Binding of Thrombin to the G-protein-linked Receptor, and Not to Glycoprotein Ib, Precedes Thrombin-mediated Platelet Activation

• Published in: The Journal of biological chemistry 1997-01, Vol.272 (3), p.1997-2004
• Main Authors: Liu, Longbin, Freedman, John, Hornstein, Adriana, Fenton, John W., Song, Yingqi, Ofosu, Frederick A.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - immunology; Binding Sites; GTP-Binding Proteins - metabolism; Humans; Hydrolysis; Platelet Activation; Platelet Glycoprotein GPIb-IX Complex - immunology; Platelet Glycoprotein GPIb-IX Complex - metabolism; Protein Binding; Receptors, Thrombin - metabolism; Thrombin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.3.1997

The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as α-thrombin-binding sites on platelets remain controversial. α-Thrombin has been proposed to bind to both GPIb and the hirudin-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 α-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 ≥0.5 nMα-thrombin for 10-60 s at 37°C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 1011 platelets/liter) after their incubation with 10 nMα-thrombin for 10 s. Parallel binding of α-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 α-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by ≤1.0 nM (but not 10 nM) α-thrombin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on α-thrombin binding to platelets or their subsequent activation. In contrast, chymotrypsin, which cleaves both GPIb and the G-protein-linked receptor, abrogated α-thrombin binding to platelets, TR-(1-41) release, and platelet activation. Furthermore, monoclonal antibodies directed against the reported α-thrombin-binding site on GPIb inhibited neither α-thrombin binding to nor activation of the platelets. Thus, α-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 α-thrombin activates platelets.