Specific cysteines in β3 are involved in disulfide bond exchange-dependent and -independent activation of αIIbβ3

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of beta 3 was suggested to be involved in activation of alpha IIb beta 3. To investigate the role of specific beta 3 cysteines in alpha IIb beta 3 expression and activation, we expressed in baby hamster kidney cells normal alpha IIb with normal beta 3 or beta 3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and beta-tail domains and assessed alpha IIb beta 3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of alpha IIb beta 3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active alpha IIb beta 3, implying that these bonds stabilize the inactive alpha IIb beta 3 conformer. Mutants of the Cys-567-Cys-581 bond in EGF-4 were inactive even after exposure to alpha IIb beta 3-activating antibodies, indicating that this bond is necessary for activating alpha IIb beta 3. Disrupting Cys-560-Cys-583 in the EGF-3/EGF-4 or Cys-608-Cys-655 in beta-tail domain resulted in alpha IIb beta 3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in alpha IIb beta 3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type alpha IIb beta 3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in alpha IIb beta 3 activation. These data suggest that different disulfide bonds in beta 3 EGF and beta-tail domains play variable structural and regulatory roles in alpha IIb beta 3.