Affinity Modulation of Platelet Integrin α(IIb)β(3) by β(3)-Endonexin, a Selective Binding Partner of the β(3) Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α(IIb)β(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. β(3)-Endonexin is a novel 111–amino acid protein that binds selectively to the β(3) tail. Since β(3)-endonexin is present in platelets, we asked whether it can affect α(IIb)β(3) function. When β(3)-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α(IIb)β(3) affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α(IIb)β(3) bound little or no PAC1. However, those transfected with GFP/β(3)-endonexin and α(IIb)β(3) bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β(3)-endonexin did not affect levels of surface expression of α(IIb)β(3) nor did it modulate the affinity of an α(IIb)β(3) mutant that is defective in binding to β(3)-endonexin. Affinity modulation of α(IIb)β(3) by GFP/β(3)-endonexin was inhibited by coexpression of either a monomeric β(3) cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β(3)-endonexin can modulate the affinity state of α(IIb)β(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 α(IIb)β(3).