A Role Of Thioredoxin-related Transmembrane Protein TMX4 In Platelet Activation And Thrombus Formation

Background:Thrombosis consists of two parts:platelet activation and coagulation system activation.Arterial thrombosis can cause a variety of clinical diseases,such as ischemic stroke and acute myocardial infarction,seriously threatening human life and health.Cardiovascular and cerebrovascular diseases remain the leading cause of death worldwide,according to a systematic analysis of the global burden of disease study reported in the Lancet in 2017.So far,there are few drugs for the effective treatment of thrombotic diseases in clinical practice.Therefore,it is particularly important to study the specific processes and mechanisms of arterial thrombosis.In recent years,a large number of studies have shown that the family of protein disulfide isomerases play an important role in thrombosis.It has been known that several secretion-type disulfide isomerases,such as PDI,ERp57,ERp72,ERp5 contribute to thrombosis.However,our understanding of the role of the transmembrane protein disulfide isomerase is very limited.Purpose:The aim of this study is to determine the role of TMX4 in platelet activation and thrombosis,and to understand its underlying mechanism.Methods:In this study,we generated TMX4 conditional knockout mice,which was verified by the methods of PCR,RT-PCR,and western blotting.Tail bleeding time of wild-type and TMX4 tissue-specific knockout mice were compared to determine the role of TMX4 in hemostasis.Flow cytometry was used to detect the expression of the major specific receptors on the surface of resting and activated wild type and TMX4 deficient platelets.Turbidimetric and fluorescent method were used to determine the role of TMX4 in platelet aggregation and ATP release.Finally,using FeCl3-induced mesenteric artery thrombosis model and laser-induced cremaster muscle micro-arterial thrombosis model,we evaluated the role of TMX4 in thrombosis and platelet accumulation in vivo.Results:(l)TMX4 was expressed on the surface of human platelets,leukocytes and endothelial cells.(2)The TMX4 gene was successfully modified by insertion of the loxp-sites of TMX4fl/fl mice.The Pf4-Cre and Tie2-Cre mice were crossed with TMX4fl/fl mice to generate tissue specific knockouts including Pf4-Cre/TMX4fl/fl mice and Tie2-Cre/TMX4fl/fl mice respectively.TMX4 gene was not expressed in platelets of both Pf4-Cre/TMX4fl/fl mice and Tie2-Cre/TMX4fl/fl mice,and in leukocytes and endothelial cells of Tie2-Cre/TMX4fl/fl mice.(3)The tail bleeding time of Pf4-Cre/TMX4fl/fl mice and Tie2-Cre/TMX4fl/fl mice was significantly longer than that of TMX4f/fl mice(p<0.0001).(4)Pf4-Cre/TMX4fl/fl mouse platelets showed significant decrease in platelet aggregation(p<0.01)and ATP release(p<0.01).(5)P-selectin expression(p<0.01)and integrin allbp3 activation(p<0.05)of TMX4-deficient platelets were reduced after thrombin stimulation.(6)In FeCl3-induced mesenteric artery injury model,the incorporation of platelets into a growing thrombus in Tie2-Cre/TMX4fl/fl mice was significantly decreased compared with the control group(p<0.001).(7)In the laser-induced cremaster muscle micro-arterial thrombosis model,platelet accumulation and fibrin formation in the vascular injury site of Tie2-Cre/TMX4fl/fl mice were significantly reduced compared with the control group(p<0.001).Conclusions:1.TMX4 is important in platelet activation.2.TMX4 supports platelet accumulation and fibrin formation in vivo.