Enhanced vitamin K dependent proteins in hemophilia

该项目利用了对维生素k依赖蛋白的膜关联的新认识。凝血级联的关键蛋白（因子VII和因子IX）对膜的亲和力较低。这种亲和力可以通过位点定向突变增加到高亲和力的维生素k依赖性蛋白。体外分析表明，这些变化对因子VII和VIIa在体外的活性有很大的影响，使用模拟生物条件的分析。在依赖组织因子的系统和不依赖组织因子的系统中都能实现增强。六个具体目标包括：1。在体外鉴定具有最高活性的人因子VIIa的位点定向突变形式。表达这些蛋白的数量适合在小鼠和兔子身上进行研究。2. 开发一种可靠的出血挑战测定方法，其数量适合于小鼠和家兔的研究。2. 开发一种可靠的血友病小鼠出血挑战试验，提供测试VIIa因子突变体功效的能力。3. 采用特异性靶标1中鉴定的突变体和特异性靶标2中鉴定的方法，测试人因子VIIa突变体在小鼠模型中的有效性。4. 表达与最佳人类因子VIIa突变相关的小鼠因子VIIa定点突变体。通过在特定目标2-3中描述的人类蛋白质分子开发的程序在小鼠中测试这些。5. 在体外测试特定目的1中描述的人活性位点阻断因子VIIa分子的抗凝血性能，以确定在兔模型中测试的最佳突变体。6. 获得其他维生素k依赖性蛋白潜在用途的初步证据，这些蛋白在Gla结构域发生突变，对膜的亲和力增加。这些可能特别包括因子IX或x。该项目使用定点蛋白突变、蛋白表达、纯化和通过蛋白化学和大量凝血试验表征。将在小鼠和家兔身上进行体内试验。

This project takes advantage of new understanding about membrane association by vitamin K-dependent proteins. Key proteins of the coagulation cascade (factors VII and IX) display low affinity for membranes. This affinity can be increased to that of high affinity vitamin K-dependent proteins by site directed mutations. In vitro assays show that these changes have a large impact on activity of factor VII and VIIa in vitro, using assays that mimic biological conditions. Enhancement is realized in both tissue factor dependent as well as tissue factor- independent systems. Six specific aims include: 1. Identify the site- directed mutant forms of human factor VIIa that have the highest activity, in vitro. Express these proteins in quantities suitable for studies in the mouse and rabbit. 2. Develop a reliable assay for bleeding challenge in quantities suitable for studies in the mouse and rabbit. 2. Develop a reliable assay for bleeding challenge in the hemophilic mouse, that provides the ability to test efficacy of factor VIIa mutants. 3. Test Efficacy of human factor VIIa mutants in the mouse model, using mutants identified in specific aim 1 and methods identified in specific aim 2. 4. Express site-directed mutants of mouse factor VIIa which correlate with the best human factor VIIa mutations. Test these in the mouse by procedures developed for the human protein molecules described in specific aims 2-3. 5. Test in vitro anti-coagulant properties of human active site-blocked factor VIIa molecules described in specific aim 1, to determine the best mutants to be tested in the rabbit model. 6. Obtain preliminary evidence for potential uses of other vitamin K-dependent proteins which have mutations in the Gla domain and increased affinity for membranes. These may include especially factors IX or X. The project uses site-directed protein mutation, protein expression, purification, and characterization by protein chemistry and numerous coagulation tests. In vivo testing will be carried out in mice and rabbits.