A novel role for thioredoxin-related transmembrane protein TMX4 in platelet activation and thrombus formation.

Background: The functions of critical platelet proteins are controlled by thiol-disulfide exchanges, which are mediated by the protein disulfide isomerase (PDI) family. It has been shown that some PDI family members are important in platelet activation and thrombosis with distinct functions. TMX4, a membrane-type PDI family member, is expressed in platelets, but whether it has a role in platelet activation remains unknown.

Objectives: To determine the role of TMX4 in platelet activation and thrombosis.

Methods: The phenotypes of TMX4-deficient mice were evaluated in tail bleeding time assay and laser-induced and FeCl₃-induced arterial injury models. The functions of TMX4 in platelets were assessed in vitro using TMX4-null platelets, recombinant TMX4 protein, and anti-TMX4 antibody.

Results: Compared with the control mice, Tie2-Cre/TMX4^fl/fl mice deficient of hematopoietic and endothelial TMX4 exhibited prolonged tail bleeding times and reduced platelet thrombus formation. Pf4-Cre/TMX4^fl/fl mice deficient of platelet TMX4 also had prolonged tail bleeding times and decreased thrombus formation, which was rescued by injection of recombinant TMX4 protein. Consistently, TMX4 deficiency inhibited platelet aggregation, integrin αIIbβ3 activation, P-selectin expression, phosphatidylserine exposure, and thrombin generation, without affecting tyrosine phosphorylation of intracellular signaling molecules Syk, LAT, PLCγ2 and calcium mobilization. Recombinant TMX4 protein enhanced platelet aggregation and reduced integrin αIIbβ3 disulfide bonds, and TMX4 deficiency decreased free thiols of integrin αIIbβ3, consistent with a potent reductase activity of TMX4. In contrast, an inactive TMX4 protein and a specific anti-TMX4 antibody inhibited platelet aggregation.

Conclusion: TMX4 is a novel PDI family member that enhances platelet activation and thrombosis.