arterioles, particularly in the shell region of thrombi. In addition, this deletion increases ADP-induced pulmonary thromboembolism. GRK2 -/- platelets also have increased platelet aggregation in response to ADP, but not to PAR4 receptor agonist, TxA 2, or convulxin. Underlying these changes in GRK2 -/- platelets is an increase in Ca 2+ mobilization, Akt phosphorylation, and Rap1 activation in response to ADP, and an attenuated rise of cAMP levels in response to ADP in the presence of prostaglandin I 2. Furthermore, platelet aggregation can be restored in GRK2 -/- platelets in response to ADP re-stimulation, indicating that GRK2 contributes to ADP receptor desensitization. To further assess the role of GRK2 in the P2Y 12 signaling pathway in vivo, we examine laser-induced thrombus formation in WT and GRK2 -/- mice treated with the P2Y 12 antagonist, cangrelor. Cangrelor treatment eliminates the phenotypic difference in platelet accumulation between WT and GRK2 -/- mice in response to injury. Using a specific GRK2 inhibitor, pharmacologic inhibition of GRK2 activity in human platelets results in an increase in platelet activation in response to ADP. Finally, our biochemical studies show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Taken together, we have demonstrated for the first time that 1) GRK2 plays a negative regulatory role in platelet activation by attenuating ADP-dependent signaling, 2) it does this by limiting P2Y 1 and P2Y 12-mediated signaling, 3) GRK2 interacts with Gβγ and functions as a signaling hub in platelets for fine-tuning GPCR signaling, and 4) although the potential inhibition of GRK2 can be beneficial for treatment of heart diseases, maintaining GRK2 activity in platelets could be beneficial for prevention of thrombotic diseases. Disclosures No relevant conflicts of interest to declare.

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The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽

10.1182/blood.v108.11.3904.3904 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3904-3904

Author(s):

Samantha Baldassarri ◽

Alessandra Bertoni ◽

Paolo Lova ◽

Stefania Reineri ◽

Chiara Sarasso ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Cannabinoid Receptors ◽

Thromboxane A2 ◽

Human Platelets ◽

Dependent Manner ◽

Cb2 Receptor ◽

Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

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Genetic and Pharmacologic Evidence Shows That Cdc42 GTPase Plays a Central Role in the Regulation of Both GPVI- and Non-GPVI-Dependent Activation of Platelets.

Blood ◽

10.1182/blood.v114.22.2997.2997 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2997-2997 ◽

Cited By ~ 1

Author(s):

Huzoor Akbar ◽

Kevin Funk ◽

Mark Berryman ◽

Joshua Raines ◽

Rehana Perveen ◽

...

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Conflicts Of Interest ◽

Therapeutic Potential ◽

Shape Change ◽

Specific Inhibitor ◽

Small Gtpases ◽

Clot Retraction ◽

Coated Surfaces ◽

Induced Aggregation

Abstract Abstract 2997 Poster Board II-975 Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton in platelets. Previously we have shown that Rac1 GTPase is involved in regulation of platelet secretion and aggregation by diverse signaling pathways (J Thromb Haemost 2007; 5: 1747-55). Others have reported that Rac1 is essential for GPVI-, but not G protein-dependent platelet aggregation (Pflugers Arch. 2009; 457:1173-85). Cdc42 was recently reported to be involved in collagen, but not collagen related peptide (CRP), a GPVI specific agonist, induced platelet aggregation (Platelets 2008; 19: 199-210). In this study we have investigated the role of Cdc42 in regulation of platelet function by using complementary approaches of (a) mouse gene targeting of Cdc42, and (b) specific inhibition of Cdc42 activity by a newly identified chemical inhibitor of Cdc42, CASIN (Cdc42 activity-specific inhibitor). Platelets from Cdc42−/− mice exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Threshold concentrations of collagen, CRP or thrombin failed to induce shape change or aggregation in platelets from Cdc42−/− mice compared with induction of shape change and maximal aggregation in platelets from Cdc42+/+ mice. Platelets from Cdc42−/− mice, as compared to Cdc42+/+ mice, exhibited a significant inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin from the dense- and alpha-granules respectively. Increasing concentrations of the agonists only partially corrected the defective aggregation and secretion responses in Cdc42−/− platelets. These data provide the genetic evidence that Cdc42 is required for collagen, CRP and thrombin mediated platelet signaling and activation. Treatment of platelets with CASIN, but not a pharmacologically inactive analog, blocked collagen induced activation of Cdc42 without detectably affecting the Rac1 activity. Human platelets pre-incubated with CASIN (10 micro-M) exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Further, treatment of platelets with CASIN (1-10 micro-M) inhibited: (a) aggregation induced by collagen, CRP, thrombin, ADP or U46619; (b) release of P-selectin and secretion of ATP induced by U46619; and (c) collagen induced phosphorylation of Akt. Addition of CASIN to platelets also blocked collagen or CRP induced aggregation in aspirinated platelets in the presence of apyrase. In other experiments, addition of CASIN to citrated platelet-rich plasma inhibited thrombin induced clot retraction. Significantly, removal of CASIN from the platelet samples by washing reversed inhibition of aggregation as well as clot retraction, reflecting a reversible suppression of Cdc42 activity by CASIN. Administration of CASIN into C57Bl/6 mice inhibited ex vivo platelet aggregation induced by collagen or ADP as well as significantly prolonged tail bleeding times. These data suggest that: (a) Cdc42 plays an essential, non-redundant role in platelet filopodia formation, spreading, secretion, aggregation and clot retraction; (b) Cdc42 is involved in GPVI, non-GPVI- and G protein-dependent signaling in platelets; (c) the pharmacologic inhibitor CASIN is capable of specifically and reversibly inhibiting Cdc42 activity in platelets, mimicking Cdc42 genetic knockout in mice. Altogether, our studies strongly implicate Cdc42 as a novel anti-platelet target, and present evidence that the Cdc42 specific small molecule inhibitor, CASIN, may have therapeutic potential. Disclosures: No relevant conflicts of interest to declare.

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An Important Role for Rac1 and RhoA In Regulating Platelet Microparticle Formation and Phosphatidylserine Exposure

Blood ◽

10.1182/blood.v116.21.2023.2023 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2023-2023

Author(s):

Michael Keegan Delaney ◽

Junling Liu ◽

Yi Zheng ◽

Xiaoping Du

Keyword(s):

Platelet Aggregation ◽

Conflicts Of Interest ◽

Human Platelets ◽

Small Gtpases ◽

Procoagulant Activity ◽

Null Mouse ◽

Signaling Mechanisms ◽

Granule Secretion ◽

Microparticle Formation

Abstract Abstract 2023 Platelets activated by physiological agonists such as thrombin and collagen shed procoagulant microparticles (MPs) and externalize the procoagulant phospholipid phosphatidylserine (PS), both of which are critical to hemostasis and play an important role in inflammation. To date, the signaling mechanisms that regulate agonist-induced MP formation and PS exposure in platelets remain unclear. In this study, we demonstrate that the small GTPases Rac1 and RhoA play important roles in regulating the procoagulant activity of platelets. Rac1 null (-/-) mouse platelets or human platelets treated with the Rac1 inhibitor NSC23766 (NSC) displayed a significant defect in MP formation and PS exposure induced by various agonists. Furthermore, Rac1-/- platelets and NSC-treated human platelets displayed a defect in procoagulant activity as demonstrated by a prolonged coagulation time following recalcification of citrated PRP. The stimulatory role of Rac1 in platelet MP formation and PS exposure is distinct from the known function of Rac1 in facilitating platelet granule secretion and secretion-dependent amplification of platelet aggregation, because supplementation of the granule content ADP rescued the defect in platelet aggregation caused by Rac1 inhibition, but failed to rescue the defect in MP formation caused by Rac1 inhibition. In contrast to Rac1, RhoA plays an inhibitory role in regulating platelet procoagulant activity, because treatment of platelets with the Rho inhibitor C3-toxin (C3) significantly enhanced agonist-induced MP formation, PS exposure, and procoagulant activity. The enhancing effect of C3 on platelet procoagulant activity is not caused by an overall enhancement of platelet activation because C3 significantly inhibited platelet secretion and aggregation. Thus, our data demonstrates that while Rac1 and RhoA both play important stimulatory roles in platelet granule secretion and aggregation, they play opposing roles in MP formation and PS exposure in platelets. Rac1 is important for stimulating platelet MP formation, PS exposure, and procoagulant activity, which is antagonized by RhoA. Disclosures: No relevant conflicts of interest to declare.

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The GPIIbIIIa Antagonist Drugs Eptifibatide and Tirofiban Inhibit Caspase-3 Activation in Thrombin- and Calcium Ionophore-Stimulated Human Platelets.

Blood ◽

10.1182/blood.v114.22.2998.2998 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2998-2998

Author(s):

Valery Leytin ◽

Asuman Mutlu ◽

Sergiy Mykhaylov ◽

David J. Allen ◽

Armen V. Gyulkhandanyan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Calcium Ionophore ◽

Human Platelets ◽

Shear Stresses ◽

Ionophore A23187 ◽

Platelet Surface ◽

Apoptotic Effects

Abstract Abstract 2998 Poster Board II-976 Introduction: The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods: The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion: We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P>0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P<0.01, n=7). However, when platelets were preincubated with eptifibatide and tirofiban before agonist treatment, these drugs significantly inhibited agonist-induced caspase-3 activation by an average of 44-50% (P<0.05, n=7). The fact that eptifibatide and tirofiban do not promote caspase-3 activation in unstimulated platelets suggests that these GPIIbIIIa antagonists do not induce transmission of pro-apoptotic transmembrane signals inside platelets through inactive GPIIbIIIa integrin. The inhibitory effect of eptifibatide and tirofiban on thrombin- and A23187-induced caspase-3 activation suggests a role of GPIIbIIIa integrin in caspase-3 activation induced by these platelet agonists. Conclusions: We have demonstrated a novel platelet-directed activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet agonists, thrombin and A23187, and the absence of adverse pro-apoptotic effects on resting platelets. Taken together with earlier reported data (Leytin et al, Br J Haematol 133: 78, 2006), the current study indicates that, aside from their well-known participation in platelet activation and aggregation, GPIIbIIIa receptors are involved in the modulation of platelet apoptosis. This GPIIbIIIa-mediated mechanism of apoptosis modulation may be very efficient given the extremely large number of GPIIbIIIa copies (≈80,000) on the platelet surface. Disclosures: No relevant conflicts of interest to declare.

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Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v122.21.3513.3513 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3513-3513

Author(s):

Wenxiu Yi ◽

Wei Li ◽

Lijie Ren ◽

Xinliang Mao ◽

Li Zhu

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Venous Blood ◽

Pi3k Pathway ◽

Dependent Manner ◽

Clot Retraction ◽

Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

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Intracellular Ca2+ rise in human platelets induced by polymorphonuclear-leucocyte-derived cathepsin G

Biochemical Journal ◽

10.1042/bj2880741 ◽

1992 ◽

Vol 288 (3) ◽

pp. 741-745 ◽

Cited By ~ 18

Author(s):

M Molino ◽

M Di Lallo ◽

G de Gaetano ◽

C Cerletti

Keyword(s):

Plasma Membrane ◽

Platelet Aggregation ◽

Platelet Activation ◽

Human Platelets ◽

Polymorphonuclear Leucocyte ◽

Cation Channel ◽

Cathepsin G ◽

Dependent Manner ◽

Bivalent Cation ◽

Platelet Suspension

Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.

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A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽

10.1182/blood.v126.23.10.10 ◽

2015 ◽

Vol 126 (23) ◽

pp. 10-10

Author(s):

Jose Perdomo ◽

Jaa Yien New ◽

Zohra Ahmadi ◽

Xing-Mai Jiang ◽

Beng H Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Immune Complexes ◽

Whole Blood ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Single Chain ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Micro Fluidics

Abstract Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions. Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec. Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1). Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition. Disclosures No relevant conflicts of interest to declare.

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