Endothelial Protein Tyrosine Phosphatase-1 Deletion Promotes Venous Thromboinflammation by SNAP23 Phosphorylation Facilitating Von Willebrand Factor Exocytosis and Extracellular Vesicle Release

Background: Endothelial activation promotes the release of procoagulant extracellular vesicles (EVs) and proinflammatory mediators, such as von Willebrand factor (VWF) from specialized storage granules. The exocytosis and release of coagulant, adhesive and inflammatory mediators from specialized endothelial storage granules, the Weibel-Palade bodies, is regulated by phosphorylation. We hypothetized that absence of protein tyrosine phosphatase-1B (PTP1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and vesicle exocytosis.

Methods: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior Vena cava (IVC) ligation to induce stasis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies.

Results: Vascular ultrasound and histological assessments revealed significantly enlarged venous thrombi with elevated Ly6G+ neutrophil counts in mice exhibiting endothelial PTP1B deletion. Intravital microscopy further confirmed elevated neutrophil recruitment post-IVC ligation. Global coagulation assays underscored abnormal coagulation system activation in End.PTP1B-KO mice. Gene expression and proteomic analyses unveiled heightened expression of endothelial activation and adhesion molecules, including P-selectin (CD62P) and VWF, in primary End.PTP1B-KO endothelial cells. In vitro, pretreatment with the NFkappaB kinase inhibitor BAY11-7082, antibodies neutralizing P-selectin glycoprotein ligand (CD162) or VWF, or RGD integrin-blocking peptides, all effectively reduced neutrophil adhesion to End.PTP1B-KO endothelial cells. Circulating levels of annexin V+ procoagulant endothelial (CD62E+) and neutrophil (Ly6G+) EVs were elevated in End.PTP1B-KO mice post-IVC ligation. Increased plasma myeloperoxidase, citrullinated histone-3 levels, and neutrophil elastase activity indicated heightened neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO EVs into C57BL/6J wild-type mice notably increased endogenous neutrophil recruitment, a response attenuated in VWF-deficient mice or by VWF-blocking antibodies. Mechanistically, reduced PTP1B binding and tyrosine dephosphorylation of SNAP23, an essential component of the endothelial membrane fusion machinery, were identified as mechanisms driving increased VWF exocytosis, and increased SNAP23 tyrosine phosphorylation in the absence of PTP1B was associated with elevated endothelial VWF release and neutrophil adhesion. Notably, RNA interference targeting SNAP23 or pharmacological inhibition of nuclear factor-kappa B activation using BAY11-7082 both prevented the increased VWF release and neutrophil adhesion in the absence of endothelial PTP1B.

Conclusions: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis and neutrophil recruitment.