Levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice

Abstract

Background: Tissue factor (TF) is present in blood in various forms, including small membrane vesicles called microparticles (MPs). Elevated levels of these MPs appear to play a role in the pathogenesis of thrombosis in a variety of diseases, including sepsis.

Objective: Measure levels of MP TF activity and activation of coagulation in control and endotoxemic mice.

Materials and methods: MPs were prepared from plasma by centrifugation. The procoagulant activity (PCA) of MPs was measured using a two-stage chromogenic assay. We also measured levels of thrombin-antithrombin and the number of MPs.

Results: Lipopolysaccharide (LPS) increased MP PCA in wild-type mice; this PCA was significantly reduced by an anti-mouse TF antibody (1H1) but not with an anti-human TF antibody (HTF-1). Conversely, in mice expressing only human TF, MP PCA was inhibited by HTF-1 but not 1H1. MPs from wild-type mice had 6-fold higher levels of PCA using mouse factor (F)VIIa compared with human FVIIa, which is consistent with reported species-specific differences in FVIIa. Mice expressing low levels of human TF had significantly lower levels of MP TF activity and TAT than mice expressing high levels of human TF; however, there were similar levels of phosphatidylserine (PS)-positive MPs. Importantly, levels of MP TF activity in wild-type mice correlated with levels of TAT but not with PS-positive MPs in endotoxemic mice.

Conclusion: These results suggest that the levels of TF-positive MPs can be used as a biomarker for evaluating the risk of disseminated intravascular coagulation in endotoxemia.

Fig. 1

Lipopolysaccharide (LPS) increases levels of microparticles (MP), MP procoagulant activity (PCA) and TAT in wild-type mice. Wild-type C57/B6J mice were given an intraperitoneal injection of either phosphate-buffered saline (PBS) or LPS and blood was collected after 6 h. (A) Total MP PCA was measured using mouse factor (F)VIIa and human FX in the presence of rat IgG (white bars) or 1H1 (black bars). (B) MP tissue factor (TF) activity was calculated by subtraction of the MP PCA generated in the presence of 1H1 from the total MP PCA generated in the presence of IgG control. (C) Levels of TAT in the plasma from mice treated with PBS (white bar) or LPS treated (black bar). (D) The number of PS-positive MPs in plasma from mice treated with PBS (white bar) or LPS (right bar) is shown. All results are shown as mean ± SD, n = 4–6 mice per group. Asterisks indicate statistically significant differences between the groups.

Fig. 2

Measurement of microparticle (MP) procoagulant activity (PCA) in wild-type mice using mouse or human factor (F)VIIa. Total MP PCA in plasma from wild-type mice treated with either phosphate-buffered saline (PBS) control (left bars) or lipopolysaccharide (LPS) (right bars) was measured using either human FVIIa (white bars) or mouse FVIIa (black bars) and human FX. The inset is an enhanced plot of the PBS control. Data are shown as mean ± SD (n = 5). Asterisks indicate a statistically significant difference between the MP PCA observed with human FVIIa.

Fig. 3

Species-specific inhibition of microparticle (MP) procoagulant activity (PCA) from wild-type and human TF (HTF) mice. (A and B) Total MP PCA in plasma from wild-type mice treated with either phosphate-buffered saline (PBS) control (left bars) or lipopolysaccharide (LPS) (right bars) was measured using human factor (F)VIIa and human FX in the presence of 1H1 or isotype IgG or HTF-1 or isotype IgG. Data are shown as mean ± SD (n = 6–7). (C and D) Total MP PCA in plasma from HTF mice treated with either PBS control (left bars) or LPS (right bars) was measured using human FVIIa and human FX in the presence of 1H1 or isotype IgG or HTF-1 or isotype IgG. Data are shown as mean ± SD (n = 4). Asterisks indicate statistically significant differences between the groups.

Fig. 4

Effect of human factor (F)VIIai and annexin V on microparticle (MP) procoagulant activity (PCA) from wild-type and human TF (HTF) mice. (A) Total PCA in MPs isolated from plasma of wild-type mice treated with either phosphate-buffered saline (PBS) control (white bar) or lipopolysaccharide (LPS) (black bars) was measured using mouse FVIIa and human FX in the presence of 1H1, isotype IgG, human FVIIai or annexin V. (B) Total MP PCA from plasma of HTF mice treated with either PBS control (white bar) or LPS (black bars) was measured using human FVIIa and human FX in the presence of HTF-1, isotype IgG, human FVIIai or annexin V. Data are shown as mean ± SD (n = 4). Asterisks indicate statistically significant differences relative to the IgG control.

Fig. 5

Levels of microparticle (MP) procoagulant activity (PCA), MP tissue factor (TF) activity, TAT and MPs in human TF (HTF) and Low TF Mice. Total MP PCA (A) and MP TF activity (B) in plasma from HTF and low mice treated with either PBS control (white bars) or LPS (black bars) was measured using human FVIIa and human FX. TF activity in the MPs was determined using HTF-1. Data are shown as mean ± SD (n = 4–5). (C) Levels of TAT in the plasma from HTF (left bars) and low TF (right bars) mice treated with phosphate-buffered saline (PBS) (white bars) or lipopolysaccharide (LPS)-treated (black bars). (D) Number of PS-positive MPs in plasma from HTF mice (left bars) and low TF mice (right bars). All results are shown as mean ± SD, (n = 4–6). Asterisks indicate statistically significant differences between the groups.

Fig. 6

Levels of microparticle (MP) tissue factor (TF) activity correlate with levels of TAT. A significant linear correlation was observed between levels of MP TF activity and TAT in endotoxemic wild-type mice (R = 0.78, P < 0.01, n = 16).