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

Abstract

To cite this article: Jurk K, Lahav J, Van Aken H, Brodde MF, Nofer JR, Kehrel BE. Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding. J Thromb Haemost 2011; 9: 2278-90.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 thrombinmediated 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.

Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the αIIbβ3 integrin

“…This finding is consistent with previous studies in which authors used the same assay and reported that the majority of surface reductase activity on nonstimulated platelets was attributable to PDI. 42,43 However, ERp57 surface reductase activity increased dramatically with thrombininduced platelet activation ( Figure 7C), with ERp57 now contributing the large majority (83%) of the disulfide reductase activity on activated platelets. The large increase of platelet surface reductase activity with platelet activation attributable to ERp57 indicates that active sites of the enzyme are in the free thiol form required to cleave the S-S bond in GSSG, the form of the active sites that also catalyze thiol-disulfide exchange reactions.…”

Staphylococcal Extracellular Adherence Protein Induces Platelet Activation by Stimulation of Thiol Isomerases

“…On the platelet surface, PDI is implicated in integrin activation, and mediates platelet adhesion, aggregation, granule secretion as well as thrombin formation on the platelet surface. [27][28][29][39][40][41] 30,42 Like PDI, ERp5 and ERp57 are involved in platelet aggregation, integrin activation, and degranulation. 30,43,44 Various molecules are known, which bind to PDI and inhibit its thiol isomerase activity.…”

Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

“…In order to evaluate the potential anticoagulant activity of PDI inhibition in humans, we measured thrombin generation in a coagulation assay dependent on PDI activity on the thrombin-platelet amplification loop (31). Following oral administration of isoquercetin, platelet-dependent thrombin generation in plasma decreased significantly in both the healthy cohort as well as in individuals with anti-phospholipid antibodies.…”