Differential role of protein kinase C delta 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.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway

Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.     

HSP27 phosphorylation is correlated with ADP-induced platelet granule secretion

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. ADP has been reported to induce heat-shock protein (HSP) 27 phosphorylation in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanisms and the roles of ADP-induced HSP27 phosphorylation in human platelets. We showed for the first time that both of decreased phosphorylation levels of HSP27 by PD98059, a MEK1/2 inhibitor and SB203580, a p38 MAPK inhibitor were correlated with the suppressed levels of platelet granule secretion but not with platelet aggregation. Furthermore, the inhibition of either the p44/p42 MAPK or p38 MAPK pathways had no effect on ADP-induced platelet aggregation. These results strongly suggest that the ADP-induced phosphorylation of HSP27 via p44/p42 MAPK and/or p38 MAPK is therefore sufficient for platelet granule secretion but not for platelet aggregation in humans.     

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.     

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.     

Dual role of platelet protein kinase C in thrombus formation: stimulation of pro-aggregatory and suppression of procoagulant activity in platelets

Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to alphaIIbbeta3 activation. Strikingly, PKC suppressed Ca(2+) signal generation and Ca(2+)-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca(2+) signaling and procoagulant activity. In murine platelets lacking G(q)alpha, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca(2+)-dependent phosphatidylserine exposure, and consequently thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

Differential Roles of the PKC Novel Isoforms,PKCδ and PKCε, in Mouse and Human Platelets

Background

Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.

Methodology/Principal Findings

In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.

Conclusions

These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid.

The role of protein kinase C (PKC) in modulating platelet activation has been examined in platelets pre-incubated with either the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the non-specific protein kinase inhibitor, staurosporine. In order to determine where in the signal transduction pathway PKC is exerting its effect platelets were activated either with a receptor-operated stimulus platelet activating factor (PAF) or by direct elevation of [Ca2+]i (ionomycin) or with arachidonic acid which is converted into thromboxane B2 (TxB2). In PAF-stimulated platelets activation of PKC inhibited both [Ca2+]i elevation and TxB2 generation but had no effect on 5-hydroxytryptamine (5-HT) release whilst staurosporine increased the duration of [Ca2+]i elevation and potentiated TxB2 generation but inhibited 5-HT release. In ionomycin-stimulated platelets modulation of PKC had no effect on [Ca2+]i elevation but in contrast to PAF-stimulated platelets PKC activation caused potentiation of TxB2 generation and 5-HT release whilst inhibition of PKC caused inhibition of TxB2 generation and 5-HT release. Modulation of PKC did not affect arachidonic acid-induced TxB2 generation. These findings suggest that in receptor activated platelets endogenously activated PKC is exerting a negative feedback role, however, when [Ca2+]i elevation is not modified by PKC activation or inhibition (such as in ionomycin stimulated platelets) the relationship between the state of PKC activation and subsequent platelet functional responses corresponds more closely. The findings from this study suggest a different relationship between PKC and TxB2 generation than between PKC and dense granule release in PAF-stimulated platelets.     

Inhibition of ADP-induced platelet activation by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole: Covalent modification of aggregin,a putative ADP receptor

ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-Cl. NBD-Cl inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-Cl also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-Cl did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-Cl blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I₂). NBD-Cl was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [¹⁴C]-NBD-Cl showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [¹⁴C]-NBD-Cl. These results (1) indicate that covalent modification of aggregin by NBD-Cl contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin. © 1996 Wiley-Liss, Inc.     

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2? mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y?? receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.     

The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets

Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.     

The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

Control of platelet protein kinase C activation by cyclic AMP

Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP. These changes were also observed when platelet cAMP was increased by prostacyclin (PGI2), forskolin, or theophylline. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and the cyclooxygenase inhibitor indomethacin also diminished the aggregation and p47 phosphorylation responses to PMA or OAG. Pre-incubation of platelets with dibutyryl cAMP significantly potentiated the inhibition of aggregation and p47 phosphorylation effected by CP/CPK and indomethacin. These results are consistent with the model that PMA- or OAG-induced activation of platelets is amplified by secreted ADP and that the response to secreted ADP is inhibited by cAMP. Furthermore, the findings that increased intracellular cAMP inhibits PMA- or OAG-induced p47 phosphorylation in excess of that due solely to CP/CPK, and that cAMP significantly potentiates the effects of ADP removal and inhibition of cyclooxygenase in blocking p47 phosphorylation suggest that cAMP also exerts non-ADP-mediated inhibitory effects on PKC in intact platelets.     

Studies on platelet functions in hirudin plasma

Comparative studies on platelet responses in citrated, hirudin and heparin plasma were carried out. The adhesion of 111In-labelled rabbit platelets to the subendothelium of rabbit aorta was more pronounced in hirudin plasma than in heparin and citrated plasma. There were no significant differences in the collagen-induced aggregation and secretion of 14C-serotonin of human blood platelets in the three plasma samples. The extent of the ADP-induced aggregation was nearly the same in the three plasma samples, however, the aggregation was reversible in hirudin plasma. Adrenaline induced a small primary aggregation in hirudin plasma whereas in citrated and in heparin plasma the aggregation was a biphasic one. Secretion of 14C-serotonin induced by ADP or adrenaline occurred in citrated plasma only. Hirudin proved to be a suitable anticoagulant for studying platelet functions at physiological calcium concentrations.     

精-甘-天冬-丝氨酸四肽对二磷酸腺苷诱导大鼠血小板活化的信号转导的影响

本工作在二磷酸腺苷（ＡＤＰ）活化的大鼠血小板上,观察精－甘－天冬－丝上肽（ＲＧＤＳ肽）对血小板聚集、蛋白磷酸化、蛋白激酶Ｃ和丝裂素活化蛋白激酶活性的影响。结果发现,５０μｍｏｌ／ＬＡＤＰ引起血小板聚集时,蛋白激酶Ｃ（ＰＫＣ０及丝裂经蛋白激酶（ＭＡＰＫ）活性增加,并引起９５和６６ｋＤ蛋白磷酸化。应用５０,１００和２００μｍｏｌ／ＬＲＧＤＳ肽与基共同孵育,呈浓度依赖地抑制ＡＤＰ引起的血小板聚集和对ＰＫ     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Tyrosine dephosphorylation, but not phosphorylation, of p130Cas is dependent on integrin alpha IIb beta 3-mediated aggregation in platelets: implication of p130Cas involvement in pathways unrelated to cytoskeletal reorganization

The newly described adapter molecule p130 Crk-associated substrate (Cas) has been reported to contribute to cytoskeletal organization through assembly of actin filaments and to be pivotal in embryonic development and in oncogene-mediated transformation. We characterized the regulation of Cas tyrosine phosphorylation in highly differentiated, anucleate platelets. Phospholipase C-activating receptor agonists, including collagen, thrombin receptor-activating peptide (TRAP), and U46619 (a thromboxane A2 analogue), and A23187 (a Ca2+ ionophore) induced rapid Cas tyrosine phosphorylation in platelets. 12-O-Tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-sn-glycerol, protein kinase C (PKC) activators, also induced Cas tyrosine phosphorylation, albeit sluggishly. Cas tyrosine phosphorylation induced by collagen or TRAP was transient in aggregating platelets; Cas became dephosphorylated in a manner dependent on integrin alpha IIb beta 3-mediated aggregation. While BAPTA-AM (an intracellular Ca2+ chelator) inhibited Cas phosphorylation induced by collagen or TRAP, Ro31-8220 (a PKC inhibitor) rather prolonged it. Under the conditions, this PKC inhibitor suppressed platelet aggregation but not intracellular Ca2+ mobilization. In contrast to Cas involvement in focal adhesions in other cells, platelet Cas phosphorylation preceded the activation of focal adhesion kinase (FAK), and blockage of alpha IIb beta 3-mediated platelet aggregation with a GRGDS peptide resulted in prolongation of stimulation-dependent Cas tyrosine phosphorylation but in suppression of FAK tyrosine phosphorylation. Furthermore, TRAP-induced Cas phosphorylation was insensitive to cytochalasin D, an actin polymerization inhibitor. The failure of FAK to associate with Cas in immunoprecipitation studies also suggests that Cas tyrosine phosphorylation is independent of FAK activation. Of the signaling molecules investigated in this study, Src seemed to associate with Cas. Finally, Cas existed mainly in cytosol and membrane cytoskeleton fractions in the resting state, and remained unchanged during platelet aggregation, when FAK translocated to the cytoskeletal fraction. Our findings on platelet Cas suggest that (i) rapid Cas tyrosine phosphorylation occurs following phosphoinositide turnover by receptor-mediated agonists and may be mediated by intracellular Ca2+ mobilization; (ii) PKC activation, by itself, may elicit sluggish Cas phosphorylation; (iii) Cas tyrosine dephosphorylation, but not phosphorylation, is dependent on integrin alpha IIb beta 3-mediated aggregation; and (iv) Cas is not involved in cytoskeletal reorganization. Anucleate platelets seem to provide a unique model system to fully elucidate the functional role(s) of Cas.