AFFINITY MODULATION OF PLATELET INTEGRIN ALPHA(IIB)BETA(3) BY BETA(3)-ENDONEXIN, A SELECTIVE BINDING PARTNER OF THE BETA(3) INTEGRIN CYTOPLASMIC TAIL

Platelet agonists increase the affinity state of integrin alpha IIb be ta(3), a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. beta(3)-endonexin is a novel 111-amino acid protein that bin ds selectively to the pg tail. Since beta(3)-endonexin is present in p latelets, we asked whether it can affect alpha IIb beta(3) function. W hen beta(3)-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and th e nucleus and could be detected on Western blots of cell lysates. PAC1 , a fibrinogen-mimetic mAb, was used to monitor alpha IIb beta(3) affi nity state in transfected cells by flow cytometry. Cells transfected w ith GFP and alpha IIb beta(3) bound little or no PAC1. However, those transfected with GFP/beta(3)-endonexin and alpha IIb beta(3) bound PAC 1 specifically in an energy-dependent fashion, and they underwent fibr inogen-dependent aggregation, GFP/beta(3)-endonexin did not affect lev els of surface expression of alpha IIb beta(3) nor did it modulate the affinity of an alpha IIb beta(3) mutant that is defective in binding to beta(3)-endonexin. Affinity modulation of alpha IIb beta, by GFP/be ta(3)-endonexin was inhibited by coexpression of either a monomeric be ta(3) cytoplasmic tail chimera or an activated form of H-Ras. These re sults demonstrate that beta(3)-endonexin can modulate the affinity sta te of alpha IIb beta(3) in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein-protein interactions at the level of the cytoplasmic tails of alpha IIb beta(3).