Active thrombin is found in the airways of patients with a variety of inflammatory lung diseases. In other organs, thrombin acts as an inflammatory mediator via thrombin receptors, PAR₁ and PAR₄ (Vergnolle et al., 2001). In the present study we examined the acute effect of thrombin receptor agonists in mice to determine whether thrombin may contribute to inflammation in lung disease.

Thrombin and thrombin receptor agonist peptides were administered intranasally (i.n.) to female balb/c mice (20 g) under isoflurane anaesthesia and neutrophil numbers were enumerated in QuickDiff-stained cytospin preparations of bronchalveolar lavage (BAL) fluid 24 hours after administration. In other experiments, alveolar macrophages were isolated from mice by BAL and the effect of thrombin receptor agonists on tumour necrosis factor-(TNF-) production were measured by ELISA after 3 hours. Results are expressed as mean ± standard error of the mean.

Thrombin (250-2500 U.kg^-1 i.n.) dose-dependently elicited a small influx of neutrophils (1.68 ± 0.3 x10⁴ cells.ml^-1 at 2500 U.kg^-1; n=11) when compared with LPS (125 µg.kg^-1 i.n.; 218.8 ± 19.0 x10⁴ cells.ml^-1; n=8). By contrast, DFP-inactivated thrombin had a much lesser effect (0.31 ± 0.2 x10⁴ cells.ml^-1; n=4; P<0.05 compared with thrombin), suggesting that the proteolytic activity of thrombin is required.

The PAR₁-activating peptide SFLLRN (20 mg.kg^-1 i.n.) did not elicit any neutrophil recruitment whereas the low potency PAR₄-activating peptide GYPGKF (20 mg.kg^-1 i.n.) elicited a small response (0.45 ± 0.2 x104 cells.ml^-1; n=6). The more potent synthetic PAR₄-activating peptide AYPGKF recruited significantlty more neutrophils (1.89 ± 0.5 x10⁴ cells.ml^-1; n=6). Previous studies have found that LPS-induced neutrophil influx is mediated by TNF- released by alveolar macrophages (Goncalves de Moraes et al., 1996). The minor inflammatory response to thrombin was inhibited by co-administering a blocking antibody against TNF- (0.31 ± 0.2 vs. control 1.76 ± 0.7 x104 cells.ml^-1; P<0.05, n=4). However, while LPS induced release of TNF- from isolated macrophages (241.2 ± 24.5% control), neither thrombin (10 U.ml^-1 107.1 ± 16.6 % control), nor the PAR₁- and PAR₄-activating peptides (100 µM; SFLLRN, 91.85 ± 16.7%; n=4; AYPGKF, 103.9 ± 6.5%, n=4) caused measurable TNF- release.

In conclusion, thrombin causes only a mild, TNF--dependent recruitment of neutrophils to the airways, most likely via proteolytic activation of PAR₄. Thus, compared to other organs where pronounced inflammation in response to extravascular thrombin is observed acutely, the responses in the airways is fairly trivial.

Vergnolle, N. et al., (2001) Trends Pharmacol. Sci. 22:146-152.
Goncalves de Moraes, V.L. et al., (1996) Br. J. Pharmacol. 117: 1792-1796.

JDM is funded by a Wellcome Trust Travelling Fellowship.