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

Abstract Background Bone 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. Objective We 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. Results Thrombus 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. Conclusions We 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.

1 Fig. S1. Normal plasma prostacyclin and nitric oxide levels in Bambi -/mice. Mouse plasma was collected from Bambi -/mice and littermates controls for measurement of the stable prostaglandin I2 (prostacyclin) derivative, 6-keto-PGF 1α (A) and nitrates and nitrites (nitric oxide, B) as described in supplemental methods. The data represent the mean ± SEM (n≥5 for each genotype). Statistical analysis was performed using unpaired student t-test (p>0.05). (C) Representative western blot of phosphorylated eNOS and eNOS in lung extracts from Bambi +/+ and Bambi -/mice (n=4). Protein levels were quantified using Image Lab 5.2.1 software (Biorad), normalised against GAPDH controls and expressed as relative intensity (peNOS/eNOS). Values are given as mean ± SEM from 2 western blots (n=4 for each genotype; right graph). Statistical analysis was performed using unpaired student t-test: non-significant (ns), p>0.05. Mice were subjected to the laser-induced thrombosis model as detailed in Figure 1. Distribution of the time to maximal thrombus size (A) and maximal thrombus size expressed in IFI platelet arbitrary units (AU) (B). (C) Graph showing the area under curve values from the platelet IFI vs time from individual thrombus. Each symbol represents one thrombus (23-25 thrombi in 3 mice for each genotype). Horizontal lines intersecting the data set represent the median. Statistical analysis was performed using Mann Whitney test: non-significant (ns): p>0.05; *p<0.05. Fig. S3. Normal plasma fibrinogen levels in Bambi -/animals Plasma from Bambi +/+ and Bambi -/mice (n=3) were collected, diluted in TBS buffer (1/100) and loaded on a 4-12% gel. Membranes were blotted and probed with a polyclonal anti-human fibrinogen-α antibody (representative western blot, left). Fibrinogen levels were quantified using Image Lab 5.2.1 software (Biorad) and normalized against total protein levels (right; mean ± SEM). Statistical analysis was performed using unpaired student t-test: nonsignificant (ns), p>0.05. Bambi flox/flox Tie2-Cre + mice (n=13) were subjected to the tail-bleeding assay. Blood was collected in PBS for the initial 10 min and blood loss was determined by quantification of haemoglobin (Hb) content in each collected sample. Each symbol represents one animal. Horizontal lines intersecting data sets represent the median. Statistical analysis was performed using Mann Whitney test: non-significant (ns), p>0.05.

Fig. S5. Increased thrombomodulin levels in endothelial cells isolated from Bambi -/mice. (A)
Representative western blot of thrombomodulin (TM) expression in endothelial cells isolated from lungs of Bambi +/+ and Bambi -/mice (MLEC). Protein levels were quantified using Image Lab 5.2.1 software (Biorad), normalised against GAPDH controls and expressed as relative (TM/GAPDH) intensities. Values are given as mean ± SEM (n≥12 from at least 4 separate isolations and 5 western blots; right graphs). Molecular weights from protein standards are indicated in kDa on each western blot. Statistical analysis was performed using unpaired student t-test with Welch's correction, *p<0.05. (B) Representative flow cytometry analysis of Bambi +/+ MLEC and Bambi -/-MLEC for thrombomodulin (left). Results are given as mean fluorescence intensities (MFI) ± SEM (n≥10 from 3 separate isolations; passages 2-8). Statistical analysis was performed using unpaired student t test, p<0.05. (C) Representative western blot of thrombomodulin (TM) expression in endothelial cells isolated from brains of Bambi +/+ and Bambi -/mice (MBEC; 5 mice per group). Protein levels were quantified using Image Lab 5.2.1 software (Biorad), normalised against GAPDH controls and expressed as relative intensities (TM/GAPDH). Values are given as mean ± SEM: Bambi +/+ MBEC (n=3 separate isolations); Bambi -/-MBEC (n=2 separate isolations). Molecular weights from protein standards are indicated in kDa on each western blot. Statistical analysis was performed using unpaired student t-test with Welch's correction: non-significant (ns), p>0.05 (right graphs). (D) APC standard curve generated for each experiment used to determine APC activity in Bambi +/+ MLEC and Bambi -/-MLEC. APC generation was quantified by determining the rate of chromogenic substrate S-2366 (0.5mM) cleavage at 405nm (cf. Fig. 6C).  -2765 (200µM) in the presence of 25µM phospholipids, the presence and absence of recombinant mouse TFPI (mTFPI; 8nM), and 40nM of polyclonal goat anti-mouse TFPI (α-TFPI) as indicated. Addition of mTFPI to the reaction led to a decrease in FXa activity by 76% ( ) compared to the control ( ). Addition of the α-TFPI antibody fully restored FXa activity ( ) demonstrating that TFPI function was fully inhibited by the addition of the antibody. Results from a representative experiment are shown (n=3).
Movie S1. Laser-induced thrombus formation in a Bambi +/+ and Bambi -/mouse: platelet and neutrophil recruitment to the vascular injury. Representative video of fluorescently-labeled platelets (green) and neutrophils (red) accumulating at the site of laser-induced injury in a cremaster muscle arteriole of a Bambi +/+ and Bambi -/mouse. Thrombus formation was studied using a combination of brightfield and fluorescence microscopy. A yellow color is seen when platelets and neutrophils are detected in the same thrombus region. Results are presented in Fig. 1. A timer is shown in the top left corner (hh:mm:ss:000) and a 10 µm scale bar in the bottom left corner.
Movie S2. Laser-induced thrombus formation in a Bambi +/+ and Bambi -/mouse: platelet and fibrin accumulation to the vascular injury. Representative video of fluorescently-labeled platelets (green) and fibrin(ogen) (red) accumulating at the site of laser-induced injury in a cremaster muscle arteriole of a Bambi +/+ and Bambi -/mouse. Thrombus formation was studied using a combination of brightfield and fluorescence microscopy. A yellow color is seen when platelets and fibrin are detected in the same thrombus region. Results are presented in Fig. 3. A timer is shown in the top left corner (hh:mm:ss:000) and a 10 µm scale bar in the bottom left corner.
Movie S3. Laser-induced thrombus formation in a Bambi +/+ and Bambi -/mouse: the effect of hirudin on thrombus stability. Representative video of fluorescently-labeled platelets (green) accumulating at the site of laser-induced injury in a cremaster muscle arteriole of a Bambi +/+ and Bambi -/mouse injected or not with hirudin. Thrombus formation was studied using a combination of brightfield and fluorescence microscopy. Results are presented in Fig. 4. A timer is shown in the top left corner (hh:mm:ss:000) and a 10 µm scale bar in the bottom left corner.
Movie S4. Laser-induced thrombus formation in a Bambi flox/flox and Bambi flox/flox Tie2-Cre + mouse: platelet and fibrin accumulation to the vascular injury. Representative video of fluorescently-labeled platelets (green) and fibrin(ogen) (red) accumulating at the site of laser-induced injury in a cremaster muscle arteriole of a Bambi +/+ and Bambi -/mouse. Thrombus formation was studied using a combination of brightfield and fluorescence microscopy. A yellow color is seen when platelets and fibrin are detected in the same thrombus region. Results are presented in Fig. 5. A timer is shown in the top left corner (hh:mm:ss:000) and a 10 µm scale bar in the bottom left corner.
Movie S5. Laser-induced thrombus formation in a Bambi -/mouse: the effect of inhibiting thrombomodulin, TFPI or both in platelet and fibrin accumulation. Representative video of fluorescently-labeled platelets (green) and fibrin(ogen) (red) accumulating at the site of laser-induced injury in a cremaster muscle arteriole of a Bambi -/mouse. When indicated, mice were injected with the following antibodies: control goat IgG (top left), goat anti-mouse thrombomodulin (α-TM; top right), goat anti-mouse TFPI (α-TFPI; bottom left), or both (α-TM + α-TFPI; bottom right). Thrombus formation was studied using a combination of brightfield and fluorescence microscopy. A yellow color is seen when platelets and fibrin are detected in the same thrombus region. Results are presented in Fig. 7. A timer is shown in the top left corner (hh:mm:ss:000) and a 10 µm scale bar in the bottom left corner.
Movie S6. Laser-induced thrombus formation in a Bambi +/+ mouse: the effect of inhibiting thrombomodulin, TFPI or both in platelet and fibrin accumulation. Representative video of fluorescently-labeled platelets (green) and fibrin(ogen) (red) accumulating at the site of laser-induced injury in a cremaster muscle arteriole of a Bambi +/+ mouse. When indicated, mice were injected with the following antibodies: control goat IgG (top left), goat anti-mouse thrombomodulin (α-TM; top right), goat anti-mouse TFPI (α-TFPI; bottom left), or both (α-TM + α-TFPI; bottom right). Thrombus formation was studied using a combination of brightfield and fluorescence microscopy. A yellow color is seen when platelets and fibrin are detected in the same thrombus region. Results are presented in Fig. 7. A timer is shown in the top left corner (hh:mm:ss:000) and a 10 µm scale bar in the bottom left corner.