ELECTRON-SPIN-RESONANCE AND FLUORESCENCE STUDIES OF THE CONFORMATIONAL ENVIRONMENT OF THE THIOL-GROUPS OF THROMBOSPONDIN - INTERACTIONS WITH THROMBIN

The free thiols of platelet thrombospondin (TSP) were modified with th iol-specific spin labels and fluorescence probes. The conformational e ffects of thrombin complexation with TSP were monitored by thiol-speci fic spin labels covalently attached to TSP and active site specific sp in labels on thrombin. The results provide evidence supporting specula tions that the thiols of the three polypeptide chains in TSP are not c onformationally identical. Studies on the effects of Ca2+ and temperat ure confirm that TSP exists in multiple conformations which are under dynamic equilibrium. The ESR spectra of spin-labeled TSP are sensitive to the proteolytic effects of thrombin in the presence and absence of calcium. Phenylsulfonyl fluoride spin labels specific for the active site of thrombin are excellent indicators of thrombin:TSP complex form ation in the absence of calcium. The anticoagulant thrombin inhibitor hirudin competes with TSP for the same binding locus on thrombin (whic h includes the requirement of an intact anion exosite). The results su ggest that the species observed here is the noncovalent complex formed during the first step of the TSP-thrombin interaction, showing also t hat thrombin activity is not essential for complex formation. ESR and fluorescence studies of thiol-labeled TSP indicate that the sulfhydryl s are not affected in the noncovalent thrombin:TSP complex, although t hey must be playing a major role in the second step, i.e., formation o f the covalent complex, through intermolecular thiol exchange.