Abstract
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.
Highlights
Tivated receptor 2 [5,6,7]
We recently showed that cell surface protein-disulfide isomerase (PDI) is associated with inactive tissue factor (TF) and targets the solvent-exposed, allosteric Cys186–Cys209 disulfide bond in TF to inhibit coagulation while maintaining direct TF1⁄7VIIa signaling [8]
In a defined system with purified proteins, we further demonstrate that PDI accelerates macromolecular substrate factor X (FX) activation of soluble TF that is known to have low procoagulant activity [38]
Summary
Materials—Bovine PDI, reduced (GSH) and oxidized (GSSG) glutathione, phenylarsenic oxide (PAO), methyl-methanethiosulfonate (MMTS), N-ethylmaleimide (NEM), and bacitracin were purchased from Sigma. Soluble TF was labeled with 5 mM PEG-mal at 37 °C for 60 min in the presence or absence of reducing agents or PDI. Efficiency of MMTS modification was determined by labeling residual free thiols with 100 M MPB for 30 min at ambient temperature, followed by Western blotting to detect biotin incorporation. FXa generation assays using supernatant containing microparticles were performed with the same VIIa and FX concentrations, and PDI was added as indicated. TF, FX/Xa, and PDI in the immunoprecipitate were detected by Western blotting
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