OC 47.1 - Neutrophil integrin α9β1 Promotes Post-stroke Deep Vein Thrombosis

Background: Stroke patients are at a high risk for developing life-threatening venous thromboembolic (VTE) events. While prophylactic anticoagulation reduces VTE, they only prevent ~half of the expected VTE events and are associated with significant risk of bleeding, suggesting critical need of novel approaches. Recently, compelling evidence has emerged that implicates neutrophils in pathogenesis of deep vein thrombosis (DVT). The integrin α9 is highly expressed on neutrophils, upregulated upon neutrophil activation, and is known to stabilize neutrophil adhesion to the activated endothelium.

Aims: In this study, we aimed to evaluate the role of neutrophil integrin α9 in modulation of post-stroke DVT.

Methods: DVT after stroke in neutrophil-specific α9-/- (α9 Mrp8Cre+) and littermate control mice was evaluated using partial ligation of inferior vena cava. Deep proteomic profiling was performed using Tandem mass tag-based mass spectrometry and the effect of VCAM-1/α9 interaction on neutrophil hyperactivation and NETosis were evaluated using in-vitro assays. Computer-aided drug design was used to identify novel small molecule integrin α9 inhibitors.

Results: Post-stroke peripheral neutrophils displayed hyperactivation and increased integrin α9 levels. In IVC stenosis model, we observed significantly increased DVT incidence in mice with stroke compared to mice without stroke (Figure 1). Importantly, significantly reduced DVT severity was observed in α9 Mrp8Cre+ mice as compared to controls (Figure 2CD). Neutrophil specific α9 deficiency resulted in reduced neutrophil content and NETosis in IVC thrombus. Detailed bioinformatic analyses of proteome revealed reduced expression of proteins associated with glycolysis, inflammasome activation and NETosis in neutrophils isolated from α9 deficient mice as compared to control mice. Mechanistically, we found that VCAM-1/α9 axis promoted neutrophil hyperactivation and NETosis via ERK phosphorylation. Using computational modelling, we identified small molecules that inhibited VCAM-1/α9 interactions and neutrophil hyperactivation.

Conclusion(s): The results of the current study support the critical role of neutrophil integrin α9 in modulation of post-stroke DVT.