Intravenous infusion of endotoxin into sheep causes diffuse pulmonary microvascular injury characterized by increased permeability, transudation of fluid into alveolar and interstitial spaces, and increased lymph flow. Many of these elements are present in patients with adult respiratory distress syndrome (ARDS). We have recently discovered that lung injury induced in sheep by endotoxin is associated with the appearance in lung lymph of high levels of a protease with trypsin-like activity. The enzyme may be related to the kallikrein family of serine proteases, although its properties differ from those of pancreatic or serum kallikrein. The time course of appearance of the enzyme and the increase in its activity corresponds to the known events of capillary injury, increased permeability to proteins and fluid, and edema. At low endotoxin levels the activity recedes with recovery.
The aim of this proposal is to explore the relationship of this enzyme to capillary endothelial injury. Principal studies will involve the lung-lymph fistula sheep preparation with pulmonary injury induced by endotoxin, oleic acid or air emboli. The time-course of enzyme activity will be established with these models. We will isolate the enzyme from sheep lymph and study its physiochemical and biochemical properties. Kinetic studies and synthetic subtrates will be used to delineate the specificity requirements of the active site of the enzyme and to identify those structural features of the substrates which conform best with the substrate recognition site of the enzyme. It is expected that the results of these studies will constitute a basis for the rational design of specific, active site directed inhibitors of the enzyme, which could be used as probes in studies dealing with the interrelationship of the enzyme with lung injury. Additional experiments of lung injury in rats will be used to explore the possibility that the same enzyme appears in alveolar fluids. If the enzyme does appear in alveolar contents, we will then search for its presence in small amounts of alveolar fluid from patients with ARDS, as an early biochemical marker of acute lung injury. It is also anticipated that by synthesis and evaluation of a series of synthetic substrates it will be possible to measure the same enzyme in serum and to follow its activity in association with developing lung injury. Collectively, these studies should lead to an understanding of the role of this protease as a biochemical marker of lung injury, and possibly, to greater knowledge about mechanisms of ARDS.
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| Orlowski, M; Lesser, M; Ayala, J et al. (1989) Substrate specificity and inhibitors of a capillary injury-related protease from sheep lung lymph. Arch Biochem Biophys 269:125-36 |
| Orlowski, M; Flick, M R; Rand, J et al. (1987) Isolation and properties of a capillary injury-related protease from lung lymph. Arch Biochem Biophys 254:156-69 |
| Lesser, M; Flick, M R; Kent, B et al. (1987) Proteolytic activity in sheep lung lymph as marker of lung capillary injury. Am Rev Respir Dis 135:643-50 |
| Chang, J C; Lesser, M; Yoo, O H et al. (1986) Increased cathepsin B-like activity in alveolar macrophages and bronchoalveolar lavage fluid from smokers. Am Rev Respir Dis 134:538-41 |
| Lesser, M; Chang, J C; Orlowski, M (1985) Cathepsin B and D activity in stimulated peritoneal macrophages. Mol Cell Biochem 69:67-73 |
