The Role Of Thioredoxin-related Transmembrane Protein TMX3 In Thrombosis And The Underlying Mechanisms

Objective Thrombotic diseases have been a serious life-threatening problem.The functions of hemostatic proteins that are critical in thrombosis are regulated by thiol-disulfide exchange.Protein disulfide isomerase(PDI)family is a group of enzymes containing thioredoxin-like-domain expressed in endoplasmic reticulum(ER),in which they catalyze disulfide formation of native peptides for protein folding.Recent studies have shown that several members of PDI family including PDI,ERp57 and ERp72 play important roles in thrombosis,regulating platelet function and coagulation activation.These results suggested that PDI family may form an integration redox network to regulate thrombosis.To better understand this regulatory network,we plan to characterize the role of each PDI family member in thrombosis using tissue-specific knockout mouse models.The results of our pilot experiments suggest that thioredoxin related transmembrane protein 3(TMX3),which is a transmembrane thiol isomerase with CGHC catalytic domains expressed in platelet and endothelial cell,is a novel member of PDI family involved in thrombosis.In this study,we aimed at determining the role of TMX3 in thrombosis and hemostasis,and characterizing the underlying mechanisms.Methods Hematopoietic and endothelial cells-specific TMX3 knockout(Tie2-cre/TMX3fl/fl)mice and platelet-specific TMX3 knockout(Pf4-cre/TMX3fl/fl)mice were generated by mating TMX3-flox(TMX3fl/fl)mice with Tie2-Cre and Pf4-Cre mice.We determined the role of TMX3 in hemostasis and thrombosis by using Tie2-cre/TMX3fl/fl mice for tail bleeding model and laser-induced cremaster arterial injury model.To study the roles of TMX3 in platelet functions,we test the effect of TMX3 deficiency on platelet aggregation,ATP secretion,JON/A binding,clot retraction,platelet adhesion and spreading.To understand the mechanism underlying the role of TMX3,we measured β3-talinl interaction,protein phosphorylation,Ca2+ mobilization,aggregation and ATP secretion induced by Ca2+ionophore A23187.By testing the effects of hydrolyzing ADP by apyrase and supplementing low concentrate of ADP on aggregation of WT and TMX3 deficient platelets,we determined whether the impaired aggregation in TMX3 deficient platelets could be attributed to reduced ADP secretion.Techniques of surface plasmon resonance and sulfhydryl labeling were used to determine the affinity between TMX3 and αⅡbβ3,and the catalytic effects of TMX3 on αⅡbβ3 thiol-disulfide exchange.To determine the role of TMX3 in procoagulant activity of platelet,we test thrombin generation induced by WT and TMX3 deficient platelets and measured phosphatidylserine(PS)expression on activated WT and TMX3 deficient platelets.To determine the role of endothelial cell-derived TMX3 in coagulation,β3-/-mice were injected with inactive TMX3 recombinant protein or control buffer through jugular vein,subjected to a laser-induced cremaster arterial injury model for measurement of fibrin formationResults Tie2-cre/TMX3fl/fl mice and Pf4-cre/TMX3fl/fl mice were generated successfully,platelets and endothelial cells from Tie2-Cre/TMX3fl/fl mice and platelets from Pf4-Cre/TMX3fl/fl mice did not express TMX3 mRNA and protein,but expressed normal levels of other PDI family members such as PDI,ERp57,ERp72 and TMX1.Complete blood count,expression and biological activity of main clotting protein,expression of main membrane receptors on platelet of Tie2-Cre/TMX3fl/fl mice are normal,while Tie2-Cre/TMX3fl/fl mice displayed a prolonged tail bleeding time,impaired platelet accumulation and fibrin formation in laser-induced cremaster arterial injury model compared with TMX3fl/fl littermates(control).These results indicated a positive role of TMX3 in regulation of hemostasis and thrombosis.The prolonged tail bleeding time of Pf4-Cre/TMX3fl/fl mice suggested that platelet-derived TMX3 played an important role in hemostasis.Compared with control platelets,TMX3-deficient platelets displayed a decrease in aggregation and ATP secretion induced by four receptor agonists(thrombin,convulxin,U46619,ADP),had significantly less binding of JON/A(recognizing active form αⅡbβ3)and less P-selectin expression induced by thrombin.Deficiency of TMX3 did not affectβ3-talin1 interaction,protein phosphorylation,Ca2+ mobilization,clot retraction,and platelet adhesion and spreading on immobilized fibrinogen.However,TMX3 deficient platelet shown a slightly reduced aggregation but a severely reduced ATP secretion induced by Ca2+ ionophore A23187.These results indicate that TMX3 is not required for αⅡbβ3 inside-out or outside-in signaling,but instead acts on platelet granule release responding to Ca2+ mobilization.The aggregation of WT platelets was reduced to a level comparable to TMX3 deficient platelet in the presence of apyrase,supplementation with a low concentration of ADP reversed the defect in platelet aggregation induced by TMX3 deficiency.These data support that the impaired aggregation of TMX3 deficient platelet is caused by the reduced ADP secretion.The affinity between TMX3 and αⅡbβ3 is relatively strong,and TMX3 catalyzes the reduction of disulfides on αⅡbβ3.The procoagulant activity and phosphatidylserine(PS)expression of TMX3 deficient platelets were weaker than that of WT platelets.In the intravital microscopy of laser-induced thrombosis assay,platelet accumulation was absent in integrin beta3 knockout mice,and infusion of TMX3 inactive mutant protein further decreased fibrin deposition,proposing that endothelial cell-derived TMX3 has a direct role in coagulationConclusion TMX3 is the first identified transmembrane thiol isomerase which is critical for hemostasis and thrombosis,and TMX3 dually supports platelet function and coagulation activation,thus,it may be a therapeutic target for thrombotic diseases.