Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III

The recent demonstration of a protein disulfide isomerase (PDI) on the surf ace of and secreted from blood platelets raises the possibility that protei ns involved in hemostasis and wound healing are also substrates of this enz yme. In this study purified preparations of platelet PDI, thrombospondin-1 (TSP), alpha-thrombin, and antithrombin III (AT) were used to demonstrate t hat PDI catalyzes formation of a TSP-thrombin-AT complex consistent with pr evious results with supernatant platelet activation. Concentrations of 1.25 mu g/ml of PDI were sufficient to concert almost 50% of thrombin to TSP-th rombin-AT complex. Complex formation requires low concentrations of a reduc ed thiol and the reaction can be prevented by N-ethylmaleimide. The complex is dissociated by reducing agents such as mercaptoethanol. Absence of Ca2 and the addition of EDTA increased the rate of complex formation, indicati ng that TSP in the Ca2+ free form is most effective. In the absence of AT a small amount of TSP-thrombin complex formed which was only 0 - 13% of maxi mal complex formation in the presence of AT. This result. in combination wi th kinetic studies showing rapid formation of thrombin-AT complex followed by conversion to ternary complex, suggests that the thrombin-AT complex is an obligatory intermediate in the reaction. Under optimal conditions over 7 0% of the thrombin is incorporated into the complex in 60 min. Heparin acce lerated the reaction largely by enhancing formation of thrombin-AT complexe s and had little effect on TSP. PDI coprecipitated with TSP from the supern atant solution of activated platelets, suggesting an association between PD I and its substrate. In summary, these data are consistent with a role for PDI-catalyzed formation of disulfide-linked complexes of TSP with other pro teins. (C) 1999 Academic Press.