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Molecular Biological and Biochemical Studies on the Control Mechanism of Blood Coagulation and Fibrinolysis

凝血和纤溶控制机制的分子生物学和生化研究

基本信息

批准号：
61480459
负责人：
KOIDE Takehiko
金额：
$ 4.61万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1988
项目状态：
已结题

项目摘要

1. Histidine-rich glycoprotein (HRG) is a plasma protein which is considered to function as a control factor in the blood coagulation and fibrinolytic system. The complete nucleotide sequence (2067 base-pairs) of cDNA for HRG was determined. The derived amino acid sequence of HRG revealed that HRG is composed of multi-domain structures, each of which may have an independent function. The detailed sequence study of HRG showed that HRG evolutionarily belongs to the cystatin (cysteine proteinase inhibitors) superfamily.2. Genomic fragments of HRG were isolated and partially characterized. The gene is about 11 kilobases in length and contains nine exons and eight introns. Locations of the introns in the gene coding for cystatin domains are identical with those of the cystatin supergene family. The gene was localized in chromosome 3.3. Heparin neutralizing ability of HRG was found to augment in the presence of Zn^<2+>. The N-terminal fragment containing two cystatin domains was isolated by limited proteolysis with chymotrypsin and identified as the heparin-binding and neutralizing domain.4. The new knowledges obtained on the structure and function of heparin-dependent anticoagulant and antifibrinolytic factors are as follows: 1) Cysteine-47 in the abnormal antithrombin III "Toyama" forms a mixed disulfide bond with a free cysteine in plasma. 2) Interaction of thrombin with heparin is more essential than that of heparin cofactor II in the heparin-dependent thrombin inhibition by heparin cofactor II. 3) Among the dextran sulfate with various sizes and S contents, the molecular species with Mr10,000 and 18% S is best as a cofactor of protein C inhibitor.

1. 富含组氨酸的糖蛋白（HRG）是一种血浆蛋白，被认为是凝血和纤溶系统中的控制因子。确定了 HRG cDNA 的完整核苷酸序列（2067 个碱基对）。 HRG的氨基酸序列揭示HRG由多结构域结构组成，每个结构域可能具有独立的功能。对HRG的详细序列研究表明，HRG在进化上属于胱抑素（半胱氨酸蛋白酶抑制剂）超家族。 2． HRG 的基因组片段被分离并部分表征。该基因长约11kb，包含9个外显子和8个内含子。编码半胱氨酸蛋白酶抑制剂结构域的基因中内含子的位置与半胱氨酸蛋白酶抑制剂超基因家族的内含子位置相同。该基因定位于3.3号染色体。发现HRG的肝素中和能力在Zn 2+ 存在下增强。胰凝乳蛋白酶有限水解，分离出含有两个半胱氨酸蛋白酶抑制剂结构域的N端片段，并鉴定为肝素结合和中和结构域。 4．关于肝素依赖性抗凝和抗纤溶因子的结构和功能的新认识如下： 1）异常抗凝血酶III“Toyama”中的半胱氨酸47与血浆中的游离半胱氨酸形成混合二硫键。 2) 在肝素辅因子 II 的肝素依赖性凝血酶抑制作用中，凝血酶与肝素的相互作用比肝素辅因子 II 的相互作用更重要。 3）在各种大小和S含量的硫酸葡聚糖中，Mr10,000和18%S的分子种类最适合作为C蛋白抑制剂的辅助因子。