Background: We have previously shown that lipopolysaccharide (LPS)-induced inflammation increased deep vein thrombosis (DVT) incidence associated with increased levels of von Willebrand factor (VWF). Venous thrombus architecture has been explored previously with conventional histology methods.

Aims: To simultaneously and quantitatively analyze the localization and interaction of ten venous thrombus constituents in control and endotoxemic mice.

Methods: The inferior vena cava stenosis model was used to induce DVT in wildtype mice, followed by intraperitoneal injections of 0.5mg/kg LPS or vehicle control. We employed Hyperion mass cytometry imaging to simultaneously analyze ten thrombus targets in these mice. Data was analyzed using Ilastik, CellProfiler, HistoCAT, and RStudio software programs.

Results: Regardless of LPS-induced endotoxemia; platelets, P-selectin, high-mobility group box 1 (HMGB1), leukocytes, VWF and fibrin localized significantly (p<0.05) in the white thrombus. Red blood cells (RBCs) were predominant in the red thrombus and neutrophil extracellular traps (NETs) were present throughout the whole thrombus. While endotoxemia did not significantly affect the overall VWF content, in those thrombi, VWF expression in the white thrombus positively correlated with HMGB1 and platelets and negatively with RBCs. VWF significantly correlated with P-selectin and fibrin in both the control and LPS cohorts. Although not statistically significant, NET-DNA correlations with VWF and fibrin were negative in the controls but positive in the LPS cohort. Correlations are shown in Figure 1. Clustering analysis identified the second and top highest VWF expression clusters among the 2 most common clusters in the control and LPS cohorts, respectively.

Correlational heatmaps of the average intensity of expression of TER119 (RBCs), CD62P (P-selectin), Ly6G (neutrophils), Ly6C (monocytes), VWF, HMGB1, NET citH3 (NET-associated citrullinated histones), aa200 (fibrin), CD41 (platelets), and NET DNA (NET-associated DNA) in Phenograph clusters in control (left) and LPS cohorts (right). Bonferroni-corrected p-values: # p<0.05, ## p<0.01, ### p<0.001, and #### p<0.0001.​

Conclusions: The majority of venous thrombus interactions, except for RBCs and NETs, occur in the white thrombus. Endotoxemia enhances VWF interactions with platelets and potentially platelet-derived HMGB1. While the VWF-fibrin thrombus scaffold remains constant, endotoxemia promotes NET interactions with VWF and fibrin. The importance of VWF’s role in venous thrombi is highlighted by the high representation of high-VWF expression clusters.

« Back to ISTH 2021 Congress

ISTH Congress Abstracts - https://abstracts.isth.org/abstract/mass-cytometry-analysis-of-venous-thrombi-demonstrates-key-roles-for-vwf-interactions-in-inflammation-associated-dvt/