Tissue factor in hemophilia

这些研究的总体目标是确定组织因子（TF）在血友病止血中的体内作用。从本项目中提出的一系列实验中获得的信息将直接与新疗法的开发有关，即针对血友病患者和抑制剂的因子VIIa （FVIIa）突变体。高剂量的FVIIa最近被FDA批准用于治疗FVIII或FIX抑制剂患者的出血，修改FVIII的膜接触部位（如项目2中提出的）可能导致FVIIa产品的开发，在低剂量（因此成本）治疗出血发作方面优于野生型FVIIa产品。然而，可以想象的是，意想不到的血栓形成性也将通过操纵Gla结构域的膜结合位点而增强。因此，为了确定这些FVII突变体的作用方式、疗效和安全性，了解其基本配体tf的体内表达是必不可少的。为此，本项目将在人类和血友病动物模型中研究TF在血管内的可用性和激活状态。首先，我们将定义TF“加密”现象的生化基础(定义为结合FVII的能力（a)，而不能表达完全的促凝活性（PCA））。特别是，膜磷脂酰丝氨酸（PS）的不对称性和TF的四级结构在调节TF PCA表达中的作用将被检查。这些实验的结果将指导设计特异性靶向活性或加密形式循环TF的抑制分子。这些抑制剂将被用作血管内TF功能可用性的探针，将在正常患者、血友病患者和获得性血友病兔模型中的两个候选“池”（细胞相关和微粒相关）中进行量化。最后，在与项目2和项目4密切相关的实验中，将使用兔模型来检测TF在生理性止血以及FVIIa野生型和突变型诱导的药理止血中的作用。

The overall goal of these studies is to define the in vivo role of tissue factor (TF) in hemostasis as in pertains to hemophilia. The information gained from the proposed series of experiments in this Project will be directly relevant to the development of new therapies, namely Factor VIIa (FVIIa) mutants, for patients with hemophilia and inhibitors. FVIIa in high doses has recently been approved by t he FDA for the treatment of bleeding experienced by patients who have an inhibitor to FVIII or FIX Modification of the membrane contact site in FVIII-as proposed in Project 2- may lead to the development of FVIIa products that are superior to wild type FVIIa with respect to treatment of bleeding episodes at a lower dose (and therefore cost). However, it is conceivable that unwanted thrombogenicity will also be enhanced by manipulation of the membrane-binding site of the Gla domain. Therefore, in order to define the mode of action, efficacy, and safety of these FVII mutants, an understanding of the in vivo expression of their essential ligand-TF-is essential. To this end, this project will study t he intravascular availability and state of activation of TF in humans, and in an animal model of hemophilia. First, we will define the biochemical basis of the phenomenon of TF 'encryption' (defined as the ability to bind FVII(a) while failing to express full pro-coagulant activity (PCA)). In particular, the roles of membrane phosphatidylserine (PS) asymmetry and TF quaternary structure in modulating the expression of TF PCA will be examined. The results of these experiments will guide the design of inhibitory molecules that specifically target either the active or encrypted forms of circulating TF. These inhibitors will be used as probes of the functional availability of intravascular TF, which will be quantified in two candidate 'pools' (cell-associated and microparticle-associated) in normals, in patients with hemophilia, and in a rabbit model of acquired hemophilia. Finally, in experiments that will dovetail closely with Projects 2 and 4, the rabbit model will be used to examine the role played by TF in physiological hemostasis, and in pharmacological hemostasis induced by wild-type and mutant forms of FVIIa.