Journal of Thrombosis and Haemostasis, 5: 1026–1033
ORIGINAL ARTICLE Glycoprotein VI oligomerization in cell lines and platelets
O. BERLANGA,1 *T.BORI-SANZ,1 *J.R.JAMES, J. FRAMPTON,* S. J. DAVIS, M. G. TOMLINSON* and S . P . W A T S O N * *Institute of Biomedical Research, Medical School, University of Birmingham, Edgbaston, Birmingham; and Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, University of Oxford, Oxford, UK
To cite this article: Berlanga O, Bori-Sanz T, James JR, Frampton J, Davis SJ, Tomlinson MG, Watson SP. Glycoprotein VI oligomerization in cell lines and platelets. J Thromb Haemost 2007; 5: 1026–33. mediated through two distinct receptor classes on the platelet
Summary. Background: Glycoprotein VI (GPVI) is a physio- surface, the integrin aIIb1 and the glycoprotein VI (GPVI)–FC logic receptor for collagen expressed at the surface of platelets receptor (FcR) c-chain receptor complex [1]. Blocking of either and megakaryocytes. Constitutive dimerization of GPVI has receptor in vitro using specific antibodies inhibits or delays, been proposed as being necessary for the interaction with respectively, collagen-induced platelet aggregation [2–4]. Simi- collagen, although direct evidence of dimerization has not been larly, platelets deficient in aIIb1 or GPVI–FcR c-chain show reported in cell lines or platelets. Objectives: To investigate loss of reactivity towards collagen in vitro [5–8]. Furthermore, oligomerization of GPVI in transfected cell lines and in platelets mice deficient in GPVI–FcR c-chain are protected against under non-stimulated conditions. Methods and results: By lethal thromboembolism [8,9], illustrating the crucial role that using a combination of molecular and biochemical techniques, the receptor plays in vivo under pathologic conditions. we demonstrate that GPVI association occurs at the surface of Although the intracellular signaling events mediated by aIIb1 transfected 293T cells under basal conditions, through an in platelets have remained elusive [10,11], the mechanism of interaction at the extracellular domain of the receptor. action of GPVI has been well documented and remains an area Bioluminescence resonance energy transfer was used to confirm of intense research. GPVI mediates platelet activation in oligomerization of GPVI under these conditions. A chemical response to collagen through a pathway that shares many crosslinker was used to detect constitutive oligomeric forms of features with those used by immune receptors such as FceRI, GPVI at the surface of platelets, which contain the Fc receptor and T-cell and B-cell antigen receptors [12]. As GPVI has no (FcR) c-chain. Conclusions: The present results directly dem- intrinsic signaling capacity, it is widely recognized that it must be onstrate GPVI–FcR c-chain oligomerization at the surface of coexpressed in association with the FcR c-chain, which acts as the platelet, and thereby add to the growing evidence that the signaling partner. Furthermore, this association is a prere- oligomerization of GPVI may be a prerequisite for binding of quisite for surface expression of GPVI on mouse platelets [13]. the receptor to collagen, and therefore for proper functioning of Froma structure–function pointofview,thereare several lines platelets upon vascular damage. of circumstantial evidence to suggest that GPVI functions as a dimer on the platelet surface. Moroi and coworkers have Keywords: BRET, dimerization, glycoprotein VI, GPVI, olig- demonstrated, using recombinant protein, that collagen binds omerization, platelets. to the dimeric but not the monomeric form of GPVI, and that only the former is able to attenuate collagen-induced platelet Introduction aggregation [14]. In contrast, both the monomeric and dimeric formsofGPVIbindtoimmobilizedconvulxinandinhibitplatelet The extracellular matrix protein collagen is the major and most aggregation induced by the snake toxin with similar concentra- thrombogenic component of the vessel wall. Circulating tion dependencies [14]. The possibility that GPVI functions as a platelets adhere to exposed collagen and undergo activation, dimer is strongly reinforced by studies analyzing the ability of a leading to thrombus formation. The interaction with collagen is series of synthetic peptides with differentially spaced GPVI- recognition motifs to activate the collagen receptor in platelets Correspondence: Steve P. Watson, Institute of Biomedical Research, [15]. Finally, structural studies of the two immunoglobulin (Ig) Medical School, University of Birmingham, Edgbaston, Birmingham domains of human GPVI have revealed the formation of a back- B15 2TT, UK. to-back dimer in the crystal structure, which is mediated through Tel.: +44 0 121 414 6514; fax: +44 0 121 415 8817; e-mail: s.p. the more membrane-proximal of the two Ig domains [16]. [email protected] As the FcR c-chain is present as a disulfide-linked homo- 1These authors contributed equally to this work. dimer, it has been proposed that each chain associates independently with GPVI [17]. In light of a recent report Received 13 May 2006, accepted 9 February 2007 indicating that the two chains of the FcR c-chain are necessary