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 glyco protein Ib (GPIb) as alpha-thrombin-binding sites on platelets remain controversial. alpha-Thrombin has been proposed to bind to both GPIb a nd the hirudin-like like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 4 1-mer peptide (TR-(1-41), where TR is alpha-thrombin receptor)) to ini tiate platelet activation. Using affinity-purified rabbit anti-human T R-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release fro m platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and inc ubated 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 plat elets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein linked receptor abrogated alp ha-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-throm bin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on cu-thrombin bindin g to platelets or their subsequent activation. In contrast, chymotryps in, which cleaves both GPIb and the G-protein linked receptor, abrogat ed alpha-thrombin binding to platelets, TR-(1-41) release, and platele t activation. Furthermore, monoclonal antibodies directed against the reported alpha-thrombin-binding site on GPIb inhibited neither alpha-t hrombin binding to nor activation of the platelets. Thus, alpha-thromb in binds to and cleaves the G-protein-linked receptor when it activate s platelets, and GPIb does not appear to serve as an important binding site when alpha-thrombin activates platelets.