Kinetics of the formation of thrombin-thrombospondin complexes: Involvement of a 77-kDa intermediate

Complexes of Thrombin with Proteins Secreted by Activated Platelets

“…Increas ing concentrations of a competitive inhibitor slowed the formation of both complexes, but there was relatively little effect on the final amount of >450 complex formed. Similarly, the major effect of varying pH (optimum about 8) was on the formation of the thrombin-PN p complex, 15 the step that requires catalytic activity of thrombin in our hypothetical model in reaction 1. In contrast, thiol-reactive reagents, lower temperature, and added Ca 2+ inhibit primarily the formation of the second complex, consis tent with the known inhibition by these of thiol-disulfide exchange reactions involving TSP.…”

“…Formation of the 77 kd complex requires a second ary substrate recognition site on thrombin that targets thrombin to substrates with a complementary site. Inhib ited thrombin was unable to form the 77 kd complex, but active site-blocked, unlabeled thrombin inhibited forma tion of the 77 kd complex by active, labeled thrombin 15,20 (Fig. 2).…”

“…There are several reactions of TSP that we consider in analysis of possible mechanisms: (1) Secreted TSP has isomerized disulfide bonds; 36 (2) TSP becomes disulfidelinked to other proteins, either other molecules of TSP 11 or protease-serpin complexes 10,15 or possibly other pro teins; (3) a disulfide bond of TSP becomes reduced. 37 Each of these is similar to a reaction catalyzed by protein disulfide isomerase.…”

“…This step requires catalytic activity as well as the anion-binding exosite of thrombin. It is inhibited by competitive inhibitors of thrombin cata lytic activity, 15 it is blocked by covalent inhibitors of thrombin 15 and it is inhibited by anything that binds to the anion-binding exosite of thrombin. 20 In the second step we hypothesize that a secreted protein disulfide isomerase (PDI) catalyzes thiol-disulfide exchange between a thiol of TSP and a disulfide bond of thrombin.…”

The thrombospondin type 1 repeat (TSR) superfamily: Diverse proteins with related roles in neuronal development

“…TSP‐1 also binds and modulates the activity of many proteases that contribute in complex ways to the formation and resolution of vascular fibrin clots. Thus, after an initial lag phase, TSP‐1 and thrombin released from activated platelets form a disulfide‐bonded complex in which the catalytic activity of thrombin is reduced (Browne et al, 1988; Chang and Detwiler, 1992). TSP‐1 also complexes with plasminogen, plasminogen activator inhibitor, or urokinase plasminogen activator to regulate the activation of plasminogen (DePoli et al, 1989; Silverstein et al, 1990; Hogg et al, 1992; Anonick et al, 1993).…”