Role Of Thiol Isomerase ERp72 In The Pathogenesis Of Venous Thrombosis

Background and ObjectiveVenous thrombosis induces venous thrombus embolism(VTE) which threatens human life and health. However, there is no effective therapeutic drug for VTE, because the underlying mechanism of venous thrombosis is not clear. In recent years, the roles of protein disulfide isomerase(PDI) family in thrombosis have been noted, some members of PDI family play important roles in activation of endothelial cells, aggregation of platelets, recruitment of leukocytes and activation of coagulation system, providing a new insight into venous thrombosis. ERp72, a 72 KDa thiol isomerase with three highly conserved homologous sequences of PDI family, is widely expressed in platelets, leukocytes and endothelial cells. In this study, we generated tissue-specific ERp72-knockout mouse, prepared recombinant ERp72 protein and inhibitory anti-ERp72 monoclonal antibody, and observed the phenotype of absence or inhibition of ERp72 in a mouse inferior vena cava(IVC) stenosis-induced venous thrombosis model. Our study is aimed at understanding the role of ERp72 in venous thrombosis and its underlying mechanism.Methods1. Tissue-specific ERp72-knockout mice were generated using Cre-loxp system by mating ERp72-flox(ERp72fl/fl) mice with Pf4-Cre and Tie2-Cre mice, genotypes of Pf4-Cre/ERp72fl/fl(platelet-specific ERp72 knockout) and Tie2-Cre/ERp72fl/fl(hematopoietic and endothelial cells-specific ERp72 knockout) mice were analyzed by polymerase chain reaction(PCR), expression level of ERp72 protein in platelet, WBC and endothelial cell were analyzed by Western blotting.2. Complete blood count of the two strains of ERp72 knockout mice and their control mice was analyzed by SIEMENS Hematology System(ADVIA 2120i). Prothrombin time(PT) and activated partial thromboplastin time(APTT) of Tie2-Cre/ERp72fl/fl mice and control ERp72fl/fl mice plasma were analyzed by Stago START 4 Coagulation Analyzer.3. Using with tissue-specific ERp72-knockout mice, we employed an IVC stenosis-induced venous thrombosis model, 48 hours after flow restriction, thrombi harvested from IVC were weighed and photographed. C57BL/6 mice were also used for IVC stenosis model, 250mg recombinant inactivated ERp72 protein(ERp72oo-oo-oo) or isopyknic control buffer were injected into jugular vein immediately after stenosis surgery, thrombi were harvested and analyzed as above described.4. In another IVC stenosis model, 6 hours after flow restriction, 400ml of citrated blood was collected from the proximal end of stenosis point for the ELISA test of plasma v WF.5. Washed platelets isolated from Pf4-Cre/ERp72fl/fl and control mouse blood were stimulated by convulxin or thrombin to test the aggregation function.6. Human PBMCs were pre-incubated with anti-ERp72 monoclonal antibody or isotype Ig G and stimulated with 100ng/ml LPS for 4 hours, and then thrombin generation assay(TGA) of the PBMCs was tested. Bone marrow monocytes isolated from Tie2-Cre/ERp72fl/fl and control mice were adjusted to 1×106/ml and stimulated by 100ng/ml LPS for 4 hours at 37℃, the expression of TF on monocyte surface were analyzed by flow cytometry(FCM).7. Recombinant wild-type ERp72 protein(ERp72ss-ss-ss) and coagulation factor FXII were incubated for 1 hour at 37℃ and labeled by MBP for 15 minutes, which was followed by non- reducing SDS-PAGE and Western blotting to analyze the free thiols of FXII.Results1. Results of PCR indicated that loxp-sites were inserted into ERp72 locus of ERp72fl/fl mice successfully, and Pf4-cre and Tie2-cre were successfully recombined into Pf4-Cre/ERp72fl/fl and Tie2-Cre/ERp72fl/fl genome respectively. Results of Western indicated that platelets from Pf4-Cre/ERp72fl/fl and Tie2-Cre/ERp72fl/fl mice did not express ERp72, the leukocytes and endothelial cells of Tie2-Cre/ERp72fl/fl mice had no ERp72 expression. All these ERp72-deficient cells expressed normal levels of other PDI family members such as PDI and ERp57.2. Complete blood count of Pf4-Cre/ERp72fl/fl mice and Tie2-Cre/ERp72fl/fl mice was comparable with that of its ERp72fl/fl littermates; PT and APTT of Tie2-Cre/ERp72fl/fl plasma showed no difference with ERp72fl/fl littermates.3. In IVC stenosis-induced venous thrombosis model, the weight of IVC thrombi harvested from Pf4-Cre/ERp72fl/fl mice showed no difference with that of control ERp72fl/fl littermates [20.58±2.20mg(n=8) vs 16.97±1.42mg(n=12), P=0.1648], however, the weight of IVC thrombi harvested from Tie2-Cre/ERp72fl/fl mice displayed a significant decrease [30.08±3.02mg(n=8) vs 16.04±3.03mg(n=7), P=0.0062]. Compared with the mice injected with control buffer, the mice injected with ERp72oo-oo-oo had little IVC thrombi [23.84±2.97mg(n=8) vs 12.04±2.32mg(n=8), P=0.0074].4. In normal condition, plasma v WF level of ERp72fl/fl mice was comparable with that of Tie2-Cre/ERp72fl/fl mice [95.30±11.66ng/ml(n=4) vs 91.80±15.46ng/ml(n=4), P=0.8648]; after 6-hour flow restriction, plasma v WF level of both group mice were increased, but Tie2-Cre/ERp72fl/fl mice had less v WF level [156.9±3.424ng/ml(n=5) vs 120.2±10.32ng/ml(n=5), P=0.0188].5. Aggregation of washed platelets isolated from Pf4-Cre/ERp72fl/fl mice was weaker than that of ERp72fl/fl platelets.6. Compared with the PBMCs incubated with isotype Ig G, after stimulated by LPS, the PBMCs incubated with anti-ERp72 monoclonal antibody showed a lower peak of thrombin generation[77.27±2.41 n M(n=3) vs 52.43±3.87 n M(n=3), P=0.0055]. After stimulated by LPS, expressions of TF on Tie2-Cre/ERp72fl/fl monocytes were also lower than that of ERp72fl/fl monocytes.7. In thiol labeling experiments, recombinant wild-type ERp72 protein(ERp72ss-ss-ss) increased the free thiol level of FXII.Conclusions1. Hematopoietic and endothelial ERp72 deficiency decrease venous thrombosis.2. ERp72 regulates venous thrombosis through v WF release, platelet aggregation, monocyte procoagulant activity and alteration of FXII redox status.