INTRINSIC SYSTEM CLOTTING: STRUCTURE AND FUNCTION

因子IX的功能域将由四个重叠定义 接近了。先天缺陷提供了数十种截然不同的突变， 其结构基础可与临床相关联。 功能和体外相互作用的严重程度和具体水平。 特异性抗血清，包括单抗和多克隆抗体 分数，有助于定位缺陷或功能。合成的 多肽可用于制备特异性抗血清，并可直接用于 抑制功能相互作用。最后，对于以下地区 合成肽不能充分反映表面结构， 合成片段可以通过表达突变的cDNA来产生 在适当的载体中，该载体已被转染到培养的 细胞系。 这一建议侧重于因子的不同功能域 IX.它们是从N端到C端的。首先，我们 会将血友病IX中的缺陷定义为异常 钙结合特性。一种合成的先导肽将是 进一步测试对细胞中GLA形成的刺激 表达系统，并与Suttie博士合作，研究其结构 体外与羧基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 通过分析它们的DNA和分离的蛋白质来表征。一个 将加大力度生产重组第IX因子 含有生长因子样区的片段(S) 探讨其止血作用。无论是在当地与海马克博士还是 与斯特恩博士在纽约，多肽、抗体和分离的 异常的IXs将被研究IX与内皮细胞的结合。 该区域的其他潜在作用将通过体外实验进行评估 凝血和结合研究及动物存活测量 重组片段。我们将确认一个特定的 外显子IV表位的抗体部分识别非GLA， 钙结合父系。利用单克隆a-IX识别一种 外显子多态将从IX扩展到VIII和Von Willebrand因子将开发用于携带者检测的快速免疫分析方法 血友病A和血管性血友病。我们会尝试 制备针对因子IXa的抗体。多肽到 IXa重链可变部分的序列将是 研究和开发新的试剂来探索细胞的功能 活性酶。严重缺陷症患者的结合抗体 将以制定本地化战略为特征 他们的缺陷。一种重链缺陷，由基因的点突变引起 Taq1裂解位点将由寡核苷酸探针定义，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.