Tissue factor promotes activation of coagulation and inflammation in a mouse model of sickle cell disease

Abstract

Sickle cell disease (SCD) is associated with a complex vascular pathophysiology that includes activation of coagulation and inflammation. However, the crosstalk between these 2 systems in SCD has not been investigated. Here, we examined the role of tissue factor (TF) in the activation of coagulation and inflammation in 2 different mouse models of SCD (BERK and Townes). Leukocytes isolated from BERK mice expressed TF protein and had increased TF activity compared with control mice. We found that an inhibitory anti-TF antibody abrogated the activation of coagulation but had no effect on hemolysis or anemia. Importantly, inhibition of TF also attenuated inflammation and endothelial cell injury as demonstrated by reduced plasma levels of IL-6, serum amyloid P, and soluble vascular cell adhesion molecule-1. In addition, we found decreased levels of the chemokines MCP-1 and KC, as well as myeloperoxidase in the lungs of sickle cell mice treated with the anti-TF antibody. Finally, we found that endothelial cell-specific deletion of TF had no effect on coagulation but selectively attenuated plasma levels of IL-6. Our data indicate that different cellular sources of TF contribute to activation of coagulation, vascular inflammation, and endothelial cell injury. Furthermore, it appears that TF contributes to these processes without affecting intravascular hemolysis.

Figure 1

TF expression in sickle cell mice. (A) TF mRNA expression and (B) procoagulant activity (PCA) in organs of WT (open bar; n = 9) and BERK mice (filled bar; n = 8). K indicates kidney; Lu, lung; H, heart; Li, liver; and S, spleen. Asterisks directly above the bars indicate statistically significant difference compared with WT controls: **P < .01 and ***P < .001. (C) TF-dependent PCA activity in the spleens of WT (open bar) and BERK mice (filled bar). *P < .05. Immunocytochemistry (D-E) or immunofluorescence (F-G) staining demonstrates TF-positive leukocytes with nuclear morphology resembling monocytes (D,F) or neutrophils (E,G). TF is shown as brown staining (top panels) or green staining (bottom panels). Nuclei are stained blue. (H) TF activity of the leukocytes isolated from the same volume of blood obtained from WT (n = 13) or BERK (n = 10) mice. **P < .01. (I) TF activity of the microparticles (MP TF) isolated from the plasma of WT (open bar; n = 6) and BERK (filled bar; n = 15) mice. (J) MP TF activity in TW^AA (open bar; n = 13) and TW^SS (filled bar; n = 17) mice.

Figure 2

Activation of coagulation in sickle cell mice. (A) Plasma TAT levels in WT (open bar; n = 6) and BERK (filled bar; n = 16) mice (both on a similar mixed genetic background). *P < .05. (B) Plasma TAT levels in WT^BM (open bar) or BERK^BM (filled bar) mice injected with either a rat anti–mouse TF (n = 5 for WT^BM; n = 14 for BERK^BM) or control rat IgG antibodies (n = 8 for WT^BM; n = 14 for BERK^BM). (C) Plasma TAT levels in TW^AA (open bar) or TW^SS (filled bar) mice injected with either a rat anti–mouse TF (n = 6 for TW^AA; n = 7 for TW^SS) or control rat IgG antibodies (n = 7 for TW^AA; n = 11 for TW^SS). Asterisks directly above the bars indicate statistically significant difference compared with WT^BM or TW^AA controls within the same treatment: **P < .01; and ***P < .001.

Figure 3

Hemolysis and red blood cell maturation. (A) Levels of hemoglobin (Hb) in WT^BM (open bar) or BERK^BM (filled bar) mice injected with either a rat anti –mouse TF (n = 5 for WT^BM; n = 14 for BERK^BM) or control rat IgG antibodies (n = 8 for WT^BM; n = 14 for BERK^BM). (B) Levels of Hb in TW^AA (open bar) or TW^SS (filled bar) mice injected with either a rat anti–mouse TF (n = 6 for TW^AA; n = 7 for TW^SS) or control rat IgG antibodies (n = 7 for TW^AA; n = 11 for TW^SS). (C-D) Plasma levels of LDH were analyzed in the same group of mice as described for panels A and B. Asterisks directly above the bars indicate statistically significant difference compared with WT^BM or TW^AA controls within the same treatment: *P < .05; **P < .01; and ***P < .001. (E) Representative density plots demonstrating levels of CD71 expression on Ter119⁺ cells (red blood cells) from TW^AA and TW^SS mice treated with 1H1 or IgG. (F) Red blood cell maturation profiles in TW^AA or TW^SS mice treated with 1H1 or IgG (n = 6-11).

Figure 4

Inflammation and endothelial cell activation in sickle cell mice. Plasma levels of various inflammatory mediators and markers of endothelial cell activation in: WT (open bar; n = 12) and BERK (filled bar; n = 22) mice (left column); WT^BM (open bar; n = 5-8) and BERK^BM (filled bar; n = 14) mice (middle column) treated with 1H1 or IgG antibodies; TW^AA (open bar; n = 6 or 7) and TW^SS (filled bar; n = 7-11) mice (right column) treated with 1H1 or IgG antibodies. Asterisks directly above the bars indicate statistically significant difference compared with wild-type controls within the same treatment: *P < .05; **P < .01; and ***P < .001.

Figure 5

Neutrophil infiltration and activation in various organs of BERK mice. Neutrophils (brown staining) are present in the lungs and livers, but not kidneys, of BERK mice. Tissue levels of MPO were determined in the organs of WT (n = 8 or 9) and BERK (n = 10-12) mice. Asterisks directly above the bars indicate statistically significant difference compared with WT group: *P < .05.

Figure 6

Effect of TF inhibition on MPO and chemokine expressions in BERK^BM mice. Tissue levels of MPO (A), MCP-1 (B), and KC (C) were analyzed in WT^BM (open bar) or BERK^BM (filled bar) mice injected with either a rat anti–mouse TF (n = 5 for WT^BM; n = 14 for BERK^BM) or control rat IgG antibodies (n = 8 for WT^BM; n = 14 for BERK^BM). Asterisks directly above the bars indicate statistically significant difference compared with WT^BM controls within the same treatment: *P < .05; **P < .01; and ***P < .001.

Figure 7

Role of endothelial cell TF in the activation of coagulation, inflammation, and endothelial cell activation in sickle cell mice. (A) Representative gel demonstrating the analysis of different hemoglobin types by cellulose acetate electrophoresis in TF^flox/flox,Tie-2 Cre mice transplanted with bone marrow from BERK mice (lines 1-3 and 9) or WT mice (lines 4 and 6-8). Upper and lower bands correspond to human sickle hemoglobin and normal mouse hemoglobin. Lines 5 and 10 contain positive controls (con) for both forms of hemoglobin. Line 11 shows the example of unsuccessful transplantation of bone marrow from BERK into TF^flox/flox,Tie-2 Cre mouse. Mice like this were excluded from experiments. A vertical line has been added to indicate 2 separate gels. (B-H) Plasma levels of TAT, IL-6, IL-18, SAP, and sVCAM-1 as well as tissue levels of MPO in lung and liver were analyzed in WT (open bar) and sickle cell mice (filled bar) with (+) or without (−) TF gene deletion in endothelial cells (EC TF Δ; n = 9-14). Asterisks directly above the bars indicate statistically significant difference compared with WT^BM controls within the same treatment: *P < .05; **P < .01; and ***P < .001.