ADHESIVE LIGAND-BINDING TO INTEGRIN ALPHA(IIB)BETA(3) STIMULATES TYROSINE PHOSPHORYLATION OF NOVEL PROTEIN SUBSTRATES BEFORE PHOSPHORYLATION OF PP125(FAK)

Tyrosine phosphorylation of multiple platelet proteins is stimulated b y thrombin and other agonists that cause platelet aggregation and secr etion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggreg ation that follows fibrinogen binding to integrin alpha(IIb)beta3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agon ists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta3 antibody (anti-LIBS6) that directly exposes the fibrinog en binding site in alpha(IIb)beta3, Proteins of 50-68 kD and 140 kD be came phosphorylated on tyrosine residues in a fibrinogen-dependent man ner. This response did not require prostaglandin synthesis, an increas e in cytosolic free calcium, platelet aggregation or granule secretion , nor was it associated with tyrosine phosphorylation of pp125FAK. Tyr osine phosphorylation of the 50-68-kD and 140-kD proteins was also obs erved when (a) fibrinogen binding was stimulated by agonists such as e pinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (C) alpha(IIb)beta3 complexes were cross-linked by anti bodies, even in the absence of fibrinogen. In contrast, no tyrosine ph osphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytocha lasin D. These studies demonstrate that fibrinogen binding to alpha(II b)beta3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reactio n may depend on the oligomerization of integrin receptors and on the s tate of actin polymerization, organizational processes that may juxtap ose tyrosine kinases with their substrates.