The mechanistic and structural role of von Willebrand factor in endotoxemia-enhanced deep vein thrombosis in mice

Abstract

Background: Although the concept of immunothrombosis has established a link between inflammation and thrombosis, the role of inflammation in the pathogenesis of deep vein thrombosis remains to be fully elucidated. Further, although various constituents of venous thrombi have been identified, their localizations and cellular and molecular interactions are yet to be combined in a single, multiplexed analysis.

Objectives: The objective of this study was to investigate the role of the von Willebrand factor (VWF) in inflammation-associated venous thrombosis. We also performed a proof-of-concept study of imaging mass cytometry to quantitatively and simultaneously analyze the localizations and interactions of 10 venous thrombus constituents.

Methods: We combined the murine inferior vena cava stenosis model of deep vein thrombosis with the lipopolysaccharide model of endotoxemia. We also performed a proof-of-concept study of imaging mass cytometry to assess the feasibility of this approach in analyzing the structural composition of thrombi.

Results: We found that lipopolysaccharide-treated mice had significantly higher incidences of venous thrombosis, an effect that was mitigated when VWF was inhibited using inhibitory αVWF antibodies. Our detailed structural analysis also showed that most thrombus components are localized in the white thrombus regardless of endotoxemia. Moreover, although endotoxemia modulated the relative representation and interactions of VWF with other thrombus constituents, the scaffolding network, comprised VWF, fibrin, and neutrophil extracellular traps, remained largely unaffected.

Conclusions: We observe a key role for VWF in the pathogenesis of inflammation-associated venous thrombosis while providing a more comprehensive insight into the molecular interactions that constitute the architecture of venous thrombi.

Keywords: animal models; image cytometry; thromboinflammation; venous thrombosis; von Willebrand factor.

FIGURE 1

Endotoxemia affects blood and plasma characteristics in mice. (A) Circulating platelet counts in blood samples collected prior to (Pre) and 24 hours after (Post) the stenosis procedure in mice receiving vehicle control (n = 16) or LPS (n = 17). Plasma levels of (B) cfDNA and (C) IL-6 at 24 hours post-stenosis in mice receiving control (n = 19) or LPS (n = 15). Pre and Post (D) VWF:Ag and (E) ratio of VWF:CB to VWF:Ag in mice receiving control (n = 17) or LPS (n = 17). (F) Pre and post-FVIII:C in mice receiving control (n = 14) or LPS (n = 17). (G) Changes in VWF:Ag from baseline to 24 hours post-stenosis in thrombosed (control n = 6, LPS n = 13) and nonthrombosed (control n = 11, LPS n = 4) mice. All assays were performed in duplicate. Pooled plasma from normal C57Bl/6 mice was used as the reference for all VWF assays. *p < .05, **p < .01, ***p < .001, ****p < .0001. cfDNA, cell-free DNA; IL-6, Interleukin 6; LPS, lipopolysaccharide; ns, not significant; VWF:Ag, plasma von Willebrand factor; VWF:CB, von Willebrand factor–collagen binding activity.

FIGURE 2

Endotoxemia increases thrombosis incidence, an outcome that is mitigated by reduced VWF activity. (A) Thrombus incidence and (B) thrombus weights (>0 mg) of vehicle control and LPS-treated mice at 24 hours post-stenosis. (C) VWF:GPIbM at 24 hours post-stenosis in vehicle control and LPS-treated mice treated with αVWF antibody (n = 10 and n = 8, respectively) or ISO (n = 6 and n = 9, respectively), expressed as a percentage of activity relative to pooled murine plasma as reference. (D) Thrombus incidence and (E) thrombus weights in control and LPS-treated mice in αVWF and ISO cohorts at 24 hours post-stenosis. ns: not significant. *p < .05, ***p < .001. ISO, isotope control antibody; LPS, Lipopolysaccharide; ns, not significant; VWF:Ag, plasma von Willebrand factor; VWF:CB, von Willebrand factor–collagen binding activity; VWF:GbIb, VWF-GPIb binding activity.

FIGURE 3

Enhanced ADAMTS13 activity does not affect endotoxemia-associated thrombosis outcomes. (A) VWF:Ag and (B) ratios of VWF:CB to VWF:Ag in blood samples collected prior to (Pre) and 24h after (Post) the stenosis procedure from mice receiving vehicle control (n = 10) or recombinant human ADAMTS13 (rhADAMTS13) (n = 11). (C) Thrombus incidence and (D) thrombus weights (>0 mg) of vehicle control and rhADAMTS13-treated mice at 24h post-stenosis. Pooled plasma from normal C57Bl/6 mice was used as the reference for all assays. **p < .01, ****p < .0001. ns, not significant; VWF:Ag, plasma von Willebrand factor; VWF:CB, von Willebrand factor–collagen binding activity.

FIGURE 4

Endotoxemia significantly increases the proportion of the red thrombus, but most thrombus components largely localize in the white thrombus. (A) Hematoxylin and eosin stains of thrombi obtained from control (left) and LPS- (right) treated mice at 24 hours post-stenosis show distinct red and white thrombi. Images are representative of thrombi from n = 7 control and n = 12 LPS mice. Scale bar, 1.0 mm. (B) Mean areas of red and white thrombi as percentages of the entire thrombus in control and LPS cohorts. (C) Heatmaps representing the areas occupied by CD41 (platelets), CD62P (P-selectin), HMGB1, Ly6C (monocytes), Ly6G (neutrophils), Ter119 (RBCs), VWF, and fibrin(ogen) as percentages of the red and white thrombi in the control (top) and LPS (bottom) cohorts. The scale on the right represents percentages. Areas occupied by (D) Ter119 and (E) fibrin(ogen) in thrombi derived from control and LPS-treated mice as percentages of red and white thrombi. *p < .05, ****p < .0001. LPS, lipopolysaccharide; ns, not significant; VWF, von Willebrand factor; VWF:Ag, plasma von Willebrand factor; VWF:CB, von Willebrand factor–collagen binding activity.

FIGURE 5

Imaging mass cytometry data analysis pipeline. Two or 3 ROIs from 3 biological replicates of thrombi from control and LPS-treated mice were stained for mass cytometry imaging. Data analysis workflow using various software is shown above. LPS, lipopolysaccharide; ROI, regions of interest.

FIGURE 6

Endotoxemia increases the relative representation of VWF and affects its localization relative to other thrombus constituents. (A) Area occupied by VWF in thrombi derived from control and LPS-treated mice as percentages of red and white thrombi. Correlational heatmaps of the average intensity of expression of Ter119 (RBCs), CD62P (P-selectin), Ly6G (neutrophils), Ly6C (monocytes), VWF, HMGB1, CitH3, fibrin(ogen), CD41 (platelets), and extracellular DNA in Phenograph clusters in (B) control and (C) LPS cohorts. t-SNE representations of the 2 most common clusters in the (D) control and (E) LPS cohorts were identified on a heatmap of VWF expression intensity. ****p < .0001. #, ##, ###, #### represent significance levels after Bonferroni multiple-tests correction. LPS, lipopolysaccharide; ns, not significant; t-SNE, T-distributed Stochastic Neighbor Embedding; VWF, von Willebrand factor.

FIGURE 7

The 2 most common clusters do not exhibit a discernible pattern. The 2 most common clusters highlighted on false color overlay images of Ter119 (red), VWF (green), and CD41 (blue) in the (A) control and (B) LPS cohorts with the zoomed areas indicated by the red square shown on the right. False color overlay images of the top 5 targets with the highest relative expressions, CD62P (red), Ly6G (green), VWF (blue), fibrin(ogen) (cyan), and CD41 (magenta), in thrombi derived from (C) control and (D) LPS cohorts with the zoomed areas indicated by the red square shown on the right. LPS, lipopolysaccharide; VWF, von Willebrand factor.

FIGURE 8

VWF associates with fibrin(ogen), but neither correlates with CitH3 and extracellular DNA, regardless of endotoxemia. False color overlay images of scaffolding components within thrombi derived from mice treated with (A) control and (B) LPS. VWF (red), fibrin(ogen) (blue), CitH3 (yellow), and extracellular DNA (green). LPS, lipopolysaccharide; VWF, von Willebrand factor.