title: Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets.
abstract: The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.
authors: Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
authors: M
authors: M
authors: Jandrot-Perrus
authors: D
authors: D
authors: Didry
authors: M C
authors: MC
authors: Guillin
authors: A T
authors: AT
authors: Nurden
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D001792:Blood Platelets / Q000032:analysis*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D008565:Membrane Proteins / Q000032:analysis; D013499:Surface Properties; D013917:Thrombin / Q000032:analysis*
chemical_list: D003432:Cross-Linking Reagents; D006629:Hirudins; D008565:Membrane Proteins; D013917:Thrombin