Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.     

Discrete intracellular Ca2+ pools coupled to two distinct Ca2+ influx pathways in human platelets

Ca(2+) influx is an important event associated with platelet activation and regulated by the content of intracellular Ca(2+). Previous studies have suggested two different Ca(2+) pools and two Ca(2+) influx pathways exist in platelets. In the present study, we have investigated the regulation of thrombin- and thapsigargin-induced Ca(2+) entry into human platelets, using fluorescent indicators to monitor Ca(2+) mobilization and membrane potential. It was found that depletion of thapsigargin-sensitive Ca(2+) stores was coupled to Ca(2+) influx through a Ca(2+)-selective pathway. Additional release of Ca(2+) from the thapsigargin-insensitive pool by thrombin caused the opening of a nonselective cation channel.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets.

[Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.     

Phosphorylation-dependent and -independent pathways of platelet aggregation.

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.     

Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.     

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.     

Inhibitory effects of phenol derivatives on bovine platelet aggregation and their effects on Ca2+ mobilization

The effects of phenol derivatives on aggregation of bovine platelets induced by ADP, thrombin, platelet activating factor, collagen and A23187 were investigated. The phenol derivatives inhibited all these induced aggregations except that by the calcium ionophore. The derivatives each inhibited the aggregations induced by ADP, thrombin, platelet activating factor and collagen, respectively, within a similar concentration range. A linear relation was found between the inhibitory potencies of the phenol derivatives and their partition coefficients between n-octanol and water (Poct values), suggesting that their interaction with hydrophobic regions of the cell was important for inhibition. Fluorescence analyses with fura-2-loaded platelets showed that in the concentration ranges in which the phenol derivatives inhibited aggregation, they also inhibited agonist-induced increases in Ca2+ both in the presence and absence of extracellular Ca2+. Moreover, a high correlation was found between the inhibitory effects of the derivatives on aggregation and their effects on Ca2+ mobilization. These results suggest that inhibition of platelet aggregation by phenol derivatives is mainly due to inhibition of the increase in cytoplasmic Ca2+ by inhibition of both intracellular Ca2+ mobilization and Ca2+ uptake.     

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.     

SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Progesterone metabolites rapidly stimulate calcium influx in human platelets by a src-dependent pathway

The effects of several steroids and their metabolites were examined for their ability to rapidly alter intracellular free calcium ([Ca(2+)](i)) in the anucleate human platelet. Earlier studies suggested that steroids had direct and rapid non-genomic effects to alter platelet physiology. The rationale for performing this study was to investigate the signal transduction events being activated by steroids. Super-physiologic concentrations (1.0-10.0microM) of beta-estradiol and several estradiol metabolites and analogs potentiated (approximately twofold) the action of thrombin to elevate [Ca(2+)](i) in platelets, whereas 10.0microM progesterone inhibited the action of thrombin by 10-15%. Progesterone and beta-estradiol by themselves did not affect [Ca(2+)](i). Progesterone metabolites can achieve high blood concentrations. Some progesterone metabolites, particularly those in the beta-conformation, were potent stimulators of Ca(2+) influx and intracellular Ca(2+) mobilization in platelets. They activated phospholipase C because their ability to increase [Ca(2+)](i) was inhibited by the phospholipase C inhibitor U-73122. The ability of pregnanediol and collagen to increase [Ca(2+)](i) was inhibited by the src tyrosine kinase inhibitor PP1, whereas the actions of thrombin and thapsigargin to increase [Ca(2+)](i) were not affected by PP1. The effects of progesterone metabolites to increase [Ca(2+)](i) were observed with concentrations as low as 0.1microM. Pregnanolone synergized with thrombin to increase [Ca(2+)](i). It is hypothesized that human platelets possess receptors for progesterone metabolites. These receptors when stimulated will activate platelets by causing a rapid increase in [Ca(2+)](i). Pregnanolone, isopregnanediol and pregnanediol were the most effective stimulators of this newly identified src-dependent signal transduction system in platelets. Progesterone metabolites may regulate platelet aggregation and hence thrombosis in vivo.     

Role of the ERK pathway in the activation of store-mediated calcium entry in human platelets

Extracellular signal-regulated kinases (ERKs), are common participants in a broad variety of signal transduction pathways. Several studies have demonstrated the presence of ERKs in human platelets and their activation by the physiological agonist thrombin. Here we report the involvement of the ERK cascade in store-mediated Ca(2+) entry in human platelets. Treatment of dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-loaded platelets with thapsigargin to deplete the intracellular Ca(2+) stores resulted in a time- and concentration-dependent activation of ERK1 and ERK2. Incubation with either U0126 or PD 184352, specific inhibitors of mitogen-activated protein kinase kinase (MEK), prevented thapsigargin-induced ERK activation. Furthermore, U0126 and PD 184352 reduced Ca(2+) entry stimulated by thapsigargin or thrombin, in a concentration-dependent manner. The role of ERK in store-mediated Ca(2+) entry was found to be independent of phosphatidylinositol 3- and 4-kinases, the tyrosine kinase pathway, and actin polymerization but sensitive to treatment with inhibitors of Ras, suggesting that the ERK pathway might be a downstream effector of Ras in mediating store-mediated Ca(2+) entry in human platelets. In addition, we have found that store depletion stimulated ERK activation does not require PKC activity. This study demonstrates for the first time a novel mechanism for regulation of store-mediated Ca(2+) entry in human platelets involving the ERK cascade.     

Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Endogenously expressed Trp1 is involved in store-mediated Ca2+ entry by conformational coupling in human platelets

Physical interaction between transient receptor potential (Trp) channels and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) has been presented as a candidate mechanism for the activation of store-mediated Ca(2+) entry. The role of a human homologue of Drosophila transient receptor potential channel, hTrp1, in the conduction of store-mediated Ca(2+) entry was examined in human platelets. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 557-571 of hTrp1, inhibited both store depletion-induced Ca(2+) and Mn(2+) entry in a concentration-dependent manner. Stimulation of platelets with the physiological agonist thrombin activated coupling between the IP(3) receptor type II and endogenously expressed hTrp1. This event was reversed by refilling of the internal Ca(2+) stores but maintained after removal of the agonist if the stores were not allowed to refill. Inhibition of IP(3) recycling using Li(+) or inhibition of IP(3)Rs with xestospongin C or treatment with jasplakinolide, to stabilize the cortical actin filament network, abolished thrombin-induced coupling between hTrp1 and IP(3)R type II. Incubation with the anti-hTrp1 antibody inhibited thrombin-evoked Ca(2+) entry without affecting Ca(2+) release from intracellular stores. These results provide evidence for the involvement of hTrp1 in the activation of store-mediated Ca(2+) entry by coupling to IP(3)R type II in normal human cells.     

Cinnamtannin B-1 from bay wood reduces abnormal intracellular Ca2+ homeostasis and platelet hyperaggregability in type 2 diabetes mellitus patients

Type 2 diabetes mellitus induces a number of cardiovascular disorders, including platelet hyperactivity and hyperaggregability, which is associated to an increased oxidant production and abnormal cytosolic Ca2+ mobilization. In the present study, we have investigated the effect of cinnamtannin B-1 obtained from bay wood on oxidants production, Ca2+ mobilization and aggregation in platelets from type 2 diabetic donors. Pretreatment of platelets with cinnamtannin B-1 reversed the enhanced oxidants production and Ca2+ mobilization, including Ca2+ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of cinnamtannin B-1 Ca2+ entry was similar in platelets from healthy and diabetic subjects. In addition, cinnamtannin B-1 reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects. We conclude that cinnamtannin B-1 exerts an effective antioxidant action in platelets from patients with type 2 diabetes mellitus and reverses the enhanced Ca2+ mobilization and hyperaggregability.     

Ca2+ influx mediated through the GPIIb/IIIa complex during platelet activation

When aequorin-loaded platelets were stimulated with thrombin, the luminescence signal of aequorin showed two peaks. From experiments with 1 mM external Ca2+ or EGTA, both one-half of the first peak and the entire second peak reflected the influx of Ca2+ from the external medium, and the remaining half of the first peak reflected the mobilization of Ca2+ from its storage site. A monoclonal antibody (TM83) that recognizes the glycoprotein IIb/IIIa (GPIIb/IIIa) complex which has binding sites for fibrinogen and the synthetic peptide GRGDSP are known to inhibit fibrinogen binding and platelet aggregation. Both eliminated the second peak of intracellular free calcium ([Ca2+]i). Similar effects were observed during activation by collagen, but not during PMA activation. It was concluded that the GPIIb/IIIa complex was intimately related to a part of the Ca2+ influx during the activation of platelets.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.