Cross‐linking of α and γ‐thrombin to distinct binding sites on human platelets

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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/polyacylamide 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 .apprxeq. 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 TosLysCH2 Cl-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.