SERPIN STRUCTURE AND THE DEVELOPMENT OF NEUTROPHIL RESISTANT SERPINS

粒细胞、蛋白酶和氧化剂与活化的中性粒细胞有关 在病理性炎症反应期间， 脓毒症中器官衰竭前的破坏和循环衰竭 和ARDS。 血管壁细胞外基质与止血通路 成分对中性粒细胞的破坏特别敏感， 弹性蛋白酶和组织蛋白酶G（catG），它们的降解与 伴有低血压和弥散性血管内凝血 在 项目3我们将serpin结构/功能的研究向两个方向扩展 开发抑制剂的实际目标是， 败血症和ARDS。 抗凝血酶III（ATIII）的最新进展 研究表明， 天然存在的α-和β-ATIII同种型的相互作用 与血管壁硫酸乙酰肝素蛋白聚糖（HSPG）。 这将是 目的1中通过测定以下异构体的结合亲和力来研究 内皮细胞，并比较其对FX激活的可及性， 在内皮细胞（其含有HSPG）上组装的复合物和 血小板和磷脂囊泡（不含）。 目标二是使 血管壁定向的弹性蛋白酶和catG抗性凝血酶抑制剂 和fXA。 一些动物和人类研究表明，高剂量 输注ATIII可逆转脓毒性DIC和相关低血压 和器官衰竭 我们假设高剂量的ATIII 由于（I）在所述细胞中存在弹性蛋白酶切割位点， 功能上重要的反应环ATIII，和（ii）耗尽的 来自商业抗凝血酶高肝素亲和性β-ATIII同种型 浓缩物。 这些考虑表明， 重组ATIII，其经工程改造具有增强的肝素亲和力， 弹性蛋白酶和catG-抗性可能有效治疗 炎症性DIC。 目的三是制备抗氧化抗弹性蛋白酶 和结合肝素的抗catG丝氨酸蛋白酶抑制剂。 肝素比丁性质 应该增加这些ee抑制剂与弹性蛋白酶的结合率， cat G，并将它们靶向血管壁， 破坏基质，维持正常的凝血调节 通路组件是重要的。 最后，Airm 4将调查 高肝素亲和性耐药 ATIII和血管壁定向的抗弹性蛋白酶和抗catG丝氨酸蛋白酶抑制剂， 内毒素血症大鼠脓毒症模型。

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.