Integrin PSI domain possesses endogenous thiol-isomerase activity. (A) Recombinant murine β3, human β1, and human β2 PSI domain (rPSI) demonstrated concentration-dependent thiol-isomerase function, as measured by the rdRNase refolding assay. (B) PDI-like function comparison between rPSI and PDI. The rPSI PDI-like activity was lower than PDI. (C) Bacitracin, a thiol-isomerase inhibitor, dose-dependently inhibited thiol-isomerase function of murine β3, human β1, and human β2 rPSI (25 μg/mL). (D) An alternative thiol-isomerase inhibitor, DTNB (100 μM), was used to further confirm the endogenous thiol-isomerase activity of the murine β3 (mβ3) PSI domain. (E) The inhibitory function of bacitracin was maintained in the presence of a protease inhibitor cocktail. (F) PSI amino-acid sequence analysis highlighting the CXXC motifs and illustrating the substitutions performed on the mutant proteins (red). (G) PSI single mutants (mutant1: C13S/C16S and mutant2: C23S/C26S, 12.5 μg/mL) and PSI double-mutant (DM PSI: C13S/C16S/C23S/C26S, 12.5 μg/mL) showed significantly less thiol-isomerase function compared with murine β3 rPSI. (H) Mutation of a cysteine located in each CXXC motif of the PSI domain (C13A and C26A [DM]) significantly decreased the thiol-isomerase activity of full-length human β3 recombinant protein. Absorbance at 284 nm is shown. (I) Full-length recombinant β3 integrin and human PSI domain catalyzed the formation of new disulfide bonds, induced rdRNase refolding, and decreased MPB incorporation into rdRNase: full-length wild-type or DM (C13A/C26A) β3 subunit (i), or recombinant human PSI domain (ii) was incubated with rdRNase, followed by incubation with MPB (100 μM) for 30 minutes at room temperature. The excess MPB was quenched by reduced glutathione (200 μM) for 30 minutes, and excess glutathione was quenched by iodoacetamide (400 µM) for 10 minutes at room temperature. The MPB-incorporated proteins were run on 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis in nonreducing conditions. After transfer, the biotinylated proteins were detected by western blotting with streptavidin–horseradish peroxidase (HRP). The figure is representative of 3 individual experiments. The square marks the area for density analysis of MPB in rdRNase with wild-type or DM full-length β3 integrin. Densitometry analysis showed a significant difference (P < .05) of MPB incorporation to rdRNase between wild-type and DM β3 integrin.