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 sodium dodecyl sulfate-stable complex of apparent mass > 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.degree. c, 50 times the rate at 22.degree. 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 SH-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 rdioactive N-ethylmaleimide/mol of protein, indicating 3 SH groups/mol. After reacting with purified glycoprotein G, thrombin developed a new Sh group. Glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin apparently 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.