Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Integrin alpha 2 beta 1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3K beta, but not of PI3K gamma or PI3K alpha. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3K beta (PI3K beta(KD)), but occurred normally in PI3K gamma(KD) platelets. Integrin alpha 2 beta 1 failed to stimulate PI3K beta in platelets from phospholipase C gamma 2 (PLC gamma 2)-knockout mice, and we found that intracellular Ca2+ linked PLC gamma 2 to PI3K beta activation. Integrin alpha 2 beta 1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLC gamma 2 and intracellular Ca2+. Whereas activation of Pyk2 occurred normally in PI3K beta(KD) platelets, stimulation of PI3K beta was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3K beta was required for alpha 2 beta 1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin alpha IIb beta 3 were reduced after inhibition of PI3K beta and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3K beta and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3K beta downstream of integrin alpha 2 beta 1, and document a novel role for Pyk2 and PI3K beta in integrin alpha 2 beta 1 promoted inside-out activation of integrin alpha IIb beta 3 and thrombus formation. (Blood. 2012;119(3):847-856)