OC 68.2 - Endothelial Toll-like receptor 4 regulates arterial thrombus growth

Background: Pattern recognition receptors affect arterial thrombosis but the cell type-specific function of these receptors remains elusive. While the microbiota promotes arterial thrombus growth via Toll-like receptor (TLR)-2, the role of other innate immune receptors remains poorly defined.

Aims: Therefore, we studied the role of TLR4 in arterial thrombus growth.

Methods: Using intravital epifluorescence microscopy, we analyzed platelet deposition in a mouse carotid artery ligation model. Thus, we studied the impact of TLR4 on arterial platelet deposition in global Tlr4-deficient and endothelial-specific Tlr4-deficient mice generated by Cre-Lox technology. Washed platelets isolated from either global Tlr4-deficient or WT mice were stimulated by thrombin and the P-selectin exposure and αIIbβ3 activation were analysed by flow cytometry. Ex vivo, thromboelastometry and VWF ELISA on plasma samples was performed.

Results: Applying the carotid artery ligation injury model to Tlr4 global knockout mice, we observed reduced platelet deposition to the ligation injury site relative to wild-type (WT) controls. By thrombin ex vivo stimulation of washed WT and Tlr4-deficient platelets, no differences were observed in platelet activation. Therefore, platelet TLR4 is not a main effector of arterial thrombus growth. To pinpoint the role of the endothelium, we studied a conditional knockout model with endothelial-specific Tlr4-deficiency. Platelet deposition to the arterial injury site was strongly impaired in Cre+ mice compared with Cre- controls, indicating that TLR4 expressed by endothelial cells is involved in arterial thrombus formation. Ex vivo clotting parameters did not differ between Tlr4 global knockout mice and controls. Consistent with reduced arterial platelet deposition in endothelial Tlr4-deficiency, we found reduced von Willebrand factor (VWF) plasma levels in endothelial-specific Tlr4-deficient mice, which is likely responsible for the observed reduction in arterial thrombus growth.

Conclusion(s): Endothelial TLR4 plays a major role in platelet deposition to the ligation injury site at the common carotid artery in vivo.