Abstract 148: Loss Of Tlr4 Signaling Inhibits Thrombus Resolution In A Non-monocyte Dependent Manner

Background: Loss of TLR4 signaling reduces Venous Thrombosis (VT) resolution. Leukocyte expression of TLR4 is elevated in humans undergoing acute to chronic DVT transformation. We hypothesized that TLR4 signaling in monocytes directs thrombus clearance. Methods: Wild type, Tlr4 -/- , LysM cre+/- Tlr4 fl/fl mice underwent VT via IVC stasis at 4/7/14d timepoints. Thrombi subtypes were identified by flow cytometry. Thrombus size was assessed, and fibrinolytic mediators were assayed in splenic/bone marrow Mφs. Whole blood from mice was stimulated with TLR2/4 and assayed for thrombolytic potential via HALO assay. Immunohistochemistry was performed for cellular infiltrate. Results: We observed elevated TLR4 cell surface expression on CD11b+/Ly6C- Mo/Mφ from wild type thrombi. Thrombus weight in Tlr4 -/ mice was elevated at 4d stasis, confirming impaired thrombus clearance. In basal conditions, circulating Mo/Mφ saw decreased Urokinase & MMP9 mRNA levels in LysM cre+/- mice. In post thrombotic conditions, we saw decreased protein levels of Urokinase and MMP9 in Cre+ bone marrow-derived Mφs. Whole blood thrombolysis efficiency increased with TLR4 agonism in Cre- mice compared to Cre+. Efficiency was restored with LPS-mediated dual TLR2/4 agonism. Total thrombus weight was unchanged. No difference was identified in monocyte infiltrate into the thrombus (Fig. 1) Conclusions: Genetic deficiency of Mo/Mφ TLR4 signaling alters induction of fibrinolytic/matrix remodeling gene expression. Ex vivo TLR4 agonism increased whole blood thrombolysis in Cre- but not Cre+ mice. Additional TLR2 agonism of Cre+ mice restored thrombolytic activity, suggesting TLR2 as a compensatory pathway that may account for the unchanged in vivo phenotype in LysMcreTlr4fl/fl mice. Further clarifying these cellular pathways may provide an alternate pathway to target VT resolution.