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

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Abstract
The role of phosphatidylinositol 3,4,5‐trisphosphate (PI3,4,5P3) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P3 was increased in platelets from mice deficient in the SH2 domain‐containing inositol 5‐phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cγ2 (PLCγ2) were unaltered in SHIP−/− platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca2+, maximal expression of P‐selectin, and potentiation of Ca2+ and aminophospholipid exposure to CRP in SHIP−/− platelets in the presence of Ca2+ (1 mM). Microinjection of PI3,4,5P3 into megakaryocytes caused a 3‐fold increase in Ca2+ in response to CRP, which was absent in X‐linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca2+ in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca2+ entry that involves PI3,4,5P3 and Btk, and which is independent of increased PLC activity.