Summary

The platelet collagen receptor, glycoprotein (GP)VI, initiates plateletaggregation at low shear stress while GPIb-IX-V, whichbinds von Willebrand factor, elicits platelet aggregation underhigh shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface,we first ascertainedthat aggregation induced by a GPVI-specific agonist,collagen-related peptide, like collagen, is markedly cross-blockedby a GPIb a -specific monoclonal antibody, SZ2. Immunoprecipitationof GPIb-IX with anti-GPIba from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblottingwith anti-GPVI demonstrated association between GPIb-IXand GPVI.This association was maintained when platelets wereactivated by thrombin. Pre-treatment of platelets with methyl-β-cyclodextrin to disrupt lipid rafts did not affect association inresting platelets under these conditions of detergent lysis.Theassociation is also independent of cytoskeletal attachment, sinceit was unaffected by treatment with N -ethylmaleimide or DNaseI,which dissociate GPIb-IX from filamin and the actin-containingcytoskeleton, respectively. Finally, the association involves aninteraction between the ectodomains of GPIba and GPVI, sincesoluble fragments of GPIb a (glycocalicin) and GPVI are co-precipitatedfrom the platelet supernatant under conditions whereGPVI is shed.A contribution of GPIb-IX-V to GPVI-induced plateletresponses, and vice versa, therefore warrants further investigation.