The role of phospholipase C in platelet responses

Degradation of inositides induced by phospholipase C in activated platelets leads to the formation of 1,2-diacylglycerol (1,2-DG) and its phosphorylated product, phosphatidic acid (PA). We have studied the relationship between activation of phospholipase C and the appearance of specific platelet responses, such as phosphorylation of proteins, shape change, release reaction and aggregation induced by different stimuli such as thrombin, platelet-activating factor, collagen, arachidonic acid (AA) and dihomogamma linolenic acid. A low degree of platelet activation induces only shape change which is associated with partial activation of phospholipase C (formation of phosphatidic acid), and phosphorylation of both a 40K molecular weight protein (protein kinase C activation) and a 20K molecular weight protein (myosin light chain). A higher degree of platelet activation induces aggregation, release of serotonin and a higher level of phospholipase C and protein kinase C activities. Metabolism of AA occurs concomitantly to aggregation and serotonin release, but AA metabolites are not related to the shape change of human platelets. Platelet shape change and the initial activation of phospholipase C induced by thrombin or platelet-activating factor is independent of the metabolites derived from cyclo-oxygenase activity. Further activation of phospholipase C which occurs during platelet aggregation and release reaction is, however, partly dependent on cyclo-oxygenase metabolites.     

The perturbation of thrombin binding to human platelets by trypsin and neuraminidase

The initial step in the interaction of thrombin with human platelets in binding of the enzyme to the platelet surface. The effects of digestion of isolated platelets with trypsin and neuraminidase on aggregation, release of serotonin and binding of thrombin have been examined.Trypsin is a powerful inducer of platelet aggregation as well as the release reaction. The aggregation effect of trypsin may be blocked with disodium ehtylenediaminetatraacetate (EDTA). Further, in the presence of EDTA, trypsin-induced release of [¹⁴C]serotonin is 15–20% lower compared to controls and the initial lag period is prolonged. Conditions were developed under which trypsin did neither aggregate nor release serotonin from platelets. Even under these conditions, trypsin caused a profound loss in the thrombin binding capacity of platelets. Thus, the trypsin-induced fall in the thrombin binding capacity and the platelet response are dissociated. This loss in the thrombin binding by trypsin is due to a lower number of binding sites available on the platelet surface and is not due to an altered affinity.Neuraminidase did not induce platelet aggregation or the release reaction. The ability of platelets to bind thrombin was also unimpaired by prior digestion with neuraminidase. Thus, the sialic acid at the platelet surface is not essential in the function of thrombin recognition by the receptor. This moiety may nontheless be a constituent of a glycoprotein which might act as the thrombin receptor.     

Shape change induced in human platelets by platelet-activating factor. Correlation with the formation of phosphatidic acid and phosphorylation of a 40,000-dalton protein

Washed human platelets that have been separated from plasma in the presence of prostacyclin are activated by the addition of platelet activating factor (PAF). Activation (shape change, serotonin release, and aggregation) correlates closely with the formation of phosphatidic acid and the phosphorylation of a 40,000-dalton protein. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation precede aggregation and are induced at lower concentrations of PAF than those required to induce release of serotonin and platelet aggregation. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation induced by PAF are not affected by trifluoperazine or indomethacin. This indicates that these responses are independent of the liberation of arachidonic acid from platelet phospholipids and the metabolism of arachidonic acid via cyclooxygenase and lipoxygenase. These responses are, however, inhibited by prostacyclin. Platelet shape change is the first measurable physiologic response to platelet agonists and may be associated with the stimulation of phospholipase C, inducing formation of 1,2-diacylglycerol and its phosphorylated product, phosphatidic acid. Transient formation of 1,2-diacylglycerol may also induce the specific activation of the protein kinase C that phosphorylates a 40,000-dalton protein.     

A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions

In human platelets stimulated by thrombin and collagen, diacylglycerol is rapidly produced from phosphatidylinositol. Concurrently, an endogenous protein having a molecular weight of about 40,000 (40K protein) is phosphorylated, and serotonin is released. These reactions are all inhibited by a prior treatment of platelets with prostaglandin E1, dibutyryl cyclic AMP, sodium nitroprusside, or with 8-bromo-cyclic GMP, which are known as potent inhibitors for platelet activation. Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) preferentially phosphorylates 40K protein. As judged by fingerprint analysis, the sites in 40K protein that are phosphorylated during the platelet activation appear to be identical with those phosphorylated by protein kinase C in a purified cell-free system. 12-O-Tetradecanoylphorbol-13-acetate, which directly activates protein kinase C by substituting for diacylglycerol, stimulates 40K protein phosphorylation and release reaction without inducing diacylglycerol formation. Tetracaine, which inhibits protein kinase C by competing with phospholipid, blocks 40K protein phosphorylation and serotonin release without inhibiting the receptor-linked diacylglycerol formation. The results indicate that thrombin and collagen activate platelets in almost similar mechanisms and that protein kinase C may lie on a common pathway which leads to the release of serotonin. However, analysis with indomethacin indicates that the role of thromboxane A2 appears to be more predominant for the action of collagen, and it is suggestive that this arachidonate metabolite activates platelets in an analogous mechanism to thrombin.     

The inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin, activates platelets by selective mobilization of calcium as shown by protein phosphorylations

We have studied the activation of human blood platelets by the inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin. The effect of thapsigargin was compared with other common agonists (calcium ionophore A23187, phorbol ester TPA and thrombin). Platelet aggregation, serotonin release, raised cytoplasmic free calcium level and phosphorylation of platelet proteins was examined in platelet-rich plasma and washed platelet suspension. In contrast to A23187 and thrombin, the platelet activation induced by thapsigargin developed slowly, with maximal response obtained after 2-3 min. Both the thapsigargin- and the A23187-induced serotonin releases were synergistically increased by TPA. Studies of the phosphorylation of platelet proteins revealed that thapsigargin and A23187 equally well induced a selective phosphorylation of two proteins with apparent molecular masses of 20 kDa and 47 kDa. These proteins, which are substrates of myosin light-chain kinase and protein kinase C respectively, are known to be involved in platelet activation. The thapsigargin-induced platelet aggregation and serotonin release was completely inhibited by class I (nimodipine), class II (verapamil) and class III (diltiazem) calcium-channel blockers. The inhibitory activity of nimodipine was abolished by the corresponding 1,4-dihydropyridine calcium-channel agonist, BAY K 8644. These results shows that the thapsigargin-induced platelet activation is mediated by an increase in the cytoplasmic free calcium level, presumably obtained by stimulation of the passive calcium transport through specific channels. These thapsigargin-sensitive channels should predominantly be located in the membranes of intracellular calcium stores rather than in the plasma membrane, because removal of extracellular calcium by EGTA had only an insignificant effect on the thapsigargin-induced rise in cytoplasmic free calcium level.     

Platelet activation by von Willebrand factor requires coordinated signaling through thromboxane A2 and Fc gamma IIA receptor

Interaction of von Willebrand Factor with glycoprotein Ib-IX-V induces platelet activation through a still poorly defined mechanism. Previous studies have suggested a possible role for the low affinity receptor for immunoglobulin, Fc gamma RIIA, in GPIb-IX-V signaling. Here we show that binding of vWF to platelets induces the tyrosine phosphorylation of Fc gamma RIIA by a Src kinase. Treatment of platelets with the anti-Fc gamma RIIA monoclonal antibody IV.3 specifically inhibits vWF-induced but not thrombin-induced pleckstrin phosphorylation and serotonin secretion. Moreover, vWF fails to induce pleckstrin phosphorylation in mouse platelets, lacking Fc gamma RIIA, and serotonin secretion is impaired. Pleckstrin phosphorylation and serotonin secretion in human platelets stimulated with vWF are blocked by the cyclooxygenase inhibitor acetylsalicylic acid. However, release of arachidonic acid and synthesis of TxA(2) induced by vWF are not affected by the anti-Fc gamma RIIA monoclonal antibody IV.3. Similarly, vWF-induced tyrosine phosphorylation of Fc gamma RIIA, as well as of Syk and PLC gamma 2, occurs normally in aspirinized platelets. Inhibition of the tyrosine kinase Syk by piceatannol does not affect vWF-induced tyrosine phosphorylation of Fc gamma RIIA but prevents phosphorylation of PLC gamma 2. Pleckstrin phosphorylation and platelet secretion induced by vWF, but not by thrombin, are also inhibited by piceatannol. Pleckstrin phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. These results indicate that PLC gamma 2 plays a central role in platelet activation by vWF and that the stimulation of this enzyme requires coordinated signals through endogenous TxA(2) and Fc gamma RIIA.     

Exogenous sn-1,2-diacylglycerols containing saturated fatty acids function as bioregulators of protein kinase C in human platelets

The ability of exogenous sn-1,2-diacylglycerols and analogs to function as bioregulators of protein kinase C in human platelets was investigated. The activation of protein kinase C in platelets is indicated by specific phosphorylation of a 40,000-dalton protein. Dihexanoylglycerol, dioctanoylglycerol (diC8), didecanoylglycerol, and sn-1-oleoyl-2-acetylglycerol were active in stimulating 40,000-dalton protein phosphorylation. Only a trace of phosphorylation was elicited by dibutyrylglycerol. Phosphorylation was not induced by analogs of diC8 in which an -H, -SH, or -Cl group replaced the free -OH, nor by monoacylglycerols or long chain diacylglycerols. Maximum phosphorylation was induced by dihexanoylglycerol, diC8, and didecanoylglycerol at concentrations from 5 to 20 microM and between 5 and 30 S after exposure of platelets to these diacylglycerols. Under conditions of maximal phosphorylation of the 40,000-dalton protein, these diacylglycerols did not induce phosphatidylinositol turnover, or platelet aggregation, or stimulate release of ATP or serotonin. A small degree of aggregation was evident with platelets isolated in the absence of prostacyclin, and release of serotonin was observed when 1 mM Ca2+ or submaximal concentrations of ionophore A23187 were included. These results are consistent with a model in which platelet activation requires the simultaneous formation of two intracellular signals, diacylglycerols and Ca2+. These diacylglycerols and diacylglycerol analogs provide useful tools to investigate the function of diacylglycerols as bioregulators in intact cells.     

Activation of platelets induced by mAb P256 specific for glycoprotein IIb-IIIa. Possible evidence for a role for IIb-IIIa in membrane signal transduction

Monoclonal antibody P256, which is specific for glycoprotein IIb-IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb-IIIa molecule was necessary for triggering aggregation since Fab' fragments did not induce aggregation as did IgG and F(ab')2 fragments; however, F(ab')2 did not induce the release as did the whole IgG. P256-induced aggregation was accompanied by release of all three granule constituents, namely dense granules, alpha-granules and lysosomes, with parallel kinetics showing half-maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P-prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5-bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium-independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo-1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb-IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.     

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.     

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.     

Are polyphosphoinositides involved in platelet activation?

In human platelets, the amounts of triphosphoinositides (TPI) and diphosphoinositides (DPI) increase after 30 sec and level off after 120 sec of thrombin stimulation. After 180 sec of thrombin challenge, TPI and DPI increase accounts for 66 and 80%, respectively. Polyphosphoinositide changes roughly parallel the release of N-acetyl-beta-D-glucosaminidase and appear as a later event compared to aggregation and serotonin secretion. It is concluded that an increased phosphorylation of polyphosphoinositides might participate in platelets to the process of stimulus-activation coupling and might be linked to thrombin receptor occupancy. A role of DPI in platelet activation is suggested by the observation that DPI promote platelet aggregation, the mechanism of which is discussed.     

Role of a JAK3-dependent biochemical signaling pathway in platelet activation and aggregation

Here we provide experimental evidence that identifies JAK3 as one of the regulators of platelet function. Treatment of platelets with thrombin induced tyrosine phosphorylation of the JAK3 target substrates STAT1 and STAT3. Platelets from JAK3-deficient mice displayed a decrease in tyrosine phosphorylation of STAT1 and STAT3. In accordance with these data, pretreatment of human platelets with the JAK3 inhibitor WHI-P131 markedly decreased the base-line enzymatic activity of constitutively active JAK3 and abolished the thrombin-induced tyrosine phosphorylation of STAT1 and STAT3. Following thrombin stimulation, WHI-P131-treated platelets did not undergo shape changes indicative of activation such as pseudopod formation. WHI-P131 inhibited thrombin-induced degranulation/serotonin release as well as platelet aggregation. Highly effective platelet inhibitory plasma concentrations of WHI-P131 were achieved in mice without toxicity. WHI-P131 prolonged the bleeding time of mice in a dose-dependent manner and improved event-free survival in a mouse model of thromboplastin-induced generalized and invariably fatal thromboembolism. To our knowledge, WHI-P131 is the first anti-thrombotic agent that prevents platelet aggregation by inhibiting JAK3.     

Erbstatin blocks platelet activating factor-induced protein-tyrosine phosphorylation, polyphosphoinositide hydrolysis, protein kinase C activation, serotonin secretion and aggregation of rabbit platelets.

The role of protein-tyrosine phosphorylation in the signal transduction of platelet activating factor (PAF) was investigated in rabbit platelets with a range of synthetic compounds that inhibit protein-tyrosine kinases. In particular, erbstatin (IC50 approximately 20 micrograms/ml) abrogated a wide range of platelet responses to PAF, including tyrosine phosphorylation of cellular proteins, polyphosphoinositide turnover, activation of membranous protein kinase C, platelet aggregation, and serotonin secretion. With about a third of the potency of erbstatin, compound RG50864 also inhibited many of these responses, whereas at 100 micrograms/ml, genistein, 670C88 and ST271 were without effect. Finally, the ability of thrombin to cause platelet aggregation and serotonin secretion was also compromised by erbstatin.     

Synergism between subthreshold concentrations of thrombin and 12-O-tetradecanoylphorbol 13-acetate in platelet activation

A comparison was made between the time courses and interdependence of platelet aggregation, serotonin release, and cytosolic free Ca2+ concentration in the same sample of platelets loaded with [14C]-serotonin and Ca2+-sensitive photoprotein aequorin. In 100 micrograms/ml aspirin-treated platelets, neither 0.01 U/ml thrombin nor 50nM TPA, an active phorbol ester, induced significant aggregation, serotonin release, or a rise in the intracellular calcium concentration. However, when these two agents were added together, marked aggregation and release were observed without a change in the cytosolic free Ca2+ concentration. No correlation was observed between the extent of the synergistic effects and time of preincubation with TPA. Potentiatory effects of protein kinase C on receptor-mediated agonists need to be considered in platelet activation.     

Differential effects of the diacylglycerol kinase inhibitor R59022 on thrombin versus collagen-induced human platelet secretion

R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the conversion of DAG to phosphatidic acid, causes an increase in endogenous DAG levels and the activity of the DAG-dependent enzyme protein kinase C. This property of the drug was utilized in the present study to assess the role of DAG, i.e., its relative importance as a potentiatory versus inhibitory mediator, in agonist-induced platelet activation. The phosphorylation of the 40-47-kDa protein by protein kinase C was monitored as an indicator of endogenous DAG levels and correlated with other agonist-induced platelet responses such as platelet aggregation, 5-hydroxytryptamine (5HT) secretion and arachidonate release, the agonists used being those that induce DAG formation, e.g., thrombin and collagen. Pretreatment of platelets with R59022 before agonist addition resulted in the potentiation of 5HT secretion as well as 45 kDa protein phosphorylation induced by thrombin and the DAG analogue, 1,2-dioctanoylglycerol (DiC8). However, collagen-induced 5HT secretion was significantly inhibited (70%) in the presence of R59022, which also had strong inhibitory effects on aggregation induced by collagen, as well as by thrombin and DiC8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC8 on the same, suggesting that even although DAG acts as a potentiatory signal in this system, the inhibitory effects of R59022 on collagen-induced aggregation can mask any effects of endogenous DAG. This inhibitory effect of R59022 on agonist-induced platelet aggregation makes it unsuitable as a tool in studying the role of DAG in platelet activation induced by agonists such as collagen as well as the 'weak' agonists (ADP, adrenaline and platelet-activating factor), where aggregation mediates other responses such as arachidonate release and secretion. Furthermore, potentiatory effects of R59022 on 5HT secretion induced by phorbol 12-myristate 13-acetate and ionomycin, which are effects unlikely to be related to inhibition of DAG kinase was observed, and these effects further underline the non-specificity in the actions of R59022 and its limitations as a tool in studying platelet stimulus-response coupling.     

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

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

Human platelet electrorotation change induced by activation: inducer specificity and correlation to serotonin release

Electrorotation of single platelets was compared with [14C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0.10(-7) S.m-1 to 1.0.10(-4) S.m-1) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.     

Inhibition of fibrinogen receptor expression and serotonin release by leupeptin and antipain

Human platelet agonists such as thrombin, ADP, and collagen stimulate the rapid expression of fibrinogen receptors. In other cell types, calcium-activated proteases have been suggested to participate in the mechanism of expression of cell surface receptors (Lynch, G., and Baudry, M. (1984) Science 224, 1057-1063). In platelets the majority of the neutral protease activity is calcium-activated protease. We examined the effects of leupeptin and antipain, two calcium-activated protease inhibitors, on the expression of platelet fibrinogen receptors. These inhibitors abolished thrombin and ADP-induced fibrinogen binding. This inhibition required the addition of leupeptin or antipain prior to the agonist and was not due to displacement of fibrinogen from its receptor or inhibition of agonist binding to platelets. Leupeptin and antipain also inhibited fibrinogen-independent thrombin-stimulated release of serotonin. These results are discussed in relation to the involvement of calcium-activated protease in early events of platelet activation.     

Weak inhibition of protein kinase C coupled with various non-specific effects make sphingosine an unsuitable tool in platelet signal transduction studies

The effects of sphingosine, the newly described inhibitor of the enzyme protein kinase C, on human platelet activation, were studied in order to gain further information on the role of protein kinase in platelet responses. Concentrations of the drug (5-20 microM) which had little effect on protein kinase C activation as measured by the phosphorylation of the 45 kDa and 20 kDa protein substrates induced by phorbol 12-myristate 13-acetate (PMA) and thrombin, strongly inhibited platelet aggregation induced by these agonists, as well as aggregation induced by ADP and ionomycin, which caused no detectable protein kinase C activation or 5-hydroxy[14C]tryptamine[( 14C]5HT) secretion. At approx. 10-fold higher concentrations (150-200 microM), sphingosine had significant inhibitory effects on PMA and thrombin-induced 45 kDa and 20 kDa protein phosphorylation. However, at these high concentrations, the drug caused extensive membrane damage/leakiness as suggested by the substantial release of [14C]5HT and [3H]adenine from pre-loaded platelets (50-70% release of both markers), and the total quenching of quin2 fluorescence by Mn2+ in the presence of the drug. Due to the increased membrane leakiness in the presence of the drug, an apparent potentiation of agonist-induced intracellular Ca2+ elevations in quin2-loaded platelets, as well as an increase in quin2 fluorescence with the drug alone (more than 50 microM) were also observed. Despite this, however, thrombin-induced [3H]arachidonate release was severely reduced in the presence of sphingosine, underlining the inhibitory effects at the membrane level. It is concluded that the weak, if any, inhibitory effects on protein kinase C at concentrations not affecting membrane integrity, as well as the inhibitory effects of sphingosine on platelet aggregation, make it an unsuitable compound as a tool for studies on platelet stimulus-response coupling.     

Modulation of platelet activation by native DNA.

Native DNA (dsDNA) was found to induce the aggregation of isolated human platelets and the release of platelet 5HT; this activation was inhibited by both theophylline and TYA, suggesting a role for cAMP and metabolic products formed from arachidonate. By contrast, nonaggregating amounts of dsDNA inhibited platelet activation induced by collagen or thrombin. This inhibition, which could be overcome by use of greater amounts of the stimulatory agents, was not associated with the loss of platelet viability. Activation of platelets by dsDNA was not observed in plasma or in isolated platelet systems to which small amounts of cell-free plasma were added. However, dsDNA maintained in plasma its ability to inhibit platelet aggregation induced by collagen and thrombin. RNA and single-stranded DNA failed to induce platelet aggregation or release of 5HT and to block the platelet activation stimulated by dsDNA. Further, dsDNA did not significantly inhibit platelet aggregation in platelet-rich plasma stimulated by ADP or epinephrine. These data implicate dsDNA as a selective and potentially important activator and modulator of platelet responsiveness.