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

1. 富组氨酸糖蛋白（HRG）是一种血浆蛋白，被认为在凝血和纤溶系统中起控制作用。测定HRG cDNA的完整核苷酸序列（2067碱基对）。HRG的氨基酸序列表明，HRG是由多结构域组成的，每个结构域可能具有独立的功能。HRG的详细序列研究表明，HRG在进化上属于半胱氨酸蛋白酶抑制剂（cystatin）超家族。分离出HRG的基因组片段并对其进行了部分鉴定。该基因长度约为11千碱基，包含9个外显子和8个内含子。基因编码胱抑素结构域的内含子位置与胱抑素超基因家族相同。该基因定位在3.3号染色体上。发现在Zn^<2+>存在时，HRG的肝素中和能力增强。含有两个胱抑素结构域的n端片段经胰凝乳蛋白酶有限蛋白水解分离得到，鉴定为肝素结合和中和结构域。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“富山”中的半胱氨酸-47与血浆中游离半胱氨酸形成混合二硫键。2)在肝素依赖性凝血酶抑制中，凝血酶与肝素的相互作用比与肝素辅助因子II的相互作用更为重要。3)在不同大小和S含量的硫酸葡聚糖中，mr10000和18% S的分子种作为蛋白C抑制剂的辅助因子效果最好。

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.