Role Of Protein Disulfide Isomerase(PDI) In Dual Regulation Of Platelet And Coagulation Activation And The Underlying Mechanisms

Background and Purpose: Thrombosis is an integrated process of two parallel events: platelet activation and coagulation. Protein disulfide isomerase(PDI) is the prototypic member of the PDI family. Several studies using anti-PDI antibody RL-90 and PDI inhibitors suggest an important role of PDI in vivo in platelet accumulation and fibrin generation. However, we found that these reagents are not specific for PDI. Moreover, we established a novel tissue-specific PDI-knockout mice, by which we found that the intravascular PDI deficiency could significantly inhibit the platelet accumulation and fibrin deposition. These new observations suggest that PDI dually requlates both platelet accumulation and fibrin formation in mice. The aim of this study was to determine the role of PDI in platelet accumulation and fibrin generation in vivo by the use of tissue-specific knock-out mice and reveal the molecular mechanism of PDI regulating the important receptors on the platelet surface and the coagulation factors, especially in determining the relative contribution of each active site of PDI in platelet function and coagulation. Thus, this study provides new insight to elucidate the molecular and cellular mechanisms of thrombosis and reveal new targets for intervention against thrombosis.Methods: We used combined approaches including recombinant PDI protein, platelet-specific PDI knock-out mice, Mx1-Cre/PDIfl/fl mice with platelet and endothelial cells lacking PDI, transgenic PDI(ss-oo) mice with PDI lacking the second CGHC active site which were obtained by screening experiment. We performed in vitro analysis of platelet aggregation,? ?-granule secretion(P-selectin expression on platelet surface) and integrin??IIb?3 activation(JON/A binding), in vivo assays of Fe Cl3-induced mesenteric artery and laser-induced arteriole thrombosis, the thrombin generation assay in vitro. Which is fully investigated about the role of PDI in dual regulation of platelet and coagulation activation and the underlying mechanism.Results:(1) RL90 could recognize or inhibit human PDI, but not for mouse PDI. RL90 inhibited the enzymatic activity of ERp57. RL90 inhibited the aggregation of platelets deficiency of PDI. RL90 inhibited P-selectin expression and integrin??IIb?3 activation in platelets deficiency of PDI.(2) Quercetin-3-O-rutinoside(Rutin) inhibited the enzymatic activity of ERp57. Rutin inhibited the aggregation of platelets deficiency of PDI.(3) Both of the recombinant PDI(oo-oo) protein and the second catalytically inactive mutant PDI(r PDI ss-oo) proten, could inhibit the human platelet aggregation induced by thrombin(P=0.0001, P=0.0001). While the wide-type PDI(r PDI ss-ss) protein and the first catalytically inactive mutant PDI(r PDI oo-ss) proten, all could enhance the platelet aggregation(P=0.001, P=0.03). r PDI(oo-oo) prolonged the tail bleeding time after infused into mice(3.601±0.4068 v.s. 7.717±1. 3, p=0.0059).(4) PDI deficiency inhibited platelet aggregation(p<0.001), integrin??IIb?3 activation(p<0.05), platelet??-granule secretion(p<0.05).??3 deficiency inhibited the binding of recombinant PDI protein to platelet surface(p=0.0162). Mice with platelets lacking PDI had long tail bleeding times(3.369±0.6597 v.s. 8.00±1.013, p=0.0003) and prolonged times to occlusion(4.914±0.3113 v.s.8.846±0.8364,p<0.0001).(5) Mx1-Cre/PDIfl/fl mice with platelet and endothelial cells lacking PDI exhibited decreased platelet accumulation(p<0.0001) and fibrin generation(p=0.033). Recombinant wild type PDI protein potentiated the thrombin generation mediated by tissue factor in vitro(p=0.0047). However, catalytically inactive mutant PDI(r PDI oo-oo) inhibited the thrombin generation dependent on activated platelet in vitro(p<0.0001).(6) We could establish transgenic PDI(ss-oo) mice and rescue the lethal PDI-/- mouse, while a parallel transgenic mice PDI(oo-ss) with an inactive first CGHC site and an active second site could not rescue the lethal PDI-/- phenotype. Thrombin-induced platelet aggregation(p<0.001), activation of the ?IIb?3 receptor(p<0.05) and P-selectin expression(p<0.05) were decreased in platelets with PDI lacking the second active site. In presence of 0.0025 U thrombin, spreading of platelets with PDI lacking the second active site, on fibrinogen-coated surfaces, was markedly inhibited(P=0.0041), compared with that of wild type platelets. Knockout-transgenic PDI(ss-oo) mice had prolonged tail bleeding times(4.142±0.675 v.s. 6.896±0.733, p=0.0054) and long times to occlusion(p=0.0047). The transgenic PDI(ss-oo) mice also had a defect in platelet accumulation(p<0.0001) and fibrin formation(p=0.0003).(7) These defects in platelet accumulation and fibrin formation in Mx1-Cre/PDIfl/fl mice and transgenic PDI(ss-oo) mice were rescued by infusion of recombinant PDI(oo-ss) containing only a functional second active site.Conclusions: PDI plays an important role in activation of platelet and coagulation. Platelets and other vascular cells contribute the source of PDI. The second active CGHC site is critical for PDI function in both platelet accumulation and fibrin generation in vivo.