Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin.
In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate 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 agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-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 vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We 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 added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.
Essex
D W
DW
Department of Medicine, Division of Hematology/Oncology, Department of Biochemistry, State University of New York Downstate Medical Center at Brooklyn 11203, USA. essex@hscbklyn.edu
Miller
A
A
Swiatkowska
M
M
Feinman
R D
RD
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D004220:Disulfides / Q000097:blood*; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000097:blood; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000097:blood; D010447:Peptide Hydrolases / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000097:blood*; D013499:Surface Properties; D013552:Swine; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D019096:Vitronectin / Q000097:blood*
D004220:Disulfides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D019096:Vitronectin; C046193:antithrombin III-protease complex; D000990:Antithrombin III; D010447:Peptide Hydrolases; D013917:Thrombin; D019704:Protein Disulfide-Isomerases; D005978:Glutathione