TYROSINE PHOSPHORYLATION OF PP125(FAK) IN PLATELETS REQUIRES COORDINATED SIGNALING THROUGH INTEGRIN AND AGONIST RECEPTORS

FAK is a focal adhesion kinase that is phosphorylated on tyrosine in a ctivated platelets. Induction of FAK phosphorylation requires both fib rinogen binding to integrin alpha(IIb)beta(3) and post-occupancy event s during agonist-induced platelet aggregation or platelet spreading on a fibrinogen matrix. To identify the signaling pathways necessary for tyrosine phosphorylation of FAK, we have examined the conditions that stimulate or inhibit this phosphorylation in platelets in which fibri nogen binding to alpha(IIb)beta(3) and platelet aggregation were induc ed directly with an anti-beta(3), Fab fragment (anti-LIBS6). Apyrase w as added to prevent effects of the endogenous platelet agonist, ADP. U nder these conditions, neither fibrinogen binding nor primary platelet aggregation was sufficient to induce FAK phosphorylation, suggesting that a second ''costimulatory'' event was required. Indeed, when epine phrine was added with fibrinogen and anti-LIBS6, large platelet aggreg ates formed and FAK phosphorylation occurred. This response was preven ted by blockade of cyclooxygenase with indomethacin or thromboxane A(2 ) receptors with SQ 30,741. A stable thromboxane A(2) analogue (U46619 ) could substitute for epinephrine as the costimulus. Epinephrine cost imulation of FAK phosphorylation was also prevented by chelation of in tracellular Ca2+ with BAPTA or selective inhibition of protein kinase C (PKC) with bisindolylmaleimide, indicating that Ca2+ and PKC are nec essary for FAK phosphorylation under these conditions. Epinephrine als o promoted FAK phosphorylation and adhesive spreading of apyrase-treat ed platelets on a fibrinogen matrix. Cytochalasin D, an inhibitor of a ctin polymerization, blocked FAK phosphorylation under all these condi tions. Thus, tyrosine phosphorylation of FAK in platelets requires coo rdinated signaling through occupied integrin and agonist receptors. Th ese separate pathways may converge to increase free Ca2+ and activate PKC and thus promote the cytoskeletal reorganization required for acti vation of FAK.