Specific cysteines in beta3 are involved in disulfide bond exchange-dependent and -independent activation of alphaIIbbeta3.
Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of beta3 was suggested to be involved in activation of alphaIIbbeta3. To investigate the role of specific beta3 cysteines in alphaIIbbeta3 expression and activation, we expressed in baby hamster kidney cells normal alphaIIb with normal beta3 or beta3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and beta-tail domains and assessed alphaIIbbeta3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of alphaIIbbeta3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active alphaIIbbeta3, implying that these bonds stabilize the inactive alphaIIbbeta3 conformer. Mutants of the Cys-567-Cys-581 bond in EGF-4 were inactive even after exposure to alphaIIbbeta3-activating antibodies, indicating that this bond is necessary for activating alphaIIbbeta3. Disrupting Cys-560-Cys-583 in the EGF-3/EGF-4 or Cys-608-Cys-655 in beta-tail domain resulted in alphaIIbbeta3 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 alphaIIbbeta3 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 alphaIIbbeta3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in alphaIIbbeta3 activation. These data suggest that different disulfide bonds in beta3 EGF and beta-tail domains play variable structural and regulatory roles in alphaIIbbeta3.
Mor-Cohen
Ronit
R
Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer 52621.
Rosenberg
Nurit
N
Landau
Meytal
M
Lahav
Judith
J
Seligsohn
Uri
U
D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics
D000911:Antibodies, Monoclonal; D004220:Disulfides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D003545:Cysteine