Characterization of a gain of function mutation of integrin α_IIbβ₃ (platelet glycoprotein IIb-IIIa)

Integrin α_IIbβ₃ (platelet glycoprotein IIb-IIIa) is a prototype of integrins involved in cellular adhesive functions. As part of a structure-function analysis of this molecule, we constructed a mutant, designated α_IIbβ₃(β_1-2), by replacing 6 amino acids within a putative ligand binding domain of the β₃ subunit with sequences derived from β₁. The alteration did not affect the capacity of β₃(β_1-2) to combine with transfected α_IIb, nor did it cause it to combine with endogenous α₅. Integrin α_IIbβ₃(β_1-2) was in a "resting" state on Chinese hamster ovary cells as judged by minimal binding of an activation-specific anti-α_IIbβ₃, PAC1. Nevertheless, cells expressing α_IIbβ₃(β_1-2) spontaneously bound fibrinogen with low affinity (K_α = (4.85 ± 0.84) × 10⁶ M^-1). Activation with an anti-β₃ antibody (monoclonal antibody 62) resulted in a 10-fold increase in fibrinogen binding affinity (K_α = (4.55 ± 0.77) × 10⁷ M^-1), which was 3-fold greater than fibrinogen binding to activated wild type α_IIbβ₃ (K_α = (1.66 ± 0.33) × 10⁷ M^-1, F= 7.46, p = 0.008). The mutant receptor also bound fibrinogen mimetic peptide ligands with enhanced affinity as measured by the conformation-specific antibody, anti-LIBS1. This indicates that the increased affinity for fibrinogen was caused by enhanced interaction of α_IIbβ₃(β_1-2) with known recognition sequences in fibrinogen. Thus, this gain of function mutant augments ligand binding function, supporting a role for this region of the β subunit in ligand binding to integrins.