Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.     

Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation. A novel synergism.

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.     

Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP.

It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2-diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+-calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i.     

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.     

Forskolin and prostacyclin inhibit fluoride induced platelet activation and protein kinase C dependent responses

Platelet activation (cytosolic [Ca2+] increase, aggregation and ATP secretion) was induced with A1F-4. This agent presumably interacts with a G protein which appears to mediate the coupling of the receptors for Ca mobilizing hormones and phospholipase C. All the A1F-4 evoked responses were inhibited by treatment with forskolin or prostacyclin, agents known to increase cellular cAMP. Thus the G protein-phospholipase C system appears to be the site of cAMP inhibition. Unexpectedly forskolin and prostacyclin also inhibited secretion and aggregation induced by the activators of protein kinase C, diglyceride and phorbol ester, suggesting that cAMP can also inhibit directly the protein kinase C dependent responses.     

Platelet responses promoted by the activation of protein kinase C or the increase of cytosolic Ca2+ are potentiated by adrenaline. Effects of cAMP and staurosporine.

We studied the action of the alpha 2 adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of adrenaline addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. Ionomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.     

Cyclic GMP and nitroprusside inhibit the activation of human platelets by fluoroaluminate

Sodium nitroprusside, an activator of the soluble guanylate cyclase, inhibits the intracellular Ca2+ mobilization, ATP secretion and aggregation of human platelets evoked by fluoroaluminate. Similar results are obtained with 8-bromo-cyclic GMP (8-Br-cGMP). Both nitroprusside and 8-Br-cGMP inhibit the protein kinase C-dependent phosphorylation of the 47 and 20 kDa proteins induced by fluoroaluminate, but not by the protein kinase C activators phorbol ester and diacylglycerol. Since fluoroaluminate interacts directly with a G protein, the present results suggest that the cGMP interferes with platelet activation at the level of G protein-phospholipase C.     

Possible mechanisms of the potentiation of blood-platelet activation by adrenaline.

Vasopressin and adrenaline in combination exert synergistic effects on platelet activity. This study investigated the effects of sub-threshold concentrations of adrenaline (0.1-1 microM) on vasopressin (10 nM-1 microM)-induced platelet aggregation, ATP secretion, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Potentiation of vasopressin-induced aggregation and ATP secretion by adrenaline was accompanied by enhanced elevation of [Ca2+]i and [32P]phosphatidic acid formation. The stimulatory effects of adrenaline on vasopressin-induced platelet activation were mimicked by the combination of the Ca2+ ionophore, ionomycin, and the protein kinase C activator, phorbol 12-myristate 13-acetate, but not by either of these agents alone. These results suggest that the potentiation of vasopressin-induced platelet activation by adrenaline is mediated via enhancement of inositol phospholipid hydrolysis and elevation of [Ca2+]i.     

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.     

Direct demonstration of involvement of protein kinase Calpha in the Ca2+-induced platelet aggregation

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin alphaIIbbeta3. However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca2+-induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of alphaIIbbeta3, suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKCalpha and beta from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCalpha. Furthermore, the addition of purified PKCalpha in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKCalpha is involved in the regulation of Ca2+-induced platelet aggregation.     

P2X1-mediated ERK2 activation amplifies the collagen-induced platelet secretion by enhancing myosin light chain kinase activation

The ATP-gated P2X1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X1 agonist alpha,beta-methylene ATP (alpha,beta-meATP) causes rapid, transient (2-5 s), and dose-dependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca2+. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca2+/CaM-dependent MLC kinase. Correspondingly, the P2X1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca2+/CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation.     

Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets.

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.     

Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases.

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.     

Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca(2+). Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCε but not PKC. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca(2+). This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca(2+) concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.     

A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signaling pathway in stimulating platelet secretion and aggregation

Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase.     

A platelet secretion pathway mediated by cGMP-dependent protein kinase

Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation.     

A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.     

Calpain I activation is not correlated with aggregation in human platelets.

Calpain-catalysed hydrolysis of platelet substrates such as cytoskeletal and calmodulin-binding proteins, and of protein kinase C, is assumed to contribute to platelet aggregation. We have measured calpain I activation by immunoblotting, and [Ca2+]i (cytoplasmic Ca2+ concn.) by fura-2 fluorescence, in parallel with measurement of aggregation, in stirred human platelets treated at different [Ca2+]ext (extend Ca2+ concns.) with A23187, leupeptin, phorbol ester and thrombin. Hydrolysis of actin-binding protein, and [3H]5-hydroxytryptamine release, were also measured in some cases. A rise in [Ca2+]i, platelet aggregation and calpain activation often occurred together. With some combinations of agonists and [Ca2+]ext, however, this correlation was clearly not maintained. It was shown: (a) that activation of calpain and its hydrolysis of platelet substrates were not strictly necessary conditions for platelet secretion and aggregation; (b) conversely, that calpain activation could occur without aggregation.     

Identification of protein kinase Calpha as an essential, but not sufficient, cytosolic factor for Ca2+-induced alpha- and dense-core granule secretion in platelets

Upon activation, platelets release many active substances. Here, we have analyzed the mechanism governing Ca(2+)-induced secretion of von Willebrand factor stored in alpha-granules and 5-hydroxytryptamine in dense-core granules in permeabilized human platelets. Both secretions were dependent on ATP and cytosol. An essential factor for both granule secretions was purified from rat brain cytosol and identified to be protein kinase Calpha (PKCalpha) by partial amino acid sequencing. Purified PKCalpha efficiently stimulated both secretions in the presence of cytosol, whereas PKCalpha alone did not support the secretion of either type of granules, suggesting that PKCalpha is not a sufficient factor. Finally, in human platelet cytosol fractionated by a gel filtration column, the stimulatory activity for dense-core granule secretion paralleled with the concentration of PKC, suggesting that PKC could also be such a stimulatory factor in platelet cytosol. Thus, we identified PKCalpha as an essential, but not sufficient, cytosolic factor for the Ca(2+)-induced secretions of both alpha- and dense-core granules in platelets.     

NAADP regulates human platelet function

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.