4-maleimido-2-2-6-6-tetramethylpiperidinooxyl and (1-oxyl-2-2-5-5-tetramethylpyrroline-3-methyl)methanethiosulfonate

The free thiols of platelet thrombospondin (TSP) were modified with thiol-specific spin labels and fluorescence probes. The conformational effects of thrombin complexation with TSP were monitored by thiol-specific spin labels covalently attached to TSP and active site specific spin labels on thrombin. The results provide evidence supporting speculations that the thiols of the three polypeptide chains in TSP are not conformationally identical. Studies on the effects of Ca2+ and temperature 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 formation in the absence of calcium. The anticoagulant thrombin inhibitor hirudin competes with TSP for the same binding locus on thrombin (which includes the requirement of an intact anion exosite). The results suggest that the species observed here is the noncovalent complex formed during the first step of the TSP--thrombin interaction, showing also that thrombin activity is not essential for complex formation. ESR and fluorescence studies of thiol-labeled TSP indicate that the sulfhydryls are not affected in the noncovalent thrombin: TSP complex, although they must be playing a major role in the second step, i.e., formation of the covalent complex, through intermolecular thiol exchange.

Topics: Animals; Binding Sites; Blood Platelets; Calcium; Cattle; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Membrane Glycoproteins; Mesylates; Peptide Fragments; Protein Conformation; Spectrometry, Fluorescence; Spin Labels; Sulfhydryl Compounds; Temperature; Thrombin; Thrombospondins