Dual Role Of The Transmembrane Protein Disulfide Isomcrasc TMX1 In Negative Regulation Of Platelet And Coagulation Activation And The Underlying Mechanisms

Background and objective:Platelets play a critical role in hemostasis and thrombosis.They constantly patrol abnormal signals from the blood vessel damage to maintain vascular balance.For this purpose,platelets must be rapidly activated.However,this efficient activation may also lead to unnecessary platelet activation and pathological thrombosis.Up to now,we have little knowledge of how platelets maintain the resting state.Therefore,the investigation into the negative regulation mechanism of platelet is particularly crucial to elucidate the mechanism of thrombosis and the pathogenesis of thrombotic diseases.There are increasing lines of evidence indicating that the protein disulfide isomerase(PDI)family enzymes such as PDI,ERP57 and ERP72,positively regulate thrombosis by reducing the disulfide bond of platelet integrin αⅡbβ3.However,whether or not the family has a negative regulation on thrombosis is not clear.Through the screening,we found that in contrast to the positive PDI family enzymes,TMX1 that is a transmembrane member of PDI family has potent oxidase activity.We will study whether TMX1 negatively regulates platelet function.Understanding of the negative regulation of thrombosis by PDI family will not only reveal redox balance regulatory network of platelet activation,but will also offer potential targets for the treatment of thrombotic diseases.Methods:1.We generated recombinant wild-type and the inactive mutant proteins of TMX1 extracellular domain,and evaluated their oxidoreductase activity by reductase activity and oxidase activity assays.2.The expression of TMX1 at mRNA and protein levels in platelet and endothelial cells were evaluated by Q-PCR and Western-blotting.Flow cytometry was used to analyze the expression of TMX1 on the surface of platelets.3.Using recombinant TMX1 and specific anti-TMX1 antibody,we evaluated the role of TMX1 on platelet function.4.We generated whole body TMX1-knock-out mice(TMX1-/-)and conditional knockout mice.5.The role of TMX1 in hemostasis of thrombosis was studied using tail bleeding time,FeCl3-induced mesenteric artery and laser-induced arteriole thrombosis models,combined with TMX1-knockout mice and TMX1 recombinant protein.6.The effect of TMX1 on redox regulation of integrin aⅡBβ3 was investigated by using of iodo-TMT and 3-(N-Maleimidylpropionyl)biocytin(MPB)sulfhydryl labeling approach.Furthermore,the cysteines in αⅡbβ3 catalyzed by TMX1 was further identified by mass spectrometry.7.Flow cytometry was used to evaluate the role of TMX1 in the binding of fibrinogenγ chain to its platelet receptors.8.The binding affinity of TMX1 to αⅡbβ3 was measured by Surface Plasmon Resonance(SPR).9.The effect of TMX1 on regulation of positive PDI molecules was investigated by reductase activity assay and sulfhydryl labeling,to further identify its role in balancing platelet function.Results:1.Both platelet and endothelial cells expressed TMX1,and its expression of TMX1 on platelet surface increased upon activation.2.Recombinant TMX1 extracellular fragment inhibits platelet aggregation,ATP release and spreading,inactive TMX1 mutant had the opposite effect in these processes.3.Anti-TMX1 antibody promotes platelet aggregation and ATP release.4.The TMX1 deficiency enhances platelet aggregation,granule content release andαⅡbβ3 activation,addition of recombinant wild-type rTMXl protein reversed these processes.5.In tail bleeding assay,we found that TMX1-/-mice had significantly shorter tail bleeding times than control TMX1+/+ mice,indicating that TMX1 inhibits hemostasis in vivo.6.Incorporation of platelets into the thrombus in TMX1-/-mice was substantially increased in FeCl3 induced mesenteric artery injury model and laser-induced arteriole thrombosis model,compared with wild-type control mice.7.rTMX1 inhibited the binding of the monovalent fibrinogen gamma-chain to activated platelets.8.Using iodo-TMT labeling assay,we found that rTMX1 oxidizes thiols in αⅡbβ3.MPB sulfhydryl labeling of mouse platelets approach showed that TMX1 deficiency in platelets increased thiols in the β3 subunit of aIIbβ3 on intact platelets surface.These data suggesting that TMX1 negatively regulates platelet function by oxidizing the disulfide bond in αⅡbβ3.9.The proteomics analysis showed that TMX1 catalyzes the formation of three pairs of disulfide bonds in αⅡbβ3(Cys508-Cys521,Cys523-Cys544,Cys608-Cys655).10.TMX1 inhibited the reductase activity of ERP57 and ERP72 in a concentrationdependent manner,and decreased their free thiols.Moreover,inhibitory anti-TMX1 antibody B01P reversed the decreased aggregation of platelet lacking ERP57 and ERP72,suggesting that TMX1 counter-balances the role of these positive PDIs in platelets function via oxidizing their disulfide bonds.Conclusion:TMX1 negatively regulates platelet activation and thrombus formation by oxidizingαⅡbβ3 and other positive PDI molecules.Background and objective:Coagulation activation forms fibrin networks to strengthen clots to stop bleeding.However,overwhelmed and uncontrolled coagulation may cause pathological thrombosis.Therefore,understanding of the negative regulatory mechanisms of coagulation is very important.An increasing line of evidence suggests that protein disulfide isomerase(PDI)family enzymes regulate coagulation pathway by targeting through different coagulation factors.For instance,PDI regulates thrombosis by interfering with the binding of coagulation factors and phospholipids.Besides,PDI regulates the activation of tissue factor by oxidizing Cys186-Cys209 in tissue factor.Using intravital thrombosis model we and others have found that PDI dually regulates platelet accumulation and fibrin deposition.However,it remains unknown whether PDI family have a negative role in regulation of coagulation.Recently we have shown that TMX1 which is a transmembrane-type member of the PDI family has a negative regulation of platelet function and thrombosis.In the current study,we aimed at determining the role of TMX1 in coagulation.Methods:1.We used laser-induced cremaster muscle artery injury model to determine the role of TMX1 in coagulation activation.2.Using thrombin generation and FXa formation assay we identified the coagulation pathway that TMX1 targeted.3.We used flow cytometry to test whether PS exposure on the surface of activated platelet and binding of clotting factors to platelet are associated with TMX1.4.Sulfhydryl labeling was used to detect the redox regulation of coagulation factors by TMX1.5.Flow cytometry was used to determinate whether TMX1 affects activity of phospholipids translocase.6.The effect of TMX1 on fibrin polymerization was detected by turbidimetric method.Results:1.In laser-induced thrombosis model,we found that β3 deficient platelets did not accumulate at the site of vascular injury due to the lack of αⅡbβ3,but the recombinant TMX1 protein still inhibited fibrin formation,suggesting that TMX1 is directly involved in coagulation activation.2.Using thrombin generation assay,we found recombinant TMX1 protein inhibited intrinsic coagulation pathway activation mediated by activated platelets in vitro,but did not inhibit tissue factor-dependent extrinsic coagulation pathway,indicating that TMX1 selectively inhibits intrinsic coagulation pathway.3.TMX1-deficiency enhanced intrinsic coagulation pathway induced by activated platelets.4.Wild-type TMX1 extracellular fragment inhibited PS exposure on activated platelet surface,anti-TMX1 antibody and TMX1 deficiency enhanced PS exposure,indicating that TMX1 negatively regulates PS exposure on activated platelet surface.5.The wild-type TMX1 extracellular fragment slightly inhibited the binding of coagulation factors to activated platelets,and the binding of TMX1-deficient platelets to coagulation factors was increased,suggesting that TMX1 negatively regulates the binding of coagulation factors to platelets.6.TMX1 deficiency enhanced the activity of floppase on surface of platelets.7.TMX1 extracellular fragment inhibited fibrin polymerization in vitro,and TMX1deficient plasma fibrin polymerization was enhanced,suggesting that TMX1 inhibits fibrin polymerization.8.Sulfhydryl labeling results showed that TMX1 extracellular fragment oxidized free thiols in the fibrinogen α chain,which is perhaps mechanism for its inhibition of fibrin polymerization.Conclusion:TMX1 inhibits the coagulation pathway,through the following mechanism:TMX1 negatively regulates the floppase activity thereby inhibiting the exposure of PS on the surface of activated platelets.TMX1 inhibits fibrin polymerization via oxidizing the alpha chain,thereby inhibiting common coagulation pathway.