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

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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.