Enhanced vitamin K dependent proteins in hemophilia

这个项目利用了对维生素K依赖蛋白与膜结合的新认识。凝血级联的关键蛋白(因子VII和因子IX)对膜的亲和力很低。这种亲和力可以通过定点突变提高到高亲和力的维生素K依赖蛋白。体外测试表明，这些变化在体外对因子VII和VIIa的活性有很大影响，使用的是模拟生物条件的测试。在组织因子依赖和组织因子非依赖系统中都实现了增强。6个具体目标包括：1.确定体外活性最高的人凝血因子VIIa定点突变形式。表达这些蛋白质的数量适合在小鼠和兔子身上进行研究。2.建立一种适合在小鼠和兔身上进行研究的可靠的出血量测定方法。2.在血友病小鼠中建立一种可靠的出血攻击试验，为检验因子VIIa突变体的疗效提供能力。3.利用特定目的1中确定的突变体和特定目的2中确定的方法，在小鼠模型中测试人因子VIIa突变体的有效性。4.表达与最佳人因子VIIa突变相关的小鼠因子VIIa的定点突变体。通过为特定目标2-3中描述的人类蛋白质分子开发的程序，在小鼠身上测试这些药物。5.检测特定目的1中描述的人活性部位封闭因子VIIa分子的体外抗凝血活性，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依赖蛋白潜在用途的初步证据，这些蛋白在GLA区域发生突变，与膜的亲和力增加。这些可能特别包括因子IX或X。该项目使用定点蛋白质突变、蛋白质表达、纯化，以及通过蛋白质化学和大量凝血试验进行表征。体内试验将在小鼠和兔子身上进行。

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.