Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity.
Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.
Versteeg
Henri H
HH
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
Ruf
Wolfram
W
D000818:Animals; D001414:Bacitracin / Q000737:chemistry; D001665:Binding Sites / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005033:Ethylmaleimide / Q000737:chemistry; D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism; D005170:Factor X / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008741:Methyl Methanesulfonate / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D015398:Signal Transduction / Q000502:physiology; D013925:Thromboplastin / Q000737:chemistry* / Q000378:metabolism
D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D010743:Phospholipids; D001414:Bacitracin; D005170:Factor X; D013925:Thromboplastin; D008741:Methyl Methanesulfonate; D015942:Factor VIIa; D019704:Protein Disulfide-Isomerases; D003545:Cysteine; D005033:Ethylmaleimide