Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly throughsecreted ADP

Platelet activation by thrombin or collagen results in secretion and synthe sis of several platelet agonists that enhance, the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-ind uced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P-2] a nd phosphatidylinositol 3,4,5-trisphosphate [PMIns(3,4,5)P-3]. In contrast, collagen induced massive production of PtdIns(3,4)P-2 and PtdIns(3,4,5)P-3 in the presence of IAS. When testing the effect of each inhibitor individu ally we found the strongest inhibition of thrombin-induced PtdIns(3,4)P-2 p roduction with the ADP scavenger system CP/CPK. Furthermore, we found a str ong synergistic effect between exogenously added ADP and thrombin on produc tion of PtdIns(3,4)P-2. In contrast to the results from I-phosphorylated ph osphoinositides, CP/CPK had little effect on thrombin-induced protein tyros ine phosphorylation. Our results show the importance of autocrine stimulati on in thrombin-induced accumulation of 3-phosphorylated phosphoinositides a nd raise the question as to whether thrombin by itself is capable of induci ng PI 3-K activation; In marked contrast to thrombin, collagen per se appea rs to be able to trigger increased production of PtdIns(3,4)P-2 and PtdIns( 3,4,5)P-3. (C) 1999 Academic Press.