The receptor tyrosine kinase EphA2 promotes thrombogenesis and impairs thrombus resolution in deep vein thrombosis

Abstract Background: Venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE), significantly contributes to global morbidity and mortality. The vascular endothelium is crucial in regulating both venous thrombus formation and resolution. Endothelial cells are known to highly express the Eph family of receptor tyrosine kinases, the largest such family in the mammalian genome. These receptors interact with their ephrin ligands on adjacent cells, mediating cell adhesion, tissue patterning, leukocyte homing, and metastasis. Our previous work demonstrated that proinflammatory cytokine-induced endothelial activation leads to increased EphA2 and ephrinA1 expression and sustained EphA2 activation. Importantly, activating EphA2 with recombinant Fc-ephrinA1 induced proinflammatory gene expression and stimulated monocyte adhesion, effects that were abrogated by EphA2 inhibition. Despite these insights, the precise role of EphA2 in DVT pathogenesis remains unclear. Objective: This study investigated the role of EphA2 in venous thrombus formation and resolution using established murine models of inferior vena cava (IVC) stenosis and stasis. Methods: DVT was induced in 12-14- week-old C57BL/6 wild type (controls) mice by partial ligation of the IVC. Unbiased RNA sequencing was performed on IVC samples 6-hours following IVC stenosis or sham surgery.To assess the role of EphA2 in DVT pathogenesis, male EphA2−/− and wild type control mice (12–14 weeks old, 19-25 g) underwent IVC stenosis surgery. Thrombus weight and incidence were evaluated 48 hours post-surgery. To study thrombus resolution, EphA2−/− and control mice were subjected to the IVC stasis model (complete ligation), with thrombus area monitored by ultrasound imaging on days 2, 7, and 14. To determine the cellular source of EphA2 that contributes to DVT pathogenesis, wild-type (WT) mice were lethally irradiated (6.5 Gy twice) and transplanted with bone marrow (BM) from either WT or EphA2−/− donors. IVC stenosis surgery was performed on these chimeric mice four weeks after transplantation. Results: Quantitative measurement of total IVC mRNA revealed distinct profiles in mice with IVC stenosis compared with mice subjected to sham surgery. IVC samples of mice with surgery exhibited increased inflammatory transcriptional genes, along with increased expression of EphA2. Notably, EphA2−/− mice exhibited a significant reduction (approximately 5-fold) in IVC thrombus weight (48-hours post-DVT) compared to WT controls. More importantly, thrombus incidence (48 hours post-DVT) was significantly lower in EphA2−/− mice (7 out of 20 mice developed a thrombus) compared to controls (15 out of 20 mice), highlighting a key role for EphA2 in thrombogenesis. In vitro mechanistic experiments revealed that silencing EphA2 using shRNA in endothelial cells resulted in reduced expression of von Willebrand factor (VWF) and increased expression of endothelial nitric oxide synthase (eNOS). Subsequent evaluation of venous thrombus resolution in the IVC stasis model revealed that the thrombus area on day 14 was significantly smaller in EphA2−/− mice (3.79 mm3) compared to WT mice (7.19 mm3). This indicates that EphA2 deficiency accelerates thrombus resolution. Furthermore, bone marrow transplant experiments showed comparable thrombus weight and incidence between WT mice transplanted with WT BM and WT mice transplanted with EphA2−/− BM, suggesting that EphA2 deficiency in bone marrow-derived cells does not impact thrombus formation or weight. Conclusions: Our findings conclusively demonstrate that EphA2 is a critical regulator of both venous thrombus formation and resolution. Specifically, EphA2 promotes thrombogenesis and impedes thrombus resolution in murine models. The positive effects of EphA2 deficiency on DVT severity appear to be mediated by EphA2 expressed on vascular endothelial cells via VWF and eNOS regulation. Therefore, targeting EphA2 presents a promising novel therapeutic strategy for the prevention and treatment of DVT.