Phosphatidylinositol 3,4,5-trisphosphate regulates Ca2+ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity

The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated, PI 3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain- containing inositol 5-phosphatase (SHIP), in response to collagen related p eptide (CRP). Tyrosine phosphorylation and activation of phospholipase C ga mma 2 (PLC gamma 2) were unaltered in SHIP-/- platelets, whereas Btk was he avily tyrosine phosphorylated under basal conditions and maximally phosphor ylated by low concentrations of CRP. There was an increase in basal Ca2+, m aximal expression of P-selectin, and potentiation of Ca2+ and aminophosphol ipid exposure to CRP in SHIP-/- platelets in the presence of Ca2+ (1 mM). M icroinjection of PI3,4,5P(3) into megakaryocytes caused a 3-fold increase i n Ca2+ in response to CRP, which was absent in X-linked immunodeficiency (X id) mice, which have a mutation in the PH domain of Btk. There was a corres ponding partial reduction in the sustained level of intracellular Ca2+ in r esponse to CRP in Xid mice but no change in PLC activity. These results dem onstrate a novel pathway of Ca2+ entry that involves PI3,4,5P(3) and Btk, a nd which is independent of increased PLC activity.