Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Phosphatidylcholine synthesis in platelets is stimulated by diacylglycerol but not by phorbol ester

The biosynthesis of phosphatidylcholine (PC) in platelets was followed by measuring the incorporation of 32Pi. Incorporation into PC was stimulated by treatment with Clostridium perfringens phospholipase C or with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. However, neither the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13-acetate or thrombin stimulated 32Pi incorporation into PC. We conclude that phorbol ester does not stimulate the hydrolysis of PC to diacylglycerol in platelets.     

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.     

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.     

Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity

Phosphoinositide breakdown in response to thrombin stimulation of human platelets results in the formation of the calcium-mobilizing messenger molecules inositol 1,4,5-trisphosphate and inositol 1,2-cyclic-4,5-trisphosphate and of diglyceride, which activates protein kinase C. We find that protein kinase C phosphorylates and thereby increases the activity of inositol 1,4,5-trisphosphate 5'-phosphomonoesterase, a phosphatase that hydrolyzes these molecules to inert compounds. The 5'-phosphomonoesterase phosphorylated using [gamma-32P]ATP comigrates on SDS-polyacrylamide gels with a protein (40 kd) phosphorylated rapidly in response to thrombin stimulation of 32PO4-labeled platelets. Peptide maps of proteolytic digests of these two phosphorylated proteins indicate that they are the same. We propose that platelet Ca2+ mobilization is regulated by protein kinase C phosphorylation of the inositol 1,4,5-trisphosphate 5'-phosphomonoesterase. These results explain the observation that phorbol ester treatment of intact human platelets results in decreased levels of inositol trisphosphate and decreased Ca2+ mobilization upon subsequent thrombin addition.     

The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.     

Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells.

In platelets, and in several other cell systems, pre-treatment with protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) results in the inhibition of receptor-mediated responses, suggesting that protein kinase C may play an important role in the termination of signal transduction. In the present study, we have attempted to locate the site of action of phorbol ester by comparing thrombin-induced (i.e. receptor-mediated) platelet activation with that induced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and NaF, two agents which by-pass the receptor and initiate platelet responses by directly modulating G-protein function. After a 10 s pre-treatment with PMA (16 nM), dense-granule secretion induced by thrombin (0.2 unit/ml), GTP[S] (40 microM) and NaF (30 mM) was potentiated, resulting in a greater than additive response to agent plus PMA. However, after a 5 min pre-treatment, thrombin-induced secretion alone was inhibited, whereas PMA plus GTP[S]/NaF-induced release remained greater than additive. [32P]Phosphatidate formation in response to all three agents, in contrast, was inhibited by 50-70% in PMA (5 min)-treated platelets. That secretion induced by these agents is a protein kinase C-dependent event was demonstrable by using staurosporine, a protein kinase C inhibitor which at concentrations of 1-10 nM inhibited (70-90%) PMA-induced as well as thrombin- and NaF-induced secretion and protein phosphorylation. In membranes from PMA-treated platelets, thrombin-stimulated GTPase activity was significantly enhanced compared with that in untreated membranes (59% versus 82% increase over basal activity). The results suggest that inhibition of receptor-mediated responses by PMA may be directed towards two sites relating to G-protein activation: (i) receptor-stimulated GTPase activity and (ii) G-protein-phospholipase C coupling. Furthermore, the lack of inhibition of NaF- and GTP[S]-induced secretion by PMA suggests that different mechanisms may be involved in thrombin-induced and G-protein-activator-induced secretion.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

Zinc deficiency in rats decreases thrombin-stimulated platelet aggregation by lowering protein kinase C activity secondary to impaired calcium uptake

Aggregation of rat platelets, when stimulated by adenosine diphosphate (ADP) or fluoride, is impaired by zinc deficiency, and the defect is associated with a decreased uptake of external Ca²⁺. Zinc deficiency also impairs the aggregatory response of platelets to phorbol myristate acetate (PMA), an activator of protein kinase C, but low zinc status decreases the PMA response only when calcium is added to the external medium. The purpose of this study was to determine the role of protein kinase C in rat platelet function and its relationship to the zinc deficiency pathology observed in platelets stimulated by thrombin (THR). The percent of maximal aggregation and the concentration of cytosolic-free Ca²⁺ were measured in washed platelets stimulated by THR and PMA. For the protein kinase C experiments platelets were obtained from rats fed a grain-based diet, and for the thrombin experiments they were from rats fed purified diets. In the latter experiments, immature male rats were fed for 2 weeks a low zinc diet (<1 mg/kg) ad libitum or a zinc adequate (100 mg/kg) diet either ad libitum or pair-fed. Zinc deficiency impaired the aggregation of platelets stimulated by 0.045 U/mL of THR by approximately 40%, and the external calcium uptake (0.03 U/mL of THR) was decreased by approximately 30%. Staurosporine, a protein kinase C inhibitor, decreased thrombin-induced aggregation in a concentration-dependent manner, but it had no effect on the external calcium uptake. While PMA had a synergistic effect with thrombin in the stimulation of platelet aggregation, it actually decreased the cytosolic-free calcium response to thrombin. It is concluded that zinc deficiency impairs thrombin-stimulated platelet aggregation and calcium uptake and that protein kinase C activity is essential for rat platelet aggregation. Protein kinase C does not stimulate calcium uptake and must act downstream of the calcium uptake defect. A model of rat platelet activation is presented depicting impaired Ca²⁺ uptake as the primary defect in zinc deficiency.     

Differential effects of chlorpromazine on secretion, protein phosphorylation and phosphoinositide metabolism in stimulated platelets.

Increasing concentrations of chlorpromazine (30-500 microM) caused a progressive lysis of gel-filtered platelets, as monitored by the extracellular appearance of cytoplasmic ([14C]adenine-labelled) adenine nucleotides. The chlorpromazine-induced lysis was markedly enhanced by thrombin and phorbol ester, and complete cytolysis was found at chlorpromazine concentrations of 100 microM and above in the presence of thrombin. At non-lytic concentrations, chlorpromazine caused a dramatic increase in the thrombin- or phorbol ester-mediated incorporation of 32P into phosphatidylinositol 4-phosphate and, to a lesser extent, into phosphatidylinositol 4,5-bisphosphate in platelets pulse-labelled with [32P]Pi. Chlorpromazine alone also caused an incorporation of 32P into the phosphoinositides. Non-lytic concentrations of chlorpromazine had no effect on the phosphorylation of the 47 kDa protein (regarded as the substrate for protein kinase C), but markedly inhibited the accompanying secretion of ATP + ADP and beta-hexosaminidase when platelets were incubated with 0.17 microM-phorbol ester or 0.1-0.2 unit of thrombin/ml. At lower concentrations of thrombin, chlorpromazine did not inhibit, but slightly enhanced, secretion. A protein of 82 kDa was phosphorylated during the interaction of platelets with thrombin and phorbol ester, and this phosphorylation was enhanced by chlorpromazine (non-lytic). These results suggest that the previously reported inhibition of protein kinase C by chlorpromazine is probably non-specific and due to cytolysis. However, since non-lytic concentrations of chlorpromazine inhibit secretion, but not protein kinase C, in platelets, activation of protein kinase C is not involved in the stimulation-secretion coupling, or chlorpromazine acts at a step after kinase activation. Possible mechanisms of this inhibition by chlorpromazine are discussed in the light of its effect on phosphoinositide metabolism and protein phosphorylation.     

12-O-Tetradecanoylphorbol 13-acetate stimulates inositol lipid phosphorylation in intact human platelets

The phorbol esters are among the most potent tumor promoters. On addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to isolated human platelets prelabelled with [32P]orthophosphate we found a rapid increase in 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. In view of similar findings with cells infected with the oncogene Rous sarcoma virus, it is suggested that inositol lipid phosphorylation might be a key event in the molecular action of phorbol esters.     

Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate

Gel-filtered human platelets that had been pre-labelled with [32P]Pi were stimulated with thrombin, ionomycin or the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The effect of the hexacationic aminoglycoside antibiotic, neomycin, on platelet physiological responses, such as aggregation and secretion, as well as changes in phosphoinositide metabolism was studied. Neomycin strongly inhibited thrombin-induced aggregation and secretion whereas the antibiotic had no effect on ionomycin- or TPA-induced platelet functions. The thrombin-induced enhancement of inositol phospholipid metabolism was strongly inhibited by the presence of neomycin whereas the TPA- or ionomycin-induced increase in inositol [32P]polyphospholipids remained unaffected. The inhibitory effect of some other aminoglycoside antibiotics was compared to that of neomycin and the data demonstrate that the inhibition of platelet secretion and phosphatidic acid production was dependent on the cationic charge of the antibiotic. It is suggested that neomycin inhibits signal transduction in platelets at a level prior to the inositol-phospholipid-specific phosphodiesterase.     

EL-4 tumor cell-induced human and rabbit platelet aggregations

EL-4 tumor cells were assayed in vitro for their ability to aggregate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets. The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets.     

Phorbol ester treatment inhibits thrombin but not stable GTP analogue-induced platelet granule secretion despite inhibition of phosphatidate formation with both agonists

Previous work has demonstrated that pre-treatment of platelets with phorbol esters that activate protein kinase C eg phorbol 12-myristate 13-acetate (PMA) results in an inhibition of inositol phospholipid breakdown and granule secretion induced by physiological agonists such as thrombin and collagen. In the present study, the effect of pre-treatment with PMA on granule secretion and [32P]-phosphatidate (PA) formation induced by the stable GTP analogue, guanosine 5'-[gamma thio] triphosphate (GTP gamma S) was examined in saponin-permeabilized platelets. A low concentration of PMA ie 1.6nM, that did not induce significant 5-hydroxytryptamine (5HT) secretion on its own, but inhibited low-dose thrombin-induced 5HT secretion totally and PA formation by 30-40% in intact as well as permeabilised platelets was chosen. Our results demonstrate a lack of inhibition of GTP gamma S (40 microM)-induced 5HT secretion by PMA in permeabilised platelets, despite significant inhibition (70%) of PA formation, suggesting that apart from the diacylglycerol pathway of secretion which may be common to thrombin and GTP analogues, secretion induced by physiological agonists such as thrombin may involve another mechanism that is inhibitable by phorbol esters.     

蛋白激酶C在血小板聚集中的作用

利用￣（３２）Ｐ－ＮａＨ２ＰＯ４标记猪血小板,以蛋白激酶Ｃ的４０ｋＤ底物为蛋白激活的标志．用血小板激动剂在聚集浓度范围内处理血小板,结果表明,除了不能使猪血小板聚集的肾上腺素外,凝血酶等激动剂都使血小板４０ｋＤ底物蛋白磷酸化明显增加,同时３８ｋＤ,２６ｋＤ蛋白质磷酸化也明显增加,且４０ｋＤ底物磷酸化与血小板聚集有平行增加关系．蛋白激酶Ｃ在血小板聚集中可能起着重要的调节作用。     

Modes of inhibitory action of 4 beta-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets

The tumor-promoting phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC50 vs thrombin = 0.5 nM, greater than 90% inhibition at 10 nM. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 nM PMA. A non-tumor-promoting phorbol ester, 4-O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated AA release, but not GTP gamma S- and AIF4(-)-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A2 by PMA pretreatment is discussed.     

Wortmannin对凝血酶诱导的人血小板聚集和磷脂酰肌醇三磷酸累积的影响

 Ｗ ｏｒｔｍ ａｎｎｉｎ 是肌醇磷脂 ３ 激酶的不可逆抑制剂．用比浊法分析血小板聚集;肌醇磷脂用３２ Ｐ 磷酸钠标记,用氯仿和甲醇抽提,用 Ｔ Ｌ Ｃ和放射自显影分析,研究了 Ｗ ｏｒｔｍ ａｎｎｉｎ 对凝血酶诱导的人血小板聚集和磷脂酰肌醇三磷酸（ Ｐ Ｉ Ｐ３）累积的影响．结果显示, Ｗ ｏｒｔｍ ａｎｎｉｎ 对凝血酶（５００ Ｕ／ Ｌ）诱导的人血小板聚集有抑制作用,这种抑制作用在一定范围内呈剂量依赖关系（２０～８０μｍ ｏｌ／ Ｌ）．凝血酶（５００ Ｕ／ Ｌ）诱导人血小板 Ｐ Ｉ Ｐ３ 的累积, Ｗ ｏｒｔｍ ａｎｎｉｎ 对此累积有抑制作用,这种抑制作用在一定范围内呈剂量依赖关系（４０～１６０ μｍ ｏｌ／ Ｌ）．结果提示： Ｗ ｏｒｔｍ ａｎｎｉｎ 可能是潜在的抗血小板药物,抑制凝血酶诱导的人血小板聚集主要与其抑制 Ｐ Ｉ Ｐ３ 的累积有关．结果也提示,肌醇磷脂 ３ 激酶在血小板活化中起重要作用．     

Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium

Signaling pathways elicited by protease-activated receptor-1 (PAR-1) agonists, thrombin receptor-activating peptide (TRAP) and thrombin, are markedly different. Here we show that TRAP-induced disaggregation of platelets is a function of extracellular calcium. Chelation of calcium with EGTA after the onset of aggregation precluded subsequent destabilization of the aggregates in TRAP-stimulated platelets, whereas disaggregation was not observed in the platelets stimulated with thrombin. TRAP-induced disaggregation was independent of the activity of the calcium-dependent thiol protease, calpain. Inhibition of phosphoinositide 3-kinase activity provoked further destabilization of the platelet aggregates in the presence of calcium; however, EGTA attenuated this effect. Activation of protein kinase C (PKC) by phorbol ester prevented disaggregation of the TRAP-stimulated platelets independent of the extracellular calcium. Two proteins of relative mobilities 67 and 75 kD were found to be significantly dephosphorylated on tyrosine in calcium-pretreated platelets as compared to the EGTA-treated platelets following continued stimulation with either TRAP or thrombin for 15 min. Inhibition of phosphoinositide 3-kinase by two pharmacologically independent inhibitors also caused dephosphorylation of p67, which was completely abrogated by chelation of extracellular calcium. Platelet activation by phorbol ester was not associated with disaggregation, although dephosphorylation of p67 was induced under this condition. SHP-1, an abundant tyrosine phosphatase in platelets, co-migrated with the p67 protein and co-localized to the actin-based cytoskeleton of aggregated platelets; however, its identity with p67 was ruled out from immunoprecipitation studies.     

The role of Na+/H+ exchanger in serotonin secretion from porcine blood platelets

This study was undertaken to evaluate whether a link exists between the activation of protein kinase C (PKC), operation of Na(+)/H(+) exchanger (NHE), cell swelling and serotonin (5-HT) secretion in porcine platelets. Activation of platelets by thrombin or phorbol 12-myristate 13-acetate (PMA), a PKC activator, initiated a rapid rise in the activity of Na(+)/H(+) exchanger and secretion of 5-HT. Both thrombin- and PMA-evoked activation of Na(+)/H(+) exchanger was less pronounced in the presence of ethyl-isopropyl-amiloride (EIPA), an NHE inhibitor, and by GF 109203X, a PKC inhibitor. Monensin (simulating the action of NHE) caused a dose-dependent release of 5-HT that was not abolished by GF 109203X or EGTA. Lack of Na(+) in the suspending medium reduced thrombin-, PMA-, and monensin-evoked 5-HT secretion. GF 109203X nearly completely inhibited 5-HT release induced by PMA-, partly that induced by thrombin, and had no effect on 5-HT release induced by monensin. EIPA partly inhibited 5-HT release induced by thrombin and nearly totally that evoked by PMA. Electronic cell sizing measurements showed an increase in mean platelet volume upon treatment of cells with monensin, PMA or thrombin. The PMA- and thrombin-evoked rise in mean platelet volume was strongly reduced in the presence of EIPA. As judged by optical swelling assay monensin and PMA produced a rapid rise in platelet volume. The swelling elicited by PMA was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Hypoosmotically evoked platelet swelling did not affect platelet aggregation but significantly potentiated thrombin-evoked release of 5-HT and ATP. Taken together, these results show that in porcine platelets PKC may promote 5-HT secretion through the activation of NHE. It is hypothesized that enhanced Na(+)/H(+) antiport may result in a rise in cell membrane tension (due to cell swelling) which in turn facilitates fusion of secretory granules with the plasma membrane leading to 5-HT secretion.