Binding of thrombin to the G-protein-linked receptor, and not to glycoprotein Ib, precedes thrombin-mediated platelet activation

The roles of the G protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as alpha-thrombin-binding sites on platelets remain controversial. alpha-Thrombin has been proposed to bind to both GPIb and the hirudin-like like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 41-mer peptide (TR-(1-41), where TR is alpha-thrombin receptor)) to initiate platelet activation. Using affinity-purified rabbit anti-human TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with greater than or equal to 0.5 nM alpha-thrombin for 10-60 s at 37 degrees C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 10(11) platelets/liter) after their incubation with 10 nM alpha-thrombin for 10 s. Parallel binding of alpha-thrombin to and activation of the platelets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein linked receptor abrogated alpha-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by less than or equal to 1.0 nM (but not 10 nM) alpha-thrombin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on cu-thrombin binding to platelets or their subsequent activation. In contrast, chymotrypsin, which cleaves both GPIb and the G-protein linked receptor, abrogated alpha-thrombin binding to platelets, TR-(1-41) release, and platelet activation. Furthermore, monoclonal antibodies directed against the reported alpha-thrombin-binding site on GPIb inhibited neither alpha-thrombin binding to nor activation of the platelets. Thus, alpha-thrombin binds to and cleaves the G-protein-linked receptor when it activates platelets, and GPIb does not appear to serve as an important binding site when alpha-thrombin activates platelets.