Activation of Protein Kinase C Is Required for the Stable Attachment of Adherent Platelets to Collagen but Is Not Needed for the Initial Rapid Adhesion Under Flow Conditions

Abstract

Abstract —We have investigated the role of protein kinase C (PKC) in the initial events of α 2 β 1 -integrin–mediated platelet adhesion to collagen under flow conditions. Although adhesion caused activation of PKC, as evidenced by pleckstrin phosphorylation, the PKC inhibitors GF 109203X and Gö 6976 had no effect on adhesion, even though they prevented pleckstrin phosphorylation. The initial kinetics and extent of platelet adhesion to collagen (<5 seconds) and tyrosine phosphorylation of p125 FAK and p72 syk were not influenced by the PKC inhibitors, whereas adhesion to polylysine was prevented. These results indicate that adhesion to collagen and polylysine involve different mechanisms and requirements for PKC activation. Pretreatment with GF 109203X destabilized collagen-adherent platelets, accelerating their detachment, which was associated with tyrosine dephosphorylation of p125 FAK . Thus, although PKC activation was not required for rapid platelet adhesion to collagen, it appears to play an important role in stabilizing the attachment of adherent platelets to collagen. We also examined the effect of PKC activation by the phorbol ester phorbol 12-myristate 13-acetate (PMA) on platelet adhesion to collagen. PMA at 100 nmol/L strongly potentiated adhesion and tyrosine phosphorylation of p125 FAK and p72 syk and activated β 1 -integrins, as determined by increased exposure of the 15/7 epitope. The PMA-stimulated adhesion was partially blocked by an anti-α 2 β 1 antibody, was completely inhibited by GF 109203X, and was not correlated with the extent of pleckstrin phosphorylation. Therefore, strong PKC activation may lead to inside-out signaling, enhancing the role of β 1 -integrins in adhesion. Pleckstrin phosphorylation does not appear to be involved in the initial phase of basic or PMA-stimulated adhesion but may help stabilize the adherent platelets.