Background: PTP1B, a prototype protein tyrosine phosphatase, negatively regulates tyrosine kinase receptor signalling, and its absence has been associated with premature endothelial senescence. Senescence is associated with an increased risk of thrombosis, particular in the venous system. The underlying mechanisms are complex and may involve endothelial dysfunction and procoagulant extracellular vesicle (EV) release.

Methods: Mice with tamoxifen-inducible, Tie2.ERT2-Cre mediated deletion of PTP1B (End.PTP1B-KO) underwent inferior Vena cava (IVC) ligation. Endothelial dysfunction and neutrophil activation were investigated by qPCR, immunofluorescence, flow cytometry, in vitro assays and intravital microscopy. Coagulation assays were performed using murine plasma or EVs isolated from it.

Results: Serial ultrasound measurements at different time points following IVC ligation revealed that endothelial PTP1B deficiency results in significantly larger venous thrombi at day 2, whereas thrombus resolution over 3 weeks was not altered. Histological analysis of thrombosed IVC segments supported the ultrasound findings and also revealed increased inflammatory leucocyte recruitment in End.PTP1B-KO mouse thrombi, including Ly6G+ neutrophils. Increased neutrophil adhesion to primary endothelial cells isolated from mice with genetic PTP1B deletion was also observed in vitro, and RT² PCR profiler array and immunofluorescence analysis demonstrated increased expression of markers of cellular activation in primary endothelial cells isolated from End.PTP1B-KO mice. Quantitative flow cytometry of annexin V+ procoagulant EVs at day 2 after IVC ligation revealed elevated circulating levels of CD62E+ endothelial- and Ly6G+ neutrophil-derived EVs in End.PTP1B-KO mice, in line with endothelial and neutrophil activation. Findings of increased myeloperoxidase levels and neutrophil elastase activity in plasma or elevated whole blood reactive oxygen species formation in response to zymosan A further supported increased neutrophil priming in End.PTP1B-KO mice, whereas plasma levels of citrullinated histone-3 (a marker of neutrophil extracellular trap formation and NETosis) did not differ. Intravital microscopy in C57BL/6 wild-type mice showed that injection of End.PTP1B-KO EVs enhanced the recruitment of endogenous neutrophils to the site of injury to a significantly greater extent than PTP1B-WT EVs, whereas neutrophil recruitment was markedly reduced in End.PTP1B-KO mice injected with C57BL/6 wild-type EVs. Activation of the coagulation cascade, both in the intrinsic and extrinsic pathway, was significantly more pronounced by End.PTP1B-KO compared to End.PTP1B-WT plasma, and findings of significantly increased factor X activation and thrombin generation were phenocopied by EVs isolated from End.PTP1B-KO mouse blood.  

Conclusions: The results of this ongoing study suggest that endothelial PTP1B deficiency promotes venous thrombosis by endothelial and neutrophil activation and the release of procoagulant EVs.