Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Abstract

Abstract. Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125 FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin C~m~3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-~3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in otm~3 Proteins of 50-68 kD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125 FA~. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, 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) t~3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or T4°°-4u). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to O/I~3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125 FAr. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.

analysis of CD9, CD32 and p67 signalling: use of degranulated platelets indicates direct involvement of CD9 and p67 in integrin activation

“…The extent of this phosphorylation correlated with the extent of aggregation, being least in the case of FcgRII crosslinking. A similar profile of protein phosphorylation is observed during integrin-mediated platelet activation (Huang et al, 1993). However, the presence of RGDS peptide (to block integrin-mediated aggregation) had no effect on the profile of tyrosine phosphorylation induced by these mAbs.…”

“…This indicates that each of the target antigens may generate some specific signals which ultimately converge to a common pathway. Many of the reactions observed in mAb-stimulated platelets were shared and resembled those observed in platelets stimulated through crosslinking of adhesion receptors by macromolecular ligands (Huang et al, 1993). A convergence of signalling of the type we propose for mAbs has been demonstrated by the phosphorylation of pp125 FAK upon ligand binding to either gpIIb/IIIa or gpIa/IIa (Haimovich et al, 1993).…”

“…This similarity in protein tyrosine phosphorylation induced by different agonists suggests a common signal. It might be argued that this signal originates from integrin engagement during aggregation since integrin-mediated signalling involves similar protein tyrosine phosphorylation (Huang et al, 1993). Therefore the response to mAbs might be related more to the induction of platelet aggregation than to a specific effect contributed by the respective target antigens.…”

The Platelet Antigens CD9, CD42 and Integrin αIIbβIIIa Can be Topographically Associated and Transduce Functionally Similar Signals

“…This suggests that the 129-kDa protein is pp125FAK, the phosphorylation of which is known to be dependent on ligand-induced cross-linking of gpIIb/IIIa (Lipfert et al, 1992;Huang et al, 1993). The phosphorylation of the 64-kDa protein was not observed in anti-PTAI -stimulated thrombasthenic platelets, but was present in thrombasthenic cells stimulated with mAb against CD9 and CD42.…”

“…4B). This suggests that the phosphorylation of the latter protein may be linked to the presence of gpIIb/IIIa, indicating that this protein might be pp12SFAK, a protein known to become tyrosine phosphorylated in a gplIb/ IIIa-dependent manner (Lipfert et al, 1992;Huang et al, 1993).…”