Defective fibrin deposition and thrombus stability in Bambi−/− mice are mediated by elevated anticoagulant function

Abstract

BackgroundBone morphogenetic and activin membrane‐bound inhibitor (BAMBI) is a transmembrane protein related to the type I transforming growth factor‐ β (TGF‐β) receptor family that is present on both platelets and endothelial cells (ECs). Bambi‐deficient mice exhibit reduced hemostatic function and thrombus stability characterized by an increased embolization.ObjectiveWe aimed to delineate how BAMBI influences endothelial function and thrombus stability.Methods Bambi‐deficient mice were subjected to the laser‐induced thrombosis model where platelet and fibrin accumulation was evaluated. Expression of thrombomodulin and tissue factor pathway inhibitor (TFPI) was also assessed in these mice.ResultsThrombus instability in Bambi −/− mice was associated with a profound defect in fibrin deposition. Injection of hirudin into Bambi +/+ mice prior to thrombus formation recapitulated the Bambi −/− thrombus instability phenotype. In contrast, hirudin had no additional effect upon thrombus formation in Bambi −/− mice. Deletion of Bambi in ECs resulted in mice with defective thrombus stability caused by decreased fibrin accumulation. Increased levels of the anticoagulant proteins TFPI and thrombomodulin were detected in Bambi −/− mouse lung homogenates. Endothelial cells isolated from Bambi −/− mouse lungs exhibited enhanced ability to activate protein C due to elevated thrombomodulin levels. Blocking thrombomodulin and TFPI in vivo fully restored fibrin accumulation and thrombus stability in Bambi −/−mice.ConclusionsWe demonstrate that endothelial BAMBI influences fibrin generation and thrombus stability by modulating thrombomodulin and TFPI anticoagulant function of the endothelium; we also highlight the importance of these anticoagulant proteins in the laser‐induced thrombosis model.

The contribution of TFPIα to the hemostatic response to injury in mice

“…Of note, a previous study by Crawley et al found that infusing a TFPI antibody that is not isoform selective into Bambi −/− mice, which have a defect in fibrin accumulation after injury, restores both fibrin accumulation and thrombus stability in an injury model similar to the one that we used. 59 Combined with our results in which only TFPIα is missing, this suggests that plasma TFPIγ and endothelial cell TFPIβ, or a combination of TFPIα, TFPIβ, and TFPIγ, are the relevant isoforms in this setting.…”

The GPIbα intracellular tail - role in transducing VWF- and Collagen/GPVI-mediated signaling

“…Next, we analyzed thrombus formation in GpIb sig/sig mice following mild laser injury to cremaster arteriole vascular wall. (Stalker, 2020, Salles-Crawley et al, 2014, Crawley et al, 2019 In this model, there was no difference between GpIb sig/sig mice and wild-type littermates in thrombus formation, as measured by the kinetics and extent, of both platelet accumulation and fibrin deposition (Figure 3B-H;…”

“…Tail bleeding time was performed as described previously. (Salles-Crawley et al, 2014, Crawley et al, 2019 Mice were anaesthetized with ketamine/medetomidine, placed on a heating pad (Harvard Apparatus) at 37°C and a 2 mm segment of the tail was sectioned with a sharp blade. The tail was immediately placed in warm PBS and the time taken for the stream of blood to stop for more than 60 seconds was defined as the bleeding time.…”

“…Thrombus formation was evaluated in the cremaster muscle microcirculation as previously described. (Salles-Crawley et al, 2014, Crawley et al, 2019 Ketamine (75mg/kg) and medetomidine (1mg/kg) was initially given as an intraperitoneally injection. The anesthesia was maintained by giving additional ketamine (12.5mg/kg) every 40 mins.…”

“…Median integrated fluorescence intensity over time from platelet or fibrin was determined and analyzed as detailed previously. (Salles-Crawley et al, 2014, Crawley et al, 2019 The operator was blinded to the genotypes during both data acquisition and analysis.…”