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

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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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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The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin

Biochemical Journal ◽

10.1042/bj3130401 ◽

1996 ◽

Vol 313 (2) ◽

pp. 401-408 ◽

Cited By ~ 20

Author(s):

Mustapha SI-TAHAR ◽

Patricia RENESTO ◽

Hervé FALET ◽

Francine RENDU ◽

Michel CHIGNARD

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Phospholipase C ◽

Platelet Activation ◽

Inositol Phosphates ◽

Cathepsin G ◽

Dependent Manner ◽

Functional Responses ◽

Kinase C

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 μM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 μM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

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The Gas6 Receptor Axl Enhances Platelet Activation Responses through Stimulation of PI3kinase- and PLC-Dependent Signaling Pathways.

Blood ◽

10.1182/blood.v104.11.630.630 ◽

2004 ◽

Vol 104 (11) ◽

pp. 630-630

Author(s):

Weston R. Gould ◽

Sangita Baxi ◽

Lisa A. Perrin ◽

Robert J. Leadley

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Intracellular Signaling ◽

Human Platelets ◽

Dense Granule ◽

Calcium Mobilization ◽

Dependent Manner ◽

Induced Aggregation ◽

Granule Release ◽

Stimulation Of

Abstract At the site of vascular injury, platelet activation is paramount in supporting formation of a platelet plug and generating a functional surface for the protein elements of coagulation. Recently, we demonstrated that the receptors for the α-granule constituent Gas6, support and enhance platelet aggregation and dense-granule release. The current study examined additional affects of Gas6 signaling in human platelets and sought to decipher intracellular signaling mechanisms initiated by stimulation of Axl, a Gas6 platelet receptor. Flow cytometry analyses indicated that all three Gas6 receptors, Axl, Sky, and Mer were present on the platelet surface. Blockade of Gas6, Sky, or Mer by specific antibodies not only inhibited TRAP- and ADP-induced platelet aggregation and dense granule release, but also prevented thrombin mediated clot retraction by as much as 55%. Furthermore, intracellular calcium mobilization in response to TRAP activation was greater than 80% inhibited in the presence of each of these blocking antibodies. A highly specific antibody directed toward Axl (< 2% cross reactivity with Sky and Mer) activated Axl leading to an enhancement of TRAP and ADP induced aggregation and degranulation. Stimulation of human platelets by this Axl agonist led to a modest, but sustained increase in calcium mobilization suggesting that Axl signaling incorporated activation of PLC. The increase in calcium mobilization was sensitive to wortmannin, demonstrating that PLC activation occurred concurrent with or downstream of PI3K. Indeed, additional experiments to ascertain the intracellular mediators of Axl activity identified a two-fold increase in specific phosphorylation of Akt downstream of PI3K as well as a similar increase in phosphorylation of PLCγ. TRAP stimulation of human platelets also increased the phosphorylation levels of Akt and PLCγ in a Gas6 dependent manner as a Gas6 blocking antibody reduced the levels of Akt and PLCγ phosphorylation by 50%. Overall, these studies suggest that Gas6 enhancement of human platelet activation occurs through the low-level stimulation of the intracellular signaling molecules Akt and PLCγ, serving at the juncture of several mediators of platelet activation. These events also increase levels of cytoplasmic calcium, further supporting an enhancement of activation observed in response to low levels of known platelet agonists. Thus, platelet Gas6 functions to support platelet activation at the very early stages of the hemostatic response to injury.

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In vitro Effects of Aprosulate sodium, a Novel Anticoagulant, On Platelet Activation: Possible Mechanism for Antiplatelet Action

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650661 ◽

1996 ◽

Vol 76 (05) ◽

pp. 786-790 ◽

Cited By ~ 1

Author(s):

Atsuhiro Sugidachi ◽

Norbert Breiter ◽

Taketoshi Ogawa ◽

Fumitoshi Asai ◽

Hiroyuki Koike

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Anticoagulant Activity ◽

Acid Amide ◽

Human Platelets ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free System ◽

Induced Aggregation

SummaryAprosulate sodium, a bis-lactobionic acid amide derivative, is a novel synthetic polyanion with potent anticoagulant activities. In the present study, the effects of aprosulate on platelet aggregation were investigated in a plasma-free system. Aprosulate inhibited thrombin (0.03-0.3 U/ml)-induced aggregation in rat washed platelets in a concentration-dependent manner, with an IC50 value of 0.38 Μg/ml. In contrast, aprosulate, at up to 10 Μg/ml, did not affect collagen (1 Μg/ml) - or ADP (3 ΜM)-induced aggregation. In fura 2-loaded platelets, aprosulate (1-10 Μg/ml) inhibited intracellular Ca2+ mobilization induced by thrombin, but not that by ADP. Protamine, a highly basic protein, abolished aprosulate-mediated inhibition of thrombin-induced platelet aggregation, suggesting that the observed inhibition is primarily due to the negative charge contained on the aprosulate molecule. In human platelets, aprosulate inhibited thrombin-induced aggregation, but failed to inhibit platelet aggregation induced by SFLLRN, a synthetic tethered ligand of a thrombin receptor. Antiplatelet profiles of aprosulate were largely similar to those of heparin, although heparin inhibited both thrombin- and collagen-induced aggregation. These in vitro studies indicate that aprosulate is capable of inhibiting thrombin-induced platelet activation and that this effect is independent of its anticoagulant activity. These results suggest that the polyanionic feature of aprosulate plays an essential role in promoting its antiplatelet activities, and that a plausible mechanism to explain the observed inhibition conferred by this agent, would be one which involves blocking the platelet-thrombin interaction.

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PLATELET-ACTIVATING FACTOR (PAF)-INDUCED INTRACELLULAR Ca MOBILIZATION IN HUMAN PLATELETS.

10.1055/s-0038-1643483 ◽

1987 ◽

Author(s):

K Matsuno ◽

F Katabami ◽

M Koyama ◽

K Abe ◽

K Sakurada ◽

...

Keyword(s):

Platelet Aggregation ◽

Light Transmission ◽

Platelet Rich Plasma ◽

Platelet Activating Factor ◽

Human Platelets ◽

Dependent Manner ◽

Platelet Suspension ◽

Fura 2 ◽

Intracellular Ca ◽

Specific Antagonist

PAF-induced intracellular Ca2+ mobilization and platelet aggregation were investigated in human platelets. Cytosolic free Ca2+ concentration ([Ca2+]cyt) was measured by using fluorescent 45Ca2+ probe quin2 and fura-2, and photoprotein aequorin. Ca2+ uptake was measured after stimulation by PAF. Platelet aggregation was studied by recording the change in light transmission with platelet rich plasma (PRP) or washed platelet suspension (WPS).These three Ca2+ -indicators could determine the elevation of [Ca2+ ]cyt that was stimulated by PAF in the presence of extra- cellular Ca2+ (quin2 method: 98.2nM to 289.7nM; fura-2 method: 102.OnM to 351.4nM; aequorin method: 4.1μM to 8.2μM). In the absence of extracellular Ca2+ , however, little elevation of [Ca2+ ]cyt was detected after stimulation by PAF. PAF could evoke the transient Ca2+ uptake.New PAF specific antagonist, ONO-6240 inhibited PAF-induced platelet aggregation at a concentration from lμM dose-dependently, whereas it didn’t inhibit collagen- and thrombin-induced platelet aggregation at a concentration of lOOyM. ONO-6240 inhibited PAF- induced increase in [Ca2+ ]cyt in a dose-dependent manner as deter mined by these Ca2+ -indicators, as well as platelet aggregation.These results suggest the increase in [Ca2+ ]cyt is responsible for platelet aggregation induced by PAF, and the increased Ca2+ is derived from external Ca2+ influx chiefly.

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Platelet CD36 Induces ERK5 Activation through a Redox-Regulated Signaling Pathway to Promote a Prothrombotic Phenotype

Blood ◽

10.1182/blood.v126.23.1033.1033 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1033-1033

Author(s):

Moua Yang ◽

Jeannette M. Vasquez-Vivar ◽

Yiliang Chen ◽

Scott J. Cameron ◽

Craig N. Morrell ◽

...

Keyword(s):

Myocardial Infarction ◽

Hydrogen Peroxide ◽

Platelet Aggregation ◽

Signaling Pathways ◽

Signaling Pathway ◽

Platelet Activation ◽

Human Platelets ◽

Dependent Manner ◽

Specific Product ◽

Redox Sensor

Abstract Atherothrombosis is a pathological event initiated by dysregulated platelet activation in the setting of unstable or ruptured atherosclerotic plaque, leading to myocardial infarction or stroke. Platelets express scavenger receptor CD36, a receptor of the innate immune system that recognizes endogenous danger signals, such as oxidized low-density lipoprotein (oxLDL), that are generated during the inflammatory and oxidative processes of atherosclerosis. Platelet CD36 induces a prothrombotic phenotype by lowering the threshold for platelet activation by low-dose agonists through multiple signaling pathways involving Src family kinases, mitogen-activated protein kinases (MAPK), Vav-family guanine nucleotide exchange factors, and a pathway that leads to the formation of reactive oxygen species (ROS). Although these signaling pathways contribute to the CD36-mediated prothrombotic phenotype, the mechanisms by which CD36 promotes thrombosis remain incompletely defined. ERK5 is a member of the MAPK family and is a redox sensor that is activated under oxidative stress conditions that can contribute to cardiovascular diseases. ERK5 was recently identified to be present and functional in platelets as a redox sensor that promotes infarct expansion in myocardial infarction. Since the role of ROS generated by CD36 in platelets is unclear, we tested the hypothesis that CD36 promotes thrombosis in atherogenic conditions by generating a redox-regulated signaling pathway requiring ERK5. Initial studies using HPLC to detect 2-hydroxyethidine, the specific product of the reaction between superoxide radical anion (O2●-) and hydroethidine, showed that CD36 induces a two-fold increase in O2●- in washed human platelets in response to oxLDL (p=0.01). We also showed that CD36 signaling generated hydrogen peroxide in human platelets, which is detected by the oxidation of the peroxide probe Amplex Red to the fluorescent product resorufin (p=0.05). We found that hydrogen peroxide has a functional role in CD36-enhanced platelet aggregation using polyethylene glycol-conjugated catalase (PEG-catalase), an enzyme catalyzing the decomposition of intracellular hydrogen peroxide to water. PEG-catalase inhibited platelet aggregation induced by oxLDL by 81.1 ± 4.3%. To determine the mechanism by which ROS promotes the CD36 signaling pathway, we stimulated human platelets with oxLDL and determined the relative phosphorylation level of ERK5 by immunoblot. We found that ERK5 was activated by oxLDL in a time- and dose-dependent manner through CD36. To investigate whether ROS can regulate ERK5 activation, we pretreated washed platelets with PEG-catalase before stimulation with oxLDL. ERK5 phosphorylation in the presence of PEG-catalase was inhibited, suggesting that hydrogen peroxide generated through CD36 signaling is critical for ERK5 activation. To test the functional relevance of ERK5 in the CD36-mediated prothrombotic phenotype, we used the small molecule pharmacologic inhibitors of ERK5 activation, BIX02188 and BIX02189, and found that pre-treatment of platelets with these compounds inhibited platelet activation, secretion, and aggregation in response to oxLDL by 50%-90%. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling via increased O2●- and hydrogen peroxide. This mechanism is mediated by increased activation of MAPK ERK5 to promote platelet activation and a prothrombotic phenotype. Disclosures No relevant conflicts of interest to declare.

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Selective Inhibition of Protease-activated Receptor 4-dependent Platelet Activation by YD-3

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613128 ◽

2002 ◽

Vol 87 (06) ◽

pp. 1026-1033 ◽

Cited By ~ 54

Author(s):

Chin-Chung Wu ◽

Tsong-Long Hwang ◽

Chang-Hui Liao ◽

Sheng-Chu Kuo ◽

Fang-Yu Lee ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Shape Change ◽

Selective Inhibition ◽

Human Platelets ◽

Cathepsin G ◽

Synthetic Compound ◽

Agonist Peptide ◽

Competitive Antagonism ◽

Slow Rise

SummaryIn the present study, the antiplatelet effect and its mechanism of a new synthetic compound YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] were examined. YD-3 inhibited the aggregation of washed human platelets caused by protease-activated receptor (PAR) 4 agonist peptide GYPGKF (IC50 = 0.13 ± 0.02 µM), but had no or little effect on that by thrombin, PAR1 agonist peptide SFLLRN, collagen or U46619. YD-3 produced a parallel, rightward shift of the concentration-response curve for GYPGKF without decreasing of the maximum platelet aggregation, indicating a competitive antagonism. In contrast to human platelets, both thrombin- and GYPGKF-induced mouse platelet shape change and aggregation were completely inhibited by YD-3. YD-3 also selectively prevented GYPGKF-induced intracellular Ca2+ mobilization in human platelets. Furthermore, in the PAR1-desensitized human platelets, thrombin induced a relatively slow rise and decay of calcium mobilization that was significantly inhibited by YD-3. In addition, the synergistic effect of SFLLRN and GYPGKF on platelet activation was prevented by YD-3. YD-3 also inhibits both fMLP-stimulated neutrophil- and purified cathepsin G-induced platelet aggregation, which has been demonstrated to be PAR4-dependent. Taken together, our results suggest that YD-3 selectively inhibits PAR4-dependent platelet activation through blockade of PAR4. To the best of our knowledge, it is the first non-peptide PAR4 antagonist.

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Ni2+ impairs thrombin-induced signal transduction by acting on the agonist and/or receptor in human platelets

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.6.c1681 ◽

1993 ◽

Vol 265 (6) ◽

pp. C1681-C1688 ◽

Cited By ~ 9

Author(s):

F. J. Azula ◽

R. Alonso ◽

A. Marino ◽

M. Trueba ◽

J. M. Macarulla

Keyword(s):

Signal Transduction ◽

Platelet Aggregation ◽

Platelet Activation ◽

Human Platelets ◽

Dependent Manner ◽

Myristate Acetate ◽

Kinase C ◽

Induced Signal ◽

Dose Dependent ◽

Protein Kinase C Activation

We have investigated the effect of NiCl2 on platelet activation induced by thrombin, phorbol 12-myristate 13-acetate, and calcium ionophores. Besides blocking Ca2+ influx, NiCl2 inhibited platelet aggregation, intracellular Ca2+ mobilization, and phospholipase C activation induced by thrombin in a dose-dependent manner. In contrast to ionomycin, NiCl2 completely blocked the platelet aggregation and intracellular Ca2+ mobilization induced by A23187. A23187 was not able to translocate Ni2+ across the plasma membrane. Ni2+ also inhibited phorbol myristate acetate-induced platelet aggregation. The results with staurosporine and low NiCl2 concentrations are in agreement in that increases in intracellular Ca2+ concentration and protein kinase C activation are necessary for full platelet activation mediated by thrombin.

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

Blood ◽

10.1182/blood.v124.21.2763.2763 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2763-2763

Author(s):

Huzoor Akbar ◽

Xin Duan ◽

Saima Saleem ◽

Ashley Kuenzi Davis ◽

Yi Zheng

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Specific Inhibitor ◽

Human Platelets ◽

Small Gtpases ◽

Ros Generation ◽

Dependent Manner ◽

Ros Production ◽

Rhoa Signaling

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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