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J. Biol. Chem., Vol. 282, Issue 42, 30434-30441, October 19, 2007

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Ligand Binding Rapidly Induces Disulfide-dependent Dimerization of Glycoprotein VI on the Platelet Plasma Membrane^*

Jane F. Arthur, Yang Shen, Mark L. Kahn, Michael C. Berndt, Robert K. Andrews, and Elizabeth E. Gardiner¹

From the Department of Immunology, Monash University, Melbourne 3004, Australia and the Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin _IIb3-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an 55-kDa soluble ectodomain fragment and an 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10–30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.

Received for publication, February 15, 2007 , and in revised form, August 6, 2007.

^* This work was supported by the National Health and Medical Research Council and the National Heart Foundation of Australia. 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: Dept. of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne 3004, Australia. Tel.: 61-3-9903-0661; Fax: 61-3-9903-0038; E-mail: elizabeth.gardiner{at}med.monash.edu.au.

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