Critical role for platelet Ral GTPases in regulating venous thrombosis in mice

Deep vein thrombosis is a major cause of morbidity and mortality globally. While its pathophysiology is complex, increasing evidence suggests a more prominent role for platelets than previously suspected. Genetic deletion of Ral GTPases, RalA and RalB, conditionally in mouse platelets (RalAB double knockout [DKO]), results in a near complete defect in P-selectin externalization upon activation, while other platelet activation responses and arterial thrombosis are preserved. Given the critical role of P-selectin in mediating platelet-neutrophil interaction and thromboinflammation, we sought to investigate whether platelet Rals would also play critical roles in venous thrombosis, a thromboinflammatory disease, using RalAB DKO mice. Deep vein thrombosis was induced by surgical partial ligation of the caudal (inferior) vena cava for 24 hours or 48 hours before venous thrombi were assessed by histology and immunofluorescence microscopy. RalAB DKO mice showed a reduction in venous thrombus formation after 24 hours and near complete ablation of venous thrombosis by 48 hours post inferior vena cava ligation. Immunofluorescence microscopy revealed that cross-sections of thrombi from wild-type mice consisted of an organized scaffolded structure of platelets surrounding leukocytes/neutrophils producing neutrophil extracellular traps (NETs) to stabilize and propagate the thrombus. This organized structure was absent in platelet-specific conditional RalAB DKO thrombi. In vitro analysis of platelet-mediated NET formation was also significantly reduced when platelets lacked RalAB or when platelets were treated with the Ral inhibitor RBC8. We identify platelet Rals as novel, potentially critical regulators of venous thrombus stability through their ability to regulate neutrophil NET formation via platelet P-selectin.