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J. Biol. Chem., Vol. 283, Issue 28, 19235-19244, July 11, 2008

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Specific Cysteines in β3 Are Involved in Disulfide Bond Exchange-dependent and -independent Activation of IIbβ3^*

Ronit Mor-Cohen, Nurit Rosenberg, Meytal Landau, Judith Lahav^¶, and Uri Seligsohn¹

From the Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer 52621, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, the Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, and the ^¶Hemostasis Laboratory, Rabin Medical Center, Belinson Campus, Petah-Tiqva 49100 and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of β3 was suggested to be involved in activation of IIbβ3. To investigate the role of specific β3 cysteines in IIbβ3 expression and activation, we expressed in baby hamster kidney cells normal IIb with normal β3 or β3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and β-tail domains and assessed IIbβ3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of IIbβ3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active IIbβ3, implying that these bonds stabilize the inactive IIbβ3 conformer. Mutants of the Cys-567–Cys-581 bond in EGF-4 were inactive even after exposure to IIbβ3-activating antibodies, indicating that this bond is necessary for activating IIbβ3. Disrupting Cys-560–Cys-583 in the EGF-3/EGF-4 or Cys-608–Cys-655 in β-tail domain resulted in IIbβ3 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 IIbβ3 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 IIbβ3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in IIbβ3 activation. These data suggest that different disulfide bonds in β3 EGF and β-tail domains play variable structural and regulatory roles in IIbβ3.

Received for publication, March 27, 2008

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 972-3-5302104; Fax: 972-3-5351568; E-mail: seligson{at}sheba.health.gov.il.

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