Protein disulfide isomerase enhances tissue factor-dependent thrombin generation

Protein disulfide isomerase (PDI) plays an important role in fibrin generation in vivo, but the underlying mechanism remains largely unknown. In this study, using thrombin generation assay (TGA), we investigated whether PDI contributes to tissue factor (TF)-mediated thrombin generation. Human peripheral blood mononuclear cells (PBMCs) were treated with 100 ng/ml lipopolysaccharide (LPS), the expression of TF on cell surface was analyzed by flow cytometry. After incubation with an inhibitory anti-TF antibody, recombinant PDI protein or a PDI inhibitor PACMA31, LPS-stimulated human PBMCs were incubated with human plasma, and thrombin generation was assessed by Ceveron Alpha TGA and a fluorescent thrombin substrate. Bone marrow mononuclear cells isolated from PDI-knockout and wild-type mice were stimulated by LPS, followed by measurement of thrombin generation. LPS stimulation increased expression of TF on PBMCs, and thrombin generation. Inhibitory anti-TF antibody almost completely suppressed thrombin generation of LPS-stimulated PBMCs, suggesting that thrombin generation was TF-dependent. Recombinant PDI protein increased thrombin generation, while PACMA31 attenuated thrombin generation. Compared with control cells, PDI-deficient marrow mononuclear cells had less capacity in thrombin generation. Taken together, these data suggest that PDI enhances TF-dependent thrombin generation.