Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding

Summary. Background: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue‐factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. Objective: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin‐mediated thrombin amplification on human platelets. Methods/results: Using a newly developed thrombin‐dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti‐TF antibodies inhibiting TF‐induced thrombin formation in platelet‐depleted plasma had no effect compared with vehicle‐treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase‐inhibitors bacitracin and anti‐PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin‐stimulated platelets exhibited increased surface‐associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin‐stimulated platelets. Conclusions: Thus, surface‐associated PDI is an important regulator of coagulation factor ligation to thrombin‐stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.