Endothelial Transforming Growth Factor-beta signaling promotes Von Willebrand Factor string formation and blood cell retention during venous thrombus formation

Background and Aim: Endothelial cells and their interaction with circulating blood and inflammatory cells play a central role in the pathogenesis of thrombosis, and defects in thrombus revascularization mediated by endothelial cells are also important for thrombus resolution. Previous work has highlighted the role of overactivated endothelial TGFbeta signaling for chronic venous thrombus non-resolution and the development of thrombofibrosis. Endothelial coagulation proteins in plasma, such as Von Willebrand Factor (VWF), contain binding motifs for active TGFbeta1, however the role of endothelial TGFbeta1 signaling during blood clot formation and thrombus formation is largely unknown.

Methods: Endothelial cells isolated from mice with genetic deletion of TGFbeta type II receptors in endothelial cells (End.TGFBRII-KO) and littermate controls were subjected to next generation sequencing (NGS). Venous thrombosis was induced by inferior Vena cava ligation, and cellular interactions during thrombus formation were observed using epifluorescence intravital microscopy. Whole blood was isolated from uninjured mice and mice on 2 day post-surgery, and whole blood cell counts, clotting times and clot contraction were analyzed in vitro.

Results: NGS analysis of primary endothelial cells revealed that out of 29.807 genes probed in total, 484 were significantly upregulated and 976 significantly downregulated in End.TGFBRII-KO cells. Among others, expression of Von Willebrand Factor (VWF) A domain containing 1 (Vwa1) was significantly increased (adjusted P value: 0.0009) in End.TGFBRII-KO mice compared to controls, and plasma levels of VWF also were significantly increased in End.TGFBRII-KO mice. Intravital microscopy in mice following inferior Vena cava ligation and transfusion of red blood cells (RBCs) from membrane Tomato-transgenic mice showed that erythrocytes adhere to endothelial cells with TGFBRII deletion much faster and stronger than in End.TGFBRII-WT littermate mice, forming erythrocyte-decorated VWF strings. Similar, but less pronounced findings were observed in mice following infusion of fluorescent platelets or neutrophils. Immunohistochemical staining of 2-day-old thrombi confirmed that RBCs robustly attach to endothelial cells lining the inner wall of the injured vein of End.TGFBRII-KO mice and higher VWF immunosignals. Blood counts did not reveal any differences in hematocrit, RBC numbers or hemoglobin content in End.TGFBRII-KO mice compared to End.TGFBRII-WT littermate controls, coagulation assays no differences in the prothrombin or activated partial thromboplastin time. However, findings of significantly shortened clotting of whole blood on glass slides in End.TGFBRII-KO mice suggested activation of the contact pathway. Also, thrombin-induced clots were significantly larger if whole blood from End.TGFBRII-KO mice was used, in line with impaired fibrin-dependent contraction and protection from fibrinolysis, and findings normalized following addition of TGFbeta-neutralizing antibodies.

Conclusions: Our findings suggest overactivated endothelial TGFbeta signaling promotes VWF release and VWF string formation resulting in increased erythrocyte, platelet and immune cell retention during venous thrombus formation thus prolonging clot lysis.