Procoagulant alveolar microparticles in the lungs of patients with acute respiratory distress syndrome

Abstract

Coagulation and fibrinolysis abnormalities are observed in acute lung injury (ALI) in both human disease and animal models and may contribute to ongoing inflammation in the lung. Tissue factor (TF), the main initiator of the coagulation cascade, is upregulated in the lungs of patients with ALI/acute respiratory distress syndrome (ARDS) and likely contributes to fibrin deposition in the air space. The mechanisms that govern TF upregulation and activation in the lung are not well understood. In the vascular space, TF-bearing microparticles (MPs) are central to clot formation and propagation. We hypothesized that TF-bearing MPs in the lungs of patients with ARDS contribute to the procoagulant phenotype in the air space during acute injury and that the alveolar epithelium is one potential source of TF MPs. We studied pulmonary edema fluid collected from patients with ARDS compared with a control group of patients with hydrostatic pulmonary edema. Patients with ARDS have higher concentrations of MPs in the lung compared with patients with hydrostatic edema (25.5 IQR 21.3-46.9 vs. 7.8 IQR 2.3-27.5 micromol/l, P = 0.009 by Mann-Whitney U-test). These MPs are enriched for TF, have procoagulant activity, and likely originate from the alveolar epithelium [as measured by elevated levels of RAGE (receptor for advanced glycation end products) in ARDS MPs compared with hydrostatic MPs]. Furthermore, alveolar epithelial cells in culture release procoagulant TF MPs in response to a proinflammatory stimulus. These findings suggest that alveolar epithelial-derived MPs are one potential source of TF procoagulant activity in the air space in ARDS and that epithelial MP formation and release may represent a unique therapeutic target in ARDS.

Fig. 1.

Negative staining electron microscopy of microparticles (MPs) from edema fluid of a patient with acute respiratory distress syndrome (ARDS). A: low magnification (direct magnification, ×53,000) of edema fluid before sucrose gradient separation. Arrows indicate MPs of varying sizes. B and C: a low-magnification (×53,000) view of the same edema fluid sample following sucrose gradient separation. B was taken from a gradient fraction with undetectable tissue factor (TF) protein, whereas C was from a fraction with high TF protein content. Arrows indicate MPs of uniform size. D: a higher magnification (direct magnification, ×230,000) of C. MPs shown in D are 32–59 nm in diameter.

Fig. 2.

Boxplot of the MP concentration in the MP fraction as measured by MP capture assay in the edema fluid of patients with hydrostatic pulmonary edema compared with those with ARDS. Horizontal bars, median; box, 25–75th percentile; error bars, 10–90th percentile. P = 0.009 vs. hydrostatic by Mann-Whitney U-test.

Fig. 3.

Protein concentration of edema fluid fractions in patients with hydrostatic edema and ARDS. In both groups, the protein concentration was 100-fold higher in the soluble fraction compared with either the 20,000 g pellet or the MPs. In all fractions, protein concentration was higher in ARDS vs. hydrostatic, but the difference did not reach statistical significance. *P < 0.001 compared with 20,000 g pellet and MPs by ANOVA with post hoc Bonferroni correction.

Fig. 4.

Distribution of procoagulant activity in the alveolar compartment in patients with hydrostatic pulmonary edema compared with ARDS. Bars represent means ± SD. *P = <0.001 by Mann-Whitney U-test. **P = 0.007 by Mann-Whitney U-test.

Fig. 5.

TF protein concentration in soluble fraction compared with MPs in edema fluid of patients with hydrostatic pulmonary edema compared with ARDS. *P = 0.015 by Mann-Whitney U-test. **P = 0.041 by Mann-Whitney U-test.

Fig. 6.

Scatterplot of the MP concentration in edema fluid vs. the TF content of the MP fraction (both log transformed) in ARDS (triangles) vs. hydrostatic edema (circles). The Spearman correlation coefficient = 0.45, P = 0.028.

Fig. 7.

Boxplot of RAGE concentration in MPs in patients with hydrostatic pulmonary edema compared with those with ARDS. ○, outlier; *P = 0.007 by Mann-Whitney U-test.

Fig. 8.

Clot time (A), TF concentration (B), and MP concentration (C) of cell-free conditioned media from A549 cells treated for varying lengths of time with serum-free culture media (control) or 20 ng/ml cytomix. *P < 0.05 vs. 0-h cytomix, †P < 0.05 vs. 0-h control by ANOVA with post hoc Tukey test.

Fig. 9.

Boxplot comparing MP concentrations in survivors (alive) vs. nonsurvivors (dead) in all patients. Patients who died had a trend towards a higher MP concentration compared with those who survived. P = 0.073 by Mann-Whitney U-test.