A collagen-related peptide regulates phospholipase C gamma 2 via phosphatidylinositol 3-kinase in human platelets

The collagen receptor glycoprotein VI (GPVI) induces platelet activation th rough a similar pathway to that used by immune receptors. In the present st udy we have investigated the role of phosphatidylinositol 3-kinase (PI 3-ki nase) in GPVI signalling. Our results show that collagen-related peptide {C RP:[GCP* (GPP*)(10)GCP*G](n); P* = hydroxyproline}, which is selective to G PVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3.4. 5)P-3] and phosphatidylinositol 3-4-bisphosphate [PI(3,4)P-2] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3- kinase, The formation of inositol phosphates and phosphatidic acid (PA), tw o markers of phospholipase C (PLC) activation, by CRP are inhibited by betw een 50 and 85% in the presence of wortmannin and LY294002. This is associat ed with inhibition of elevation of intracellular Ca2+ ([Ca2+](i)) and aggre gation, Wortmannin and LY294002 also partially inhibit elevation of Ca2+ by CRP in murine megakaryocytes, Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4,5)P- 3, but not the R28C (Arg(28) --> Cys) mutant which binds to PI(3,4,5)P-3, w ith low affinity, also inhibits elevation of [Ca2+](i) in megakaryocytes, s uggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylat ion of PLC gamma 2, These results demonstrate that PY 3-kinase is required for full activation of PLC gamma 2 by GPVI in platelets and megakaryocytes.