Human Platelet Secretion Research Articles

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

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

The CEACAM1-derived peptide QLSN impairs collagen-induced human platelet activation through glycoprotein VI.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates collagen-mediated platelet activation through its cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). However, the function of CEACAM1's extracellular cleavage fragments is currently unknown. In the present study, we used mass spectrometry (MS) to identify 9 cleavage fragments shed by matrix metallopeptidase 12 (MMP-12), and then we synthesized peptides with sequences corresponding to the fragments. QLSNGNRTLT (QLSN), a peptide from the A1-domain of CEACAM1, significantly attenuated collagen-induced platelet aggregation. QLSN also attenuated platelet static adhesion to collagen. Additionally, QLSN reduced human platelet secretion and integrin αIIbβ3 activation in response to glycoprotein VI (GPVI)-selective agonist, convulxin. Correspondingly, QLSN treatment significantly decreased convulxin-mediated phosphorylation of Src, protein kinase B (Akt), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) in human platelets. These data indicate that the CEACAM1-derived peptide QLSN inhibits GPVI-mediated human platelet activation. QLSN could potentially be developed as a novel antiplatelet agent.

Comparative study of platelet aggregation and secretion induced by Bothrops jararaca snake venom and thrombin

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 μg/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1−/−) platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced β-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

The disulfide isomerase ERp72 supports arterial thrombosis in mice

AbstractSeveral CGHC motif–containing disulfide isomerases support thrombosis. We here report that endoplasmic reticulum protein 72 (ERp72), with 3 CGHC redox-active sites (ao, a, and a′), supports thrombosis. We generated a new conditional knockout mouse model and found that Tie2-Cre/ERp72fl/fl mice with blood and endothelial cells lacking ERp72 had prolonged tail bleeding times and decreased platelet accumulation in laser-induced cremaster arteriole injury and FeCl3-induced mesenteric arterial injury. Fibrin deposition was decreased in the laser injury model. Both platelet and fibrin accumulation defects were fully rescued by infusion of recombinant ERp72 containing functional a and a′ CGHC motifs (ERp72(oo-ss-ss)). Infusion of ERp72 containing inactivated a and a′ CGHC motifs (ERp72(ss-oo-oo)) inhibited platelet accumulation and fibrin deposition in wild-type mice. Infusion of ERp72(oo-ss-ss) into β3-null mice increased fibrin deposition in the absence of platelets. ERp72-null platelets had defective aggregation, JON/A binding, P-selectin expression, and adenosine triphosphate (ATP) secretion. The aggregation and ATP secretion defects were fully rescued by ERp72(oo-ss-ss) but partially rescued by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). Aggregation and ATP secretion of human platelets was potentiated by ERp72(oo-ss-ss) but inhibited by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). These data suggest that both the a and a′ active sites are required for platelet function. ERp72 bound poorly to β3-null mouse platelets, and the addition of ERp72(oo-ss-ss) to human platelets generated thiols in αIIbβ3, suggesting a direct interaction of ERp72 with αIIbβ3. Defective aggregation of ERp72-null platelets was recovered by ERp72, but not other thiol isomerases. In summary, ERp72 plays a critical role in platelet function and coagulation through the a and a′ CGHC motifs.

NF-κB Links TLR2 and PAR1 to Soluble Immunomodulator Factor Secretion in Human Platelets.

The primary toll-like receptor (TLR)-mediated immune cell response pathway common for all TLRs is MyD88-dependent activation of NF-κB, a seminal transcription factor for many chemokines and cytokines. Remarkably, anucleate platelets express the NF-κB machinery, whose role in platelets remains poorly understood. Here, we investigated the contribution of NF-κB in the release of cytokines and serotonin by human platelets, following selective stimulation of TLR2 and protease activated receptor 1 (PAR1), a classical and non-classical pattern-recognition receptor, respectively, able to participate to the innate immune system. We discovered that platelet PAR1 activation drives the process of NF-κB phosphorylation, in contrast to TLR2 activation, which induces a slower phosphorylation process. Conversely, platelet PAR1 and TLR2 activation induces similar ERK1/2, p38, and AKT phosphorylation. Moreover, we found that engagement of platelet TLR2 with its ligand, Pam3CSK4, significantly increases the release of sCD62P, RANTES, and sCD40L; this effect was attenuated by incubating platelets with a blocking anti-TLR2 antibody. This effect appeared selective since no modulation of serotonin secretion was observed following platelet TLR2 activation. Platelet release of sCD62P, RANTES, and sCD40L following TLR2 or PAR1 triggering was abolished in the presence of the NF-κB inhibitor Bay11-7082, while serotonin release following PAR1 activation was significantly decreased. These new findings support the concept that NF-κB is an important player in platelet immunoregulations and functions.

Nicergoline inhibits human platelet Ca(2+) signalling through triggering a microtubule-dependent reorganization of the platelet ultrastructure.

Recently, we demonstrated that a pericellular Ca(2+) recycling system potentiates agonist-evoked Ca(2+) signalling and granule secretion in human platelets and hypothesized a role for the membrane complex (MC) in orchestrating the accumulation of Ca(2+) in the pericellular region. Previous work has demonstrated that treatment with high concentrations of nicergoline may disrupt the MC through an ability to trigger a re-organization of the dense tubular system. Experiments were therefore performed to assess whether nicergoline-induced changes in platelet ultrastructure affects thrombin-evoked Ca(2+) fluxes and dense granule secretion. Thrombin-evoked Ca(2+) fluxes were monitored in Fura-2- or Fluo-5N-loaded human platelets, or using platelet suspensions containing Fluo-4 or Rhod-5N K(+) salts. Fluorescence microscopy was utilized to monitor microtubule structure and intracellular Ca(2+) store distribution in TubulinTracker- and Fluo-5N-loaded platelets respectively. Dense granule secretion was monitored using luciferin-luciferase. Nicergoline treatment inhibited thrombin-evoked Ca(2+) signalling and induced alterations in the microtubule structure and the distribution of intracellular Ca(2+) stores in platelets. Nicergoline altered the generation and spreading of thrombin-induced pericellular Ca(2+) signals and almost completely prevented dense granule secretion. Stabilization of microtubules using taxol reversed most effects of nicergoline on platelet Ca(2+) signalling and partially reversed its effects on dense granule secretion. Nicergoline-induced alterations to platelet ultrastructure disrupt platelet Ca(2+) signalling in a manner that would be predicted if the MC had been disrupted. These data suggest that nicergoline may be a useful prototype for the discovery of novel MC-disrupting anti-thrombotics.

A critical role for the transient receptor potential channel type 6 in human platelet activation.

While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE) remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6) mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR)-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules), integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.

Protein phosphatase inhibitor calyculin – A modulates activation markers in TRAP-stimulated human platelets

Platelet activation is accompanied with the phosphorylation of a number of proteins on serine (Ser) and threonine (Thr) residues. The phosphorylation level of these proteins is dependent upon the protein kinase/phosphatase activity ratio. The aim of this study was to investigate the consequences of inhibiting protein phosphatase 1 (PP1) and 2A (PP2A) on platelet functions. Protein phosphatases were inhibited by preincubation of platelet rich plasma (PRP) samples with calyculin-A (CLA). Subsequently, platelets were activated by thrombin-receptor activating peptide (TRAP) and platelet aggregation, platelet-derived microparticle (PMP) formation, surface expressions of P-selectin (CD62), lysosome-associated membrane protein (CD63), glycoprotein Ib and IIb were examined. Phosphatase activity was determined by using phosphorylated 20 kDa myosin light chain (P-MLC20) as substrate. In CLA-treated platelets substantial decrease of P-MLC20 phosphatase activity was observed. CLA significantly suppressed TRAP-induced surface expression of P-selectin and CD63 in a concentration-dependent manner as compared to non-treated samples and moderately decreased platelet aggregation. In TRAP-activated samples, 50 nM of CLA pretreatment completely abolished the level of PMPs and the prevention of GPIb downregulation was also observed; however, no difference was found in GPIIb expression. In conclusion, PP1 and PP2A-catalyzed dephosphorylation processes have crucial roles in PMP formation and in the regulation of alpha-granule and lysosome secretion in human platelets.

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

Tyrosine phosphorylation / dephosphorylation balance is involved in thrombin-evoked microtubular reorganisation in human platelets

We have investigated the intracellular mechanisms involved in microtubular remodelling by thrombin and its possible involvement in platelet aggregation and secretion. Platelet stimulation with thrombin induces a time- and concentration-dependent regulation of the microtubular content, which was found to be maximally effective at the concentration 0.1 U/ml. Thrombin (0.1 U/ml) evoked an initial decrease in the microtubule content detectable at 5 seconds (sec) and reached a minimum 10 sec after stimulation. The microtubular content then increased, exceeding basal levels again approximately 30 sec after stimulation. Inhibition of tyrosine phosphatases using vanadate abolished thrombin-induced microtubular depolymerisation while inhibition of tyrosine kinases by methyl-2,5-dihydroxycinnamate prevented microtubule polymerisation. Thrombin activates the cytosolic Bruton's tyrosine kinase (Btk) and Src proteins. Inhibition of Btk or Src by LFM-A13 or PP1, respectively, abolished thrombin-induced microtubular polymerisation, while maintaining intact its ability to induce initial depolymerisation. Microtubular disruption by colchicine significantly reduced thrombin-induced platelet aggregation and ATP secretion. Similar results were observed after inhibition of microtubular disassembly by paclitaxel. These findings indicate that thrombin induces microtubular remodelling by modifying the balance between protein tyrosine phosphorylation and dephosphorylation. The former seems to be required for microtubular polymerisation, while tyrosine dephosphorylation is required for microtubular depolymerisation. Both, initial microtubular disassembly and subsequent polymerisation are required for thrombin-induced platelet aggregation and secretion in human platelets.

Stimulatory Roles of Nitric-oxide Synthase 3 and Guanylyl Cyclase in Platelet Activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.

The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.

Differential Role of Protein Kinase Cδ Isoform in Agonist-induced Dense Granule Secretion in Human Platelets

Several platelet agonists, including thrombin, collagen, and thromboxane A(2), cause dense granule release independently of thromboxane generation. Because protein kinase C (PKC) isoforms are implicated in platelet secretion, we investigated the role of individual PKC isoforms in platelet dense granule release. PKCdelta was phosphorylated in a time-dependent manner that coincided with dense granule release in response to protease-activated receptor-activating peptides SFLLRN and AYPGKF in human platelets. Only agonists that caused platelet dense granule secretion activated PKCdelta. SFLLRN- or AYPGKF-induced dense granule release and PKCdelta phosphorylation occurred at the same respective agonist concentration. Furthermore, AYPGKF and SFLLRN-induced dense granule release was blocked by rottlerin, a PKCdelta selective inhibitor. In contrast, convulxin-induced dense granule secretion was potentiated by rottlerin but was abolished by Go6976, a classical PKC isoform inhibitor. However, SFLLRN-induced dense granule release was unaffected in the presence of Go6976. Finally, rottlerin did not affect SFLLRN-induced platelet aggregation, even in the presence of dimethyl-BAPTA, indicating that PKCdelta has no role in platelet fibrinogen receptor activation. We conclude that PKCdelta and the classical PKC isoforms play a differential role in platelet dense granule release mediated by protease-activated receptors and glycoprotein VI. Furthermore, PKCdelta plays a positive role in protease-activated receptor-mediated dense granule secretion, whereas it functions as a negative regulator downstream of glycoprotein VI signaling.

Platelet α-granule secretion and its modification by SC-57101A, a GPIIb/IIIa antagonist

Platelet–agonist interaction results in aggregatory and secretory responses. While the activation of glycoprotein (GP) IIb/IIIa plays an essential role in platelet aggregation, its role in granule secretion is not clear. The present study was performed to examine the effect of 3-[[[[1-[4-(aminoiminomethyl) phenyl]-2-oxo-3 S-pyrrolidinyl]amino]carbonyl]amino]-propanoate monohydrochloride salt (SC-57101A), a GPIIb/IIIa antagonist, on platelet α-granule secretion responses to collagen, ADP, and thrombin receptor activating peptide (TRAP). Both SC-57101A and prostaglandin E 1 (PGE 1) inhibited collagen-, ADP-, and TRAP-induced platelet aggregation in a concentration-dependent manner. SC-57101A inhibited the collagen- and ADP-induced release of platelet-derived growth factor (PDGF) and β-thromboglobulin (β-TG) from platelets, but not TRAP-induced secretion of these granule contents. On the other hand, PGE 1 inhibited the release of PDGF and β-TG from platelets activated with all the agonists used. ADP and TRAP elicited P-selectin expression in the absence of platelet aggregation, while collagen produced no such reaction. SC-57101A only moderately inhibited P-selectin expression induced by ADP and had no inhibitory effect on that induced by TRAP. The inhibition of ADP-induced secretion of α-granule contents by SC-57101A was abolished when platelets were pretreated with aspirin. These results suggest that GPIIb/IIIa activation plays a minor role, if any, in α-granule secretion in human platelets.

Platelet Activation by von Willebrand Factor Requires Coordinated Signaling through Thromboxane A2 and FcγIIA Receptor

Interaction of von Willebrand Factor with glycoprotein Ib-IX-V induces platelet activation through a still poorly defined mechanism. Previous studies have suggested a possible role for the low affinity receptor for immunoglobulin, Fc gamma RIIA, in GPIb-IX-V signaling. Here we show that binding of vWF to platelets induces the tyrosine phosphorylation of Fc gamma RIIA by a Src kinase. Treatment of platelets with the anti-Fc gamma RIIA monoclonal antibody IV.3 specifically inhibits vWF-induced but not thrombin-induced pleckstrin phosphorylation and serotonin secretion. Moreover, vWF fails to induce pleckstrin phosphorylation in mouse platelets, lacking Fc gamma RIIA, and serotonin secretion is impaired. Pleckstrin phosphorylation and serotonin secretion in human platelets stimulated with vWF are blocked by the cyclooxygenase inhibitor acetylsalicylic acid. However, release of arachidonic acid and synthesis of TxA(2) induced by vWF are not affected by the anti-Fc gamma RIIA monoclonal antibody IV.3. Similarly, vWF-induced tyrosine phosphorylation of Fc gamma RIIA, as well as of Syk and PLC gamma 2, occurs normally in aspirinized platelets. Inhibition of the tyrosine kinase Syk by piceatannol does not affect vWF-induced tyrosine phosphorylation of Fc gamma RIIA but prevents phosphorylation of PLC gamma 2. Pleckstrin phosphorylation and platelet secretion induced by vWF, but not by thrombin, are also inhibited by piceatannol. Pleckstrin phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. These results indicate that PLC gamma 2 plays a central role in platelet activation by vWF and that the stimulation of this enzyme requires coordinated signals through endogenous TxA(2) and Fc gamma RIIA.

Protein kinase C–catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion

Protein kinase C (PKC)-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1delta catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A(2) analog STA(2) resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca(++) ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2-loaded platelets with no change in cytosolic Ca(++). The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC(20)) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC(20) with similar IC(50) values. These findings suggest that the activation of PKC by PMA induces MLC(20) phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI. STA(2)-induced MLC(20) phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA(2)-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor Y-27632 and GF109203X led to a further decrease in STA(2)-induced MLC(20) phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696. Inhibition of STA(2)-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC(20) phosphorylation. Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase-mediated regulation of myosin phosphatase, during agonist-induced platelet secretion. (Blood. 2001;97:3798-3805)

Elevation of cyclic AMP decreases phosphoinositide turnover and inhibits thrombin-induced secretion in human platelets

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-specific phospholipase C (PLC) by ill-defined mechanism(s). Signal transduction steps downstream of PLC are inhibited by elevated cAMP, suggesting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [ 32P] i-prelabeled platelets, forskolin increased intracellular cAMP (104 nmol/10 11 cells at 10 −5 M forskolin) and [ 32P]phosphatidylinositol 4-phosphate (Δ[ 32P]PIP) (30% at 10 −7–10 −6 M forskolin). The thrombin-induced (0.1 U/ml) increase in production of [ 32P]PA, ‘overshoots’ in [ 32P]PIP and [ 32P]PIP 2 ([ 32P]phosphatidylinositol 4,5-bisphosphate), and the increase in [ 32P]PI and secretion of ADP+ATP were abolished by forskolin (10 −7 M). Forskolin stimulated total [ 32P]P i uptake in resting platelets (48%), increased 32P incorporation into PIP (110%), and inhibited 32P incorporation into PI (50%). The latter inhibition was most likely considerably greater due to the forskolin-induced stimulation of [ 32P]P i uptake. The changes in radioactive PA, PIP and PIP 2 are regarded as being proportional with their masses in the prelabeled platelets, while the increase in PI (phosphatidylinositol) is regarded as a change in specific radioactivity, and hence in its synthesis. The results suggest that cAMP elevation inhibits the flux in the polyphosphoinositide cycle through both inhibition of PIP 5-kinase and PI synthesis. The inverse relation between forskolin-produced ΔPIP and [ 32P]PA production suggests that the PLC reaction is inhibited by elevated cAMP through reduction of substrate (PIP 2) resynthesis, and not by inhibition of the PLC enzyme.

Accumulation of Unphosphorylated Calponin in the Submembranous Cytoskeletons of Arachidonic Acid-stimulated Human Platelets

Calponin, a basic smooth-muscle protein capable of binding to F-actin, tropomyosin and calmodulin in vitro, was tested for its expression and subcellular localization in resting and stimulated human platelets. Using immunoblotting techniques calponin was revealed as a single protein band with a molecular weight of 34 kDa. Although calponin has been shown to be proteolytically degraded by calpain, in the presence of the calpain inhibitor E-64 and EGTA a significant hydrolysis of calponin could not be detected. Upon stimulation with 10 microM arachidonic acid calponin became increasingly incorporated into Triton X-100 insoluble cytoskeletal fractions reaching a plateau after 15 s. The accumulation of calponin in the cytoskeletons of stimulated platelets paralleled the polymerization of actin into newly formed microfilaments. Immunofluorescence microscopy revealed a submembranous co-localization of calponin and actin in aggregated platelets. Since isolated calponin is phosphorylated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II thereby losing its inhibitory effect on the actomyosin MgATPase activity, we examined whether changes in cell shape due to platelet stimulation are accompanied by a phosphorylation of calponin. By performing immunoblotting analysis on either resting or stimulated platelets phosphorylation of calponin on tyrosine, serine or threonine residues could not be demonstrated. In line, [32P]radiolabeling experiments were unable to detect phosphate incorporation into calponin. These observations support the hypothesis that calponin plays a physiological role in regulating contraction and secretion of human platelets even in the absence of its phosphorylation.

An antibody against the exosite of the cloned thrombin receptor inhibits experimental arterial thrombosis in the African green monkey.

Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation. The immune IgG (IgG 9600) inhibited thrombin-stimulated aggregation and secretion of human platelets. In contrast, it had no effect on platelet activation induced by other agonists including ADP, collagen, or the thrombin receptor-derived peptide SFLLR-NH2. IgG 9600 also inhibited thrombin-induced aggregation of African Green monkey (AGM) platelets. By Western blot analysis, the IgG identified a protein of approximately 64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antibody on arterial thrombosis was evaluated in an in vivo model of platelet-dependent cyclic flow reductions (CFRs) in the carotid artery of the AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth monkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thrombin was completely inhibited during the 120-minute postbolus observation period in all four animals. There was a twofold increase in bleeding time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin inhibitor recombinant hirudin also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effect on CFRs, ex vivo platelet aggregation, bleeding time, or APTT. These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.

Possible Involvement of Protein Kinase C and Low Molecular Weight GTP-Binding Proteins in Thrombin-Induced Histamine Secretion in Human Platelets

Thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA) caused histamine secretion from human platelets. To clarify the intracellular signalling mechanism of thrombin-induced histamine secretion, the effects of pertussis toxin (PTX) and botulinus toxin (BTX) on thrombin- and TPA-induced histamine secretion were examined in human platelets. The secretion by thrombin was sensitive to BTX, but not PTX. The secretion by TPA was also inhibited by BTX. These results suggest that protein kinase C and low molecular weight G-proteins sensitive to BTX are involved in histamine secretion.