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

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的控制因子。测定了该基因的全长核苷酸序列(2067个碱基对)。对HRG的氨基酸序列分析表明，HRG由多个结构域组成，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在进化上属于半胱氨酸蛋白酶抑制物超家族。分离了HRG的基因组片段，并进行了部分鉴定。该基因全长约11kb，包含9个外显子和8个内含子。编码半胱氨酸氨基转移酶结构域的基因内含子的位置与半胱氨酸氨基转移酶超基因家族的位置相同。该基因定位在3.3号染色体上。结果表明，加入锌离子后，肝素中和能力增强。用胰凝乳酶限制性蛋白水解法分离得到含有两个半胱氨酸氨基转移酶结构域的N-末端片段，鉴定为肝素结合区和中和区。对肝素依赖的抗凝血剂和抗纤溶因子的结构和功能的新认识如下：1)抗凝血酶III异常的富山中的半胱氨酸-47与血浆中的游离半胱氨酸形成混合二硫键。2)在肝素辅助因子II对肝素依赖性凝血酶的抑制中，凝血酶与肝素的相互作用比肝素辅助因子II更为重要。3)在不同大小和S含量的硫酸葡聚糖中，以Mr 10000和18%S作为蛋白C抑制物的辅助因子的分子种类最好。

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.