Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

Selective secretion of protein kinase C isozymes by thrombin-stimulated human platelets

The protein kinase C (PKC) was secreted from thrombin-stimulated human platelets in a time- and dose-dependent manner. The PKC specific inhibitors Ro31-8220 (0.05 microM) and GF 109203X (0.5 microM) totally inhibited the secreted kinase activity. Western blot analysis of the secretory components showed reactivity to PKCalpha, PKCbetaII, and PKCdelta antibodies, but not to PKCbetaI, and p42/44 MAPK, although they were present in lysed platelets. The fractionation of platelets secreted components showed that PKC activity increased in both soluble and microparticle fractions after thrombin treatments. This is the first report demonstrating that activated human platelets selectively secrete protein kinase C isozymes. Protein kinase C secreted by platelets in this unique manner may have an extracellular role in the plasma, and may regulate cellular functions, including remodeling of vascular endothelial cells.     

Tumor necrosis factor signal transduction. Cell-type-specific activation and translocation of protein kinase C

We have investigated the changes in protein kinase C (PKC) activity after treatment of several cell lines with TNF. Binding studies with [3H]phorbol dibutyrate (PBt2) on whole cells revealed rapid and transient activation of PKC in Jurkat, K562, and U937 cells with a maximum of phorbol ester binding at 6 min after TNF treatment. As shown by Scatchard analysis, the TNF-induced increase of [3H]PBt2 binding reflected increments of phorbol ester binding site numbers rather than greater binding affinities. Upon subfractionation of TNF-treated U937 cells a transient increase of PBt2 binding in the membrane fraction was accompanied by a long term loss of PBt2-binding in the cytosol, indicating a TNF-induced translocation of PKC from the cytosol to the cell membrane. With histone III-S as a substrate, the determination of specific PKC activity revealed similar kinetics of PKC translocation in U937 cells. TNF also induced PKC translocation in K562 and Jurkat cells. However, although TNF caused long term down-regulation of cytosolic PKC activity in U937 cells, the cytosolic PKC activity only transiently decreased in both Jurkat and K562 cells and then recovered to near basal levels. In the human nonmalignant fibroblast cell line CCD18, PKC was not activated by TNF. Our data suggest that PKC activation may play a major role in TNF signal transduction in some, but not all target cells.     

Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C

The activation of protein kinase C in human platelets by phorbol-12, 13- dibutyrate (PDBu) results in the phosphorylation of a 40,000 dalton protein. This phosphorylation is time- and concentration-dependent. Maximal phosphorylation is rapid and is not affected by indomethacin or prostacyclin. PDBu does not promote activation of the phosphodiesteratic cleavage (phospholipase C) of the inositol phospholipids and the subsequent formation of 1,2-diacylglycerol or its phosphorylated product, phosphatidic acid. If platelets exposed to PDBu are subsequently stimulated with thrombin, this stimulus does not initiate further 40,000 dalton protein phosphorylation but will promote the formation of phosphatidic acid and also the phosphorylation of a 20,000 dalton protein (myosin light chain). However, prostacyclin will prevent the subsequent stimulation of phosphatidic acid synthesis by thrombin in a concentration-dependent manner. The fact that prostacyclin can affect the response to thrombin, even in the presence of phorbol ester, supports the idea that the enzymes related to the formation of phosphatidic acid or inhibition of its synthesis are not related to the phosphorylated 40K protein.     

IL-4 receptor signal transduction in human monocytes is associated with protein kinase C translocation.

IL-4 regulates B cell differentiation and monocyte functions. Protein kinases, such as kinase C (PKC), transduce receptor signals. The involvement of PKC in IL-4R signaling was investigated in human monocytes. Treatment with IL-4 (10 ng/ml) for 10 min resulted in a significant redistribution of the PKC activity from cytosol to nuclear fraction. Total PKC activity localized in the nuclear fraction of IL-4-treated and control monocytes was, respectively, 68 and 19%. In contrast, similar PKC activity was found in membrane fraction of IL-4-treated and control cells. The kinetics of IL-4-mediated redistribution of PKC activity to the nuclear fraction were rapid. Within 30 s of IL-4 exposure, 29% of the total PKC activity localized in the nuclear fraction as compared to 15% in control monocytes and increased to 69% at 10 min. The PKC activity in the nuclear fraction appears to be a sequestered form. Extraction with Triton X-100 and additional sonication were required for functional assay of PKC activity. Additional support for PKC involvement in IL-4R signaling is provided by the dose-dependent effect of IL-4 on PKC activity and the abrogation of this effect after heat denature and immunoabsorption of IL-4. Furthermore, electron microscope examination and subcellular marker enzyme assays excluded significant contamination of the nuclear fraction by plasma membranes or subcellular organelles and IL-4 altering membrane disruption. The data presented indicate that IL-4R signaling in human monocytes involves PKC translocation to a nuclear fraction.     

Spacial isolation of protein kinase C activation in thrombin stimulated human platelets

Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.     

Attenuation of protein kinase C and cAMP-dependent protein kinase signal transduction in the neurogranin knockout mouse

Neurogranin (Ng) is a brain-specific, postsynaptically located protein kinase C (PKC) substrate, highly expressed in the cortex, hippocampus, striatum, and amygdala. This protein is a Ca(2+)-sensitive calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. To investigate the role of Ng in neural function, a strain of Ng knockout mouse (KO) was generated. Previously we reported (Pak, J. H., Huang, F. L., Li, J., Balschun, D., Reymann, K. G., Chiang, C., Westphal, H., and Huang, K.-P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11232-11237) that these KO mice displayed no obvious neuroanatomical abnormality, but exhibited deficits in learning and memory and activation of Ca(2+)/CaM-dependent protein kinase II. In this report, we analyzed several downstream phosphorylation targets in phorbol 12-myristate 13-acetate- and forskolin-treated hippocampal slices from wild type (WT) and KO mice. Phorbol 12-myristate 13-acetate caused phosphorylation of Ng in WT mice and promoted the translocation of PKC from the cytosolic to the particulate fractions of both the WT and KO mice, albeit to a lesser extent in the latter. Phosphorylation of downstream targets, including mitogen-activated protein kinases, 90-kDa ribosomal S6 kinase, and the cAMP response element binding protein (CREB) was significantly attenuated in KO mice. Stimulation of hippocampal slices with forskolin also caused greater stimulation of protein kinase A (PKA) in the WT as compared with those of the KO mice. Again, phosphorylation of the downstream targets of PKA was attenuated in the KO mice. These results suggest that Ng plays a pivotal role in regulating both PKC- and PKA-mediated signaling pathways, and that the deficits in learning and memory of spatial tasks detected in the KO mice may be the result of defects in the signaling pathways leading to the phosphorylation of CREB.     

Thrombin- and histamine-induced signal transduction in human endothelial cells. Stimulation and agonist-dependent desensitization of protein phosphorylation

Treatment of human endothelial cells with thrombin, histamine, or dioctanoylglycerol (DiC8), a synthetic diacylglycerol, resulted in the rapid and transient phosphorylation of a Mr = 29,000 protein (P29) in a dose-dependent manner. Various tumor promoters also promoted P29 phosphorylation while the adenylate cyclase activator, forskolin, did not. The level of phosphorylation with all three agonists was similar (2.5-4 fold), and analysis of P29 by two-dimensional gel electrophoresis revealed identical patterns in each case. Receptor specificity was demonstrated for the histamine-stimulated changes; pyrilamine (10(-6) M; H1) but not cimetidine (10(-4); H2) blocked the response. The thrombin effect was active site-dependent. Phosphorylation induced by thrombin and histamine occurred within 1 min, peaked between 5 and 10 min, and returned to control levels by 1 h. DiC8-induced phosphorylation occurred more slowly but was also reduced by 1 h while phorbol ester treatment prolonged phosphorylation for at least 4 h. Treatment of these cells with thrombin or histamine for 1 h desensitized P29 to further phosphorylation by the homologous agonist although secondary phosphorylation could occur with heterologous compounds. However, if the primary agonist was removed following the onset of a desensitized state, secondary phosphorylation of P29 could be stimulated by the same compound. These same results were observed with two other phosphoproteins Mr = 18,000 (P18) and 80,000 (P80) which became more highly phosphorylated in response to thrombin treatment and with histamine/thrombin-stimulated prostaglandin I2 production. In contrast, homologous down-regulation of P29 phosphorylation was not observed with DiC8-treated cells, and the decline in phosphorylated P29 was associated with the loss of functional DiC8. The protein kinase inhibitors staurosporine and H-7 blocked P18 and P80 phosphorylation by thrombin but had no effect on P29 phosphorylation by histamine, thrombin, or DiC8 suggesting distinct pathways leading to the phosphorylation of these different proteins. These data suggest that multiple and independent thrombin/histamine-induced events are susceptible to receptor occupancy-dependent homologous down-regulation.     

Inhibition of sphingosine kinase in vitro and in platelets. Implications for signal transduction pathways.

Sphingosine kinase was partially purified and characterized from rat brain microsomes. A new assay, utilizing octyl-beta-D-glucopyranoside and sphingosine mixed micelles, was developed to quantitate formation of the sphingosine-1-phosphate product. The assay was proportional with respect to time and protein, displayed Michaelis-Menten kinetics, and was subject to surface dilution in regard to the sphingosine substrate. Investigations into substrate specificity showed that the enzyme is specific for the erythro-enantiomers of sphingosine and dihydrosphingosine. Neither of the threo-enantiomers were phosphorylated in this system, but both were found to be potent competitive inhibitors of sphingosine kinase activity. Human platelet sphingosine kinase activity displayed substrate and inhibitor specificities similar to the rat brain enzyme. A mixture of DL-threo-dihydrosphingosine competitively inhibited sphingosine kinase activity in a dose dependent manner in isolated platelets. DL-Threo-dihydrosphingosine caused a prolongation of the inhibition of thrombin-induced protein kinase C-dependent 40 (47)-kDa protein phosphorylation in platelets. D-, L-, or DL-Threo-dihydrosphingosine may be useful as a tool to investigate D-Erythrosphingosine metabolism and the function of sphingosine-1-phosphate in signal transduction processes.     

Cytosolic Ca2+ pulses and protein kinase activation in the signal transduction pathways leading to the plant oxidative burst

The oxidative burst, the rapid production of O₂- and H₂O₂ by plant cells in response to pathogens and Stressors, is a critical step in plant disease resistance and is controlled by several different elicitor-initiated signaling pathways. While different defense elicitors appear to activate disparate initial steps in signaling the oxidative burst, all of the elicitors tested thus far appear to stimulate pathways that converge on the same three core signaling intermediates: 1) the Ca₂₊-independent activation of a mitogen-activated protein kinase (MAPK) family member, 2) the influx of Ca₂₊ into the cytosol, deriving most critically from an internal compartment, and 3) the Ca²⁺-dependent activation of additional protein kinases including a second MAPK homologue and possibly calcium dependent protein kinases (CDPKs). Data from several recent reports are summarized to place these signaling events into a complete and updated model of signaling to the plant oxidative burst.     

Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes

Activation of the signal transduction pathways mediated by protein kinase A (PKA) or protein kinase C (PKC) led to different responses of several serum inducible genes including the jun gene family, c-fos, c-myc, krox 20 and krox 24. Whereas all of these genes were stimulated by the phorbol ester TPA, a chemical activator of protein kinase C, they were differently regulated upon cAMP stimulation of the PKA dependent pathway. The proto-oncogenes jun B, c-fos, and to a lesser extent jun D were stimulated by increasing the intracellular concentration of cAMP, whereas the TPA stimulation of c-jun and c-myc was inhibited under these conditions. Krox 20 and krox 24 were insensitive to this second messenger. This study allowed us to classify these growth stimulated genes into three distinct groups distinguished by their sensitivity to elevated concentrations of intracellular cAMP. The inhibition of c-jun and c-myc expression in the presence of increased cAMP levels may be at least partially responsible for the growth inhibitory effect of this agent in Balb/c-3T3 cells.     

Receptor- and phorbol-ester-mediated redistribution of protein kinase C in human platelets. Evidence that aggregation promotes degradation of protein kinase C.

Translocation of Ca2+/phospholipid-dependent protein kinase (PKC) activity from cytosolic to membrane fractions was assessed in washed human platelet suspensions. Phorbol myristate acetate (PMA) induced a rapid loss of PKC activity from the cytosolic compartment in stirred platelets, which was not accompanied by measurable increases in membrane-associated activity, but was paralleled by a decrease in total cellular enzyme activity (cytosol plus membrane). When platelet aggregation was prevented by not stirring, (i) cytosolic activity was decreased by PMA, (ii) significant and maintained (1-15 min with PMA) increases in membrane-bound PKC were detected, and (iii) the decline in total enzyme activity was markedly slower. In stirred platelets, total and specific inhibition of PMA-induced aggregation by a fibrinogen-derived peptide (RGDS, i.e. Arg-Gly-Asp-Ser) promoted maximal increases in membrane-associated PKC in the presence of PMA and completely prevented the loss in cellular activity. Thrombin and collagen both induced a decrease in cytosolic PKC and a loss of total activity, but a significant rise in membrane activity was seen only with collagen; ADP had no detectable effect on enzyme distribution. These results demonstrate an agonist-induced redistribution of PKC and indicate that platelet aggregation may play an important role in the proteolysis, and hence persistence, of membrane-associated PKC. This observation has implications for the potency and duration of PKC-mediated responses induced by agonists and exogenous PKC activators.     

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.     

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.     

Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.     

The signal Peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein

A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretory pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins.     

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.