## Abstract

We have investigated factors affecting the activation of phospholipase C in human platelets. Prior exposure of platelets to phorbol esters that stimulate protein kinase C inhibits the activation of phospholipase C in response to a variety of receptor-directed agonists, including <latex>$\alpha$</latex>- and <latex>$\gamma$</latex>-thrombin and thromboxane A<latex>$_2$</latex> analogues. Such activation has been assayed by measurements of accumulated InsP<latex>$_3$</latex> (including Ins(1,4,5)P<latex>$_3$</latex> and Ins(1,3,4)P<latex>$_3$</latex>) and PtdOH. Inhibition is not overcome by Ca<latex>$^{2+}$</latex> ionophores, and substances that block or mimic Na<latex>$^+$</latex>-H<latex>$^+$</latex> exchange neither block nor mimic these inhibitory effects. Cyclic AMP and cyclic GMP, other agents known to inhibit phospholipase C activation, do not accumulate in platelets exposed to phorbol esters. Although a portion of the effects of phorbol ester on InsP<latex>$_3$</latex> accumulation may be explained by 5-phosphomonoesterase activity, it is likely that more direct effects on phospholipase C are being exerted as well, and contribute the major inhibitory route. We have examined the susceptibility of adenylyl cyclase-associated G<latex>$_i$</latex> and `G<latex>$_p$</latex>'-activated phospholipase C to inhibitory ADP-ribosylation by pertussis toxin-derived enzyme (S<latex>$_1$</latex> protomer) administered to saponin-permeabilized platelets. The effects of <latex>$\alpha$</latex>-thrombin on adenylyl cyclase can be inhibited by up to 50% by S<latex>$_1$</latex>, at which point inhibition of phospholipase C is barely detectable. Thromboxane A<latex>$_2$</latex> analogues, which do not affect adenylyl cyclase (G<latex>$_i$</latex>), stimulate phospholipase C; this effect is not impaired by S<latex>$_1$</latex>. We therefore propose that the inhibitory effects of phorbol esters on the activation of phospholipase C are not mediated primarily by effects on G<latex>$_i$</latex>.