Gas6 Promotes Pro-Inflammatory (Ly6C^hi) Monocyte Recruitment in Venous Thrombosis

Recent studies have demonstrated that innate immune cells, i.e. neutrophils and monocytes, provide the initiating stimulus for venous thrombus development. Gas6 was found to promote inflammation by inducing interactions between the endothelium and innate immune cells. In addition, we recently showed that Gas6 was involved in venous thrombosis by inducing tissue factor expression in the endothelium. Since Gas6 is expressed in monocytes, we hypothesize that Gas6 may be involved in monocyte recruitment during venous thrombosis. Venous thrombosis was induced in the inferior vena cava of wild type (WT) and Gas6 deficient (-/-) mice using 5% FeCl₃. Using ultrasonography, we found that global monocyte depletion by clodronate resulted in the formation of smaller thrombi. Selective depletion of the pro-inflammatory (Ly6C^hi) monocyte subset, using an anti-CCR2 antibody, also induced the formation of smaller clots. In addition, MOMA-2 (monocyte-macrophage marker) staining showed a reduced number of monocytes in thrombi from Gas6^-/- mice. More importantly, immunofluorescent staining revealed that fewer Ly6C^hi monocytes were recruited to the thrombi of Gas6^-/- mice compared to WT. However, Ly6C^low (patrolling) monocytes were equivalently recruited between Gas6^-/- and WT mice. In vitro, mRNA expression of CCR2 was increased by thrombin in WT but not in Gas6^-/- monocytes. The mRNA and protein expression of the CCR2 ligand, CCL2, was also increased by thrombin in WT but not in Gas6^-/- endothelial cells. CCL2 secretion, as demonstrate by ELISA, was induced by thrombin treatment in WT but not in Gas6^-/- endothelial cells. Conditioned media from WT or Gas6^-/- endothelial treated by thrombin was used for monocyte migration experiments. The conditioned media from WT endothelial cells treated with thrombin increased migration of WT monocytes compared to media from untreated or Gas6^-/- endothelial cells. Our results demonstrate an important role for Ly6C^hi monocytes in thrombus formation and that Gas6 is specifically involved in the recruitment of these monocytes through the expression of CCL2 and CCR2.