Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin

In this study, purified preparations of platelet protein disulfide isomeras e (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to de monstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes . Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agen ts such as mercaptoethanol. No vitronectin-thrombin complex formed in the a bsence of AT, indicating that the thrombin-AT complex is an obligate interm ediate in the reaction. Under optimal conditions, the majority of the throm bin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronect in for thrombin-AT in the low-Ca2+ environment that favors the active form of thrombospondin. The results presented here may also explain previous stu dies showing that vitronectin-thrombin-AT complexes form better in plasma ( which contains PDI) than with purified proteins (where PDI was not used). W e were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that adde d purified PDI can function in a plasma environment. Complex formation in p lasma was inhibited by inhibitors of PDI. PDI was released from the platele t surface in a soluble form at high pH (around the physiologic range), sugg esting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thromb in-AT.