Abstract

The exudative phase of ALI (acute lung injury) is characterized by loss of the endothelial/alveolar barrier, disruption of local water homeostasis, and intra-alveolar activation of blood coagulation. In previous studies, ALI exudates sampled by BAL (bronchoalveolar lavage) demonstrated increased tissue factor and antiproteinase activities compared to BAL from healthy lungs. This study applied osmotic stress techniques to explore the hypothesis that local water activity influences the procoagulant and antiproteinase activities recovered in human BAL fluids. Samples were prepared from 8 injured and 7 normal lungs by sequentially centrifuging BAL fluid at 200 and 40,000g to remove cells and surfactant particles, respectively. Factor Xa generation and inhibition were measured by chromogenic assay under standard (buffer pH 7.4, 0.13 NaCl, 1.3 mM CaCl2, 0.5mg/ml ovalbumin) and osmotic stress (~0.6 atm in standard buffer with polyethylene glycol 8000, ~26 Å radius) conditions by adding either purified factor X (200 nM) or factor Xa (50 nM) to the samples. Factor Xa generation curves had a sigmoidal shape, and the yields in 25 min-long reactions were variable, as expected, and ranged from 4 to 47 nM. Average activity values were 16-fold higher in ALI than in normal lungs, while tissue factor antigen levels measured by ELISA were 6-fold higher than in normal and ranged from 3.5 to 108 ng/ml. Under osmotic stress, the yields and rates increased in samples from healthy lungs, while the responses in ALI samples were mixed. Four showed decreases; 2 increases and 2 showed no change. Antiproteinase capacity, measured as the decrease in factor Xa activity, was variable but higher in ALI samples, and in both ALI and normal lungs, it increased in response to osmotic stress. These results imply that local hydration and dehydration directly influence procoagulant/anticoagulant activities in bronchoalveolar exudates, regardless of other factors, and that impaired alveolar fluid clearance may contribute directly to coagulation imbalances in ALI.

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