In human platelets, prostaglandin E1 and forskolin inhibit 5-hydroxytryptamine-induced phospholipase C, C-kinase and myosin light-chain kinase activity in a concentration-dependent way. Phospholipase C activation, however, was only partly inhibited, and this at higher concentrations than the protein kinases. Direct activation of the C kinase either by exogenous synthetic diacylglycerol or by 12-O-tetradecanoylphorbol 13-acetate was antagonized by prostaglandin E1 and forskolin. Since C-kinase activation is one of the key events in excitatory signal transduction in the platelets, we suggest that the inhibitory effect of agents that increase platelet cyclic AMP on platelet secretion and aggregation might reside in their capacity to antagonize C-kinase activity.

The signal transducing system coupled to the serotonin-S2 receptor on platelets involves metabolism of inositol-containing phospholipid, elevation of intracellular free Ca2+ and activation of protein kinase C. Evidence for coupling of the serotonin-S2 receptor to the same signal transducing system in brain and smooth muscle tissue is reviewed.

SummaryR 68 070 or (E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]- methylen]amino]oxy] pentanoic acid (Janssen Research Foundation, Belgium) combines specific thromboxane A2 (TXA2) synthetase inhibition with TXA2/prostaglandin endoperoxide receptor blockade in one molecule.In vitro, the compound specifically inhibits the production of TXB2 from [14C] arachidonic acid by washed human platelets (IC50 = 8.2 × 10-9 M) and by platelet microsomes (IC50 = 3.6 × 10-9 M), of MDA (IC50 = 1.91 × 10-8 M) and of TXB2 (IC50 = 1.47 × 10-8 M) by thrombin-coagulated human platelet-rich plasma (P.R.P.) and whole blood respectively and increases the levels of PGD2, PGE2, PGF2α and 6-keto-PGF1α. The activity of cyclo-oxygenase-, prostacyclin synthetase-, 5-, 12- and 15-lipoxygenase-enzymes are not affected. Additionally, R 68 070 inhibits human platelet aggregation in P.R.P. induced by U 46619 3 × 10-7 M to 2 × 10-6 M (IC50 = 2.08 × 10-6 M to 2.66 × 10-5 M), collagen 0.5 to 2 μg/ml (IC50 = 2.85 × 10-6 M to 4.81 × 10-5 M), arachidonic acid 7.5 × 10-4 M to 2 × 10- M (IC50 = 2.1 × 10-8 M to 3.3 × 10-8 M) and the U 46619 (1 × 10-7 M)-induced accumulation of [32P] phosphatidic acid (IC50 = 5.24 × 10-7 M) in washed human platelets. Collagen (0.75 μg/ml)-induced ATP release (IC50 = 4.1 × 10-6 M), ADP (1 to 2.5 × 10-6 M)-induced second wave aggregation (IC50 = 3.19 × 10-6 M) in P.R.P. as well as the collagen (1 μg/ml)-induced adhesion/aggregation reaction in human whole blood (IC50 = 1.02 × 10-5 M) are reduced as well by the compoun.Primary platelet reactions induced by serotonin, ADP, PAF, or A 23187, platelet adenylate cyclase- and cAMP phosphodiesterase-activity, and platelet inhibitory activities of PGD2, PGI2, PGE2, PGE1 are not modified by R 68 070.This biochemical profile is compatible with a dual mechanism of action of R 68 070, namely TXA2 synthetase inhibition at low concentrations, plus additionally TXA2/prostaglandin endoperoxide receptor blockade at higher concentrations

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Simultaneous addition to platelets of submaximal amounts of excitatory agonists acts synergistically in provoking secretory and aggregatory responses. By measuring changes in intracellular free Ca* + concentration, inositol phospholipid metabolism and protein phosphorylation, we verified whether synergism could be evidenced at the level of signal transduction. Challenging platelets with epinephrine only induced minor changes on the measured parameters. However, when added together with serotonin, epinephrine amplified mobilisation of intracellular Ca2 + , PA formatio n, PIP formation, protein kinase C and myosin light chain kinase activity as compared to the alterations induced by serotonin alone. It is concluded that synergistic effects on simultaneous addition of serotonin and epinephrine might originate at the level of signal transduction.

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

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Five serotonin-S2 antagonists, ketanserin, pipamperone, methysergide, ritanserin, and LSD, were tested for their ability to inhibit signal transduction coupled to the serotonin-S2 receptor by measuring the serotonin-induced [32P] phosphatidic acid formation. The five drugs inhibited the response in a non-competitive manner. Since drugs such as ketanserin and pipamperone were found to dissociate rapidly from serotonin-S2 receptor sites in binding studies, slowly reversible binding can hardly explain the apparent non-competitive inhibition of the biochemical effect. The authors, therefore, propose a new model of interaction between the serotonin-S2 receptor macromolecule and the signal transducing system that is compatible with the present and previous experimental observations.