Human platelets stimulated by epinephrine undergo enhanced turnover of phosphatidylinositol 4,5-bisphosphate, accumulate inositol trisphosphate, diacylglycerol, and phosphatidic acid, and phosphorylate a 47-kDa protein.All of these phenomena indicate stimulation of phospholipase C. These responses are blocked completely by inhibitors of a2-adrenergic receptors (yohimbine), cyclooxygenase (aspirin or indomethacin), phospholipase A [2-(p-amylcinnamnoyl)amino-4-chlorobenzoic acid (ONO-RS-082)], Na+/H+ exchange [ethylisopropylamiloride (EIPA)], fibrinogen binding to glycoprotein [lb/I11a (antibody A2A9), Ca2+/Mg' binding (EDTA), or removal of fibrinogen. Epinephrine evokes (i) an increased turnover of ester-linked arachidonic acid in aspirintreated platelets that is inhibited by ONO-RS-082, EDTA, yohinbine, or the absence of fibrinogen and (ii) a rapid cytoplasmic alkalinization that is inhibited partially by blockage of cyclooxygenase activity and completely by A2A9 or EIPA. In contrast, when incubated with subaggregatory concentrations of the prostaglandin H2/thromboxane A2 analogue [(15S)-hydroxy-1 ia,9a-(epoxymethano)prosta-5,13-dienoic acid (U46619) and epinephrine, aspirin-treated platelets show a potentiation of phospholipase C activation that is unaffected by the above inhibitors. We propose that epinephrine, in promoting exposure of glycoprotein Ilb/ia sites for fibrinogen binding, leads to a cytoplasmic alkalinization, which, in conjunction with local shifts in Ca2 , promotes low-level activation of phospholipase A. The resulting free arachidonic acid is converted to cyclooxygenase products, which, potentiated by epinephrine, activate phospholipase C. This further amplifies the initial stimulatory response.Human platelets undergo aggregation and secretion of granule contents following exposure to epinephrine in the presence of fibrinogen and Ca2". Accompanying such activation is an enhanced turnover of phosphatidylinositol catalyzed by phospholipase C (PLC), indicated in earlier studies by stimulated incorporation of [3H]glycerol (1, 2), diacylglycerol accumulation, and activation of protein kinase C (3). However, human platelet adrenergic receptors have been characterized as being primarily of the a2 subclass (4, 5), which, in other tissues, is not linked with phosphatidylinositol phosphate turnover (6).A resolution of this paradox may lie in a report that epinephrine-stimulated generation of [32P]phosphatidic acid (PtdOH), an indirect indicator of PLC activation, can be inhibited by aspirin (7). This implies a possible role for the cyclooxygenase-catalyzed oxygenation of arachidonic acid in mediating the process of PLC activation in response to epinephrine. Recently, investigators have found that epinephrine promotes arachidonic acid mobilization (a precondition for cyclooxygenase action) in a manner dependent upon Na+/H+ exchange (8). This mobilization was quantitated by measuring thromboxane B2 (TXB2), the stable metabolite of thromboxane A2, which is, in turn, formed by way of cyclooxygenase. ...

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.

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Thrombin stimulates phospholipase C and inhibits adenylate cyclase in human platelets. We have studied the effect of purified S1 monomer, the ADP-ribosylating subunit of pertussis toxin, on these receptor-coupled G-protein-dependent activities. ADP-ribosylation of a 41 kDa protein is associated with a marked decrease in the ability of thrombin to inhibit cyclic AMP formation, but has little effect on phospholipase C. Therefore adenylate cyclase and phospholipase C appear to be modulated by different G-proteins.

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