INTRINSIC SYSTEM CLOTTING: STRUCTURE AND FUNCTION

因子 IX 的功能域将由四个重叠的部分定义 接近。 先天性缺陷产生了数十种不同的突变体， 其结构基础可以与临床相关 功能性体外相互作用的严重性和具体水平。 特异性抗血清，包括单克隆抗体和多克隆抗体 分数，有助于定位缺陷或功能。 合成的 肽可用于制备特异性抗血清并直接 抑制功能相互作用。 最后，对于以下地区： 合成肽不足以反映表面结构， 可以通过表达突变的 cDNA 来产生合成片段 转染到培养物中的适当载体中 细胞系。 该提案侧重于因子的不同功能域 九． 这些从 N 端到 C 端呈现。 首先，我们 将血友病 IX 的缺陷定义为异常 钙结合特性。 合成的前导肽将是 进一步测试了细胞中 Gla 形成的刺激 表达系统并与 Suttie 博士合作，因其结构 体外与羧化酶系统相互作用的要求。 其次，涉及第四外显子的血友病缺陷将是 通过对其 DNA 和分离蛋白质的分析来表征。 一个 将投入大量精力生产重组因子 IX 含有生长因子样区域的片段，以便 探讨其止血作用。 两人都在当地与 Heimark 博士一起 与纽约斯特恩博士一起研究肽、抗体和分离的 将研究异常 IX 的 IX 与内皮细胞的结合。 该区域的其他潜在作用将通过体外评估 凝血和结合研究以及动物存活测量 重组片段。 我们将确认具体 外显子 IV 表位的抗体部分识别非 Gla， 钙结合父系。 使用单克隆 a-IX 识别 外显子多态性将从 IX 扩展到 VIII 和 von 血友病因子开发用于携带者检测的快速免疫测定法 A 型血友病和冯·维勒布兰德病。 将会做出尝试 制备针对因子 IXa 的特异性抗体。肽 IXa 重链可变部分中的序列将是 研究并开发了新试剂来探测其功能 活性酶。 严重缺陷患者的结合抗体 将制定本地化战略 他们的缺陷。 由于点突变导致的重链缺陷 Taq1 切割位点将由寡核苷酸探针确定，以 与血友病突变的功能数据进行比较。

Functions domains of factor IX will be defined by four overlapping approaches. Congenital defects provide dozens of distinct mutants, the structural bases of which can be correlated to clinical severity and specific levels of functional, in vitro interactions. Specific antisera,including monoclonal antibodies and polycolonal fractions, assist in localizing defects or functions. Synthetic peptides can be used to prepare specific antisera and to directly inhibit functional interactions. Finally, for regions in which synthetic peptides inadequately reflect surface structure, synthetic fragments can be produced by expression of mutated cDNAs in an appropriate vector which has been transfected into a cultured cell line. This proposal focuses upon distinct functional domains of factor IX. These are presented from the N to the C terminus. First, we will define the defect in a hemophilic IX which as abnormal calcium-binding properties. A synthetic leader peptide will be tested further for both stimulation of Gla formation in cell expression systems and with Dr. Suttie, for its structural requirements for interaction with the carboxylase system in vitro. Secondly, hemophilic defects involving the fourth exon will be characterized by analyses of their DNA and isolated proteins. A major effort will mounted to produce recombinant factor IX fragments containing the growth factor-like regions(s) in order to probe its role in hemostasis. Both locally with Dr. Heimark and with Dr. Stern in New York, peptides, antibodies and isolated abnormal IXs will be studied for IX binding to endothelial cells. Other potential roles of this region will be assessed by in vitro clotting and binding studies and animal survival measurements of the recombinant fragments. We will confirm that a specific antibody fraction to an exon IV eptiope recognizes a non-Gla, calcium binding sire. The use of a monoclonal a-IX recognizing an exonic polymorphism will be extended from IX to VIII and von Willebrand factor to develop rapid immunoassays for carrier testing in hemophilia A and von Willebrand disease. Attempts will be made to prepare antibodies specific for factor IXa. Peptides to sequences in the variable portion of IXa's heavy chain will be studied and new reagents developed to probe the function of the active enzyme. Binding antibodies in patients with severe defects will be characterized to develop strategies for localization of their defects. A heavy chain defect due to a point mutation in a Taq1 cleavage site will be defined by oligonucleotide probes, to compare with functional data of the hemophilic mutation.