Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange.
When 125I-labeled thrombin was incubated with washed human platelets or with the supernatant solution of activated platelets, it formed a NaDodSO4-stable complex of apparent mass greater than 450 000 daltons. Formation of the complex was temperature dependent; with 20 nM thrombin incubated with the supernatant solution of ionophore-activated platelets, the initial rate of formation of the stable complex was 1 nM thrombin/min at 37 degrees C, 50 times the rate at 22 degrees C. Thrombin with all free amino groups methylated was still reactive. Active-site-blocked thrombin formed the complex only slowly. The complex that formed with active thrombin was not dissociated by hydroxylamine in urea. Reduction with 2-mercaptoethanol dissociated the complex, and its formation was blocked by the sulfhydryl-blocking agents iodoacetamide and 4,4'-dithiodipyridine. The complex was thus unlike those of thrombin and alpha 2-macroglobulin or antithrombin III, but it had characteristics of a disulfide-linked complex. Of the secreted proteins, albumin and glycoprotein G adhered to an activated thiol-Sepharose column, indicating that they contained free thiol groups. Purified glycoprotein G and thrombin formed a complex similar to the complex formed when thrombin was incubated with the supernatant solution of activated platelets. The purified glycoprotein bound 2.6 mol of radioactive N-ethylmaleimide/mol of protein, indicating three sulfhydryl groups per mole. After reacting with purified glycoprotein G, thrombin developed a new sulfhydryl group. It is concluded that glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin form a dissociable complex that leads to a covalent complex by thiol-disulfide exchange of a thiol group on glycoprotein G and a disulfide on thrombin.
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*; D019699:Thrombospondins
D006023:Glycoproteins; D046911:Macromolecular Substances; D019699:Thrombospondins; D000001:Calcimycin; D013917:Thrombin