Neutrophil KLF2 Regulates Arterial and Venous Thrombosis

Experimental, clinical and pathological studies support an important link between inflammation and thrombosis. Although accumulating evidence suggests that cells of the innate immune system contribute to the thrombotic process, the identity of nodal molecular determinants operative in immune cells remains poorly understood. Our previous work in mice bearing global deletion of the transcription factor KLF2 identifiedthis factor as a critical mediator of thrombosis). Here, using cell-specific KLF2 deleted murine models (endothelial, platelet, and myeloid- deleted KLF2) we identify myeloid KLF2 as the major determinant of thrombosis. Complete blood counts and coagulation assays in myeloid KLF2 deleted mice (MY-K2-KO) were similar to those in control LysM cre mice. Using two models of vascular thrombosis (carotid artery thrombosis assay, Rose Bengal model and complete inferior vena cava ligation model of venous thrombosis) we observed a robust prothrombotic phenotype in the MY-K2-KO mice as compared to control Cre mice. As LysM Cre deletes in both neutrophils and monocyte/macrophages, we next sought to determine whether one subset was dominantly responsible for the observed phenotype. Adoptive transfer of KLF2-KO neutrophils in control mice conferred both the arterial and venous pro-thrombotic phenotype. Conversely, neutrophil depletion with Ly6G antibody (>90% depletion achieved) reversed the prothrombotic phenotype in MY-K2-KO mice. By contrast, depletion of monocytes/macrophages using clodronate liposome injection did not affect arterial or venous thrombosis. Next, we evaluated the effect of KLF2 deficiency on critical neutrophil functions that may contribute to the pro-thrombotic phenotype. As compared to controls, MY-K2-KO neutrophils demonstrated more pronounced recruitment to the endothelium. In microflow chamber assays, MY-K2-KO neutrophils showed decreased rolling and increased P-selectin induced adhesion. The importance of this was affirmed by the observation that adoptive transfer of MY-K2-KO neutrophils incubated with PSGL-1 antibody was unable to generate a prothrombotic phenotype in control mice. In addition, MY-K2-KO neutrophils demonstrated enhanced neutrophil extracellular traps generation associated with increased myeloperoxidase and neutrophil elastase expression. And finally, MY-K2-KO neutrophils (but not monocytes) demonstrated increased tissue factor expression and activity. Collectively, these studiesidentify neutrophil KLF2 as a critical determinant of both arterial and venous thrombosis and suggest that modulation of neutrophil KLF2 may be a fruitful approach to the management of thrombosis.