Integrin α IIbβ 3 plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of α IIbβ 3 to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins' cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of α IIbβ 3 to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between α IIb and β 3 cytoplasmic tails, we showed that its binding site in the membrane-proximal β 3 715-730 segment is cryptic and becomes exposed as a result of binding of isolated α IIbβ 3 to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with α IIbβ 3 in resting platelets or suspended α IIbβ 3-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only β 3 but also the membrane-proximal 989-1000 segment of the α IIb cytoplasmic tail binds the skelemin fragment. Finally, the same residues, α 3 Val 990, α IIb Arg 995, and β 3 His 722, involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of α IIbβ 3 by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between α IIb and β 3 cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.
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