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

Background
Deep vein thrombosis (DVT) 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 DKO), results in a near complete defect in P-selectin externalisation upon activation, while other platelet activation responses and arterial thrombosis are preserved.

Objective
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.

Methods
DVT was induced by surgical partial ligation of the caudal (inferior) vena cava for 24-h or 48-h before venous thrombi were assessed by histology and immunofluorescence microscopy.

Results
RalAB DKO mice show a reduction in venous thrombus formation after 24-h, and near complete ablation of venous thrombosis by 48-h post IVC ligation. Immunofluorescence microscopy revealed that cross sections of thrombi from wildtype mice consist of an organised scaffolded structure of platelets surrounding leukocytes/neutrophils producing NETs to stabilize and propagate the thrombus. This organised 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. Conclusion: 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.