Granule, proteinases and oxidants relesd from activatedneutrophils during pathological inflammatory reactions contribute to the tissue destruction and circulatory collapse preceding organ failure in sepsis and ARDS. Vessel wall extracellular matrix and hemostatic pathway components are particularly sensitive to destruction by neutrophil elastase and cathepsin G (catG), and their degradation is associated with hypotension and disseminated intravascular coagulation (DIC). In Project 3 we will extend our serpin structure/funciton research twoards the practical goal of developing inhibitors which would be useful in sepsis and ARDS. Recent developments in antithrombin III (ATIII) research suggest potentially important funcitonal differences in the interactions of the naturally occurring alpha- and beta-ATIII isoforms with vessel wall heparan sulfate proteoglycans (HSPGs). This will be investigated in Aim 1 by determining isoform binding affinities for endothelial cells, and comparing their accessibility to fX-activating complex assembled on endothelial cells (which contain HSPGs) and on platelets and phospholipid vesicles (which do not).
Aim 2 is to make vessel wall directed, elastase- and catG-resistant inhibitors of thrombin and fXa. Several animal and human studies have shown that high dose infusion of ATIII can reverse septic DIC and associated hypotension and organ failure. We hypothesize that high doses of ATIII are required due to (I) the preseence of an elastase cleavage site in the funcitonally important reactive loop of ATIII, and (ii) depletion of the high-heparin-affinity beta-ATIII isoform from commercial antithrombin concentrates. These considerations suggest that reduced amounts of a recombinant ATIII engineered to have enhanced heparin affinity and elastase- and catG- resistance may be effective for treating inflammatory DIC.
Aim 3 is to make oxidation-resistant antielastase and anti-catG serpins that bind heparin. The heparin bidning property should increase association rates ofthesee inhibitors with elastase and cat G, and target them to the vessel wall where blocking extracellular matrix destruction and maintaining normal regulation of coagulation pathway assemblies is important. Finally, Airm 4 is to investigate the therapeutic potenital of the high-heparin-affinity neutrophil-resistant ATIII and the vessel wall directed anti-elastase and anti-catG serpins in an endotoxemic rat model of sepsis.
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