Cytosolic Phospholipase A2α (cPLA2α) Functions at the Nexus of Bidirectional Integrin Signaling in Platelets.

cPLA2 catalyzes the release of arachidonic acid from the sn-2 position of platelet membrane glycerophospholipids, leading via cyclooxygenase to the production of thromboxane A2 (TXA2), a platelet agonist. One function of TXA2 and other platelet agonists is to activate the fibrinogen-binding function of integrin αIIbβ3 through a series of biochemical steps known as inside-out signaling. While some platelet agonists stimulate cPLA2 activity and TXA2 production during inside-out signaling, others appear to require fibrinogen binding to αIIbβ3 and subsequent outside-in signaling through the integrin to elicit TXA2 production. However, the precise relationship between cPLA2 and αIIbβ3 is unclear. Here we have discovered that cPLA2α, the principal cPLA2 isoform in platelets, interacts with αIIbβ3 and participates in both inside-out and outside-in integrin signaling. A pool of cPLA2α was constitutively associated with αIIbβ3 in human and murine platelets, as determined by co-immunoprecipitation with antibodies to cPLA2α or αIIbβ3. Although the mode of interaction of cPLA2α with αIIbβ3 is unknown, vimentin, an intermediate filament protein known to bind to cPLA2α and integrin β tails, co-immunoprecipitated with both cPLA2 and αIIbβ3 from platelets. Selective induction of fibrinogen binding to platelets by MnCl₂ caused a detectable increase in cPLA2 activity in both whole platelet extracts and αIIbβ3 immunoprecipitates. However, this response was not observed in platelets from mice deficient in cPLA2α or in human platelets pre-treated with pyrrophenone, a selective cPLA2α inhibitor. Integrin-dependent activation of cPLA2α had functional consequences. First, thromboxane A2 produced in response to fibrinogen binding to αIIbβ3 was required for the recruitment of activated protein kinase Cβ to the αIIbβ3 signaling complex, an event known to be required for cytoskeletal reorganization and full platelet spreading on fibrinogen. Second, platelets from cPLA2α-deficient mice exhibited reduced spreading on fibrinogen compared to platelets from wild-type littermate controls (P < 0.05). Similarly, human platelets pre-treated with either pyrrophenone or a thromboxane A2 receptor antagonist spread poorly on fibrinogen compared to vehicle-treated control platelets (P < 0.05). Third, cPLA2α-deficient platelets stimulated by the GP VI collagen receptor agonist, CRP, exhibited decreased binding of soluble fibrinogen (P < 0.05). These studies establish that cPLA2α can function in association with αIIbβ3 to produce a pool of TXA2 that promotes integrin-dependent activation of protein kinase Cβ, cytoskeletal reorganization and platelet spreading. Given the previous demonstration of prolonged bleeding times and protection from thromboembolism in cPLA2α-deficient mice, these results imply that normal platelet function requires cPLA2α activation both upstream and downstream of αIIbβ3.