Amino acid residues of heparin cofactor II required for stimulation of thrombin inhibition by sulphated polyanions.

A variety of sulphated polyanions in addition to heparin and dermatan sulphate stimulate the inhibition of thrombin by heparin cofactor II (HCII). Previous investigations indicated that the binding sites on HCII for heparin and dermatan sulphate overlap but are not identical. In this study we determined the concentrations (IC50) of various polyanions required to stimulate thrombin inhibition by native recombinant HCII in comparison with three recombinant HCII variants having decreased affinity for heparin (Lys-173-->Gln), dermatan sulphate (Arg-189-->His), or both heparin and dermatan sulphate (Lys-185-->Asn). Pentosan polysulphate, sulphated bis-lactobionic acid amide, and sulphated bis-maltobionic acid amide resembled dermatan sulphate, since their IC50 values were increased to a much greater degree (>/=8-fold) by the mutations Arg-189-->His and Lys-185-->Asn than by Lys-173-->Gln (Gln and Lys-185-->Asn (>/=6-fold) than by Arg-189-->His (

Deletion mutagenesis of heparin cofactor II: defining the minimum size of a thrombin inhibiting serpin.

Heparin cofactor II (HCII) is a 66 kDa plasma glycoprotein that belongs to the serpin superfamily of protease inhibitors. Its natural target is thrombin. HCII inhibits thrombin in both a progressive reaction, and in an accelerated reaction catalyzed by a glycosaminoglycan, dermatan sulphate (DS). Both modes of inhibition result in the formation of a stable, denaturation-resistant complex. Using a cDNA clone encoding rabbit HCII recently isolated and characterized in our laboratory, we have employed deletion mutagenesis to identify amino-terminal regions of the molecular which are essential to the progressive reaction. PCR was employed to produce four deletion constructs: delta 58, delta 81, delta 106, and delta 169, all in an in vitro transcription vector plasmid background. Transcription of the full-length construct, and of the four deletion constructs, followed by in vitro translation in rabbit reticulocyte lysate, was used to produce the corresponding HCII-related polypeptides. The delta 106 and delta 169 mutants failed to react with thrombin, even in the presence of DS. In contrast, the delta 58 and delta 82 mutants retained the ability to form complexes with thrombin, although the rate of complex formation was decreased for the latter mutant compared to the full-length recombinant HCII; no acceleration of complex formation in the presence of 20 micrograms/ml DS was noted for either truncated recombinant HCII. Alignment of the rabbit HCII primary structure with secondary structural elements found in alpha 1 antitrypsin and other serpins showed that the non-functional delta 106 mutant lacks helix A, while the functional delta 82 mutant contains this element. Our results suggest that helix A is an essential part of a functional serpin, and define the limits of the amino-terminal region of HCII which is not essential for thrombin inhibition.

Molecular cloning and expression of rabbit heparin cofactor II: a plasma thrombin inhibitor highly conserved between species.

Heparin cofactor II (HCII), a circulating plasma protein that inhibits thrombin, is a member of the serine proteinase (serpin) family of proteins. The extent to which HCII structure is conserved across species lines was investigated, by obtaining cDNA clones encoding rabbit HCII. Overlapping clones corresponding to rabbit HCII were obtained by the combined use of hybridization screening of a rabbit liver cDNA library, and by rapid amplification of cDNA ends (RACE). The consensus sequence obtained spans 2178 nucleotides, and is comprised of a 5' untranslated region of 77 nucleotides, an open reading frame of 1440 nucleotides, and a 3' untranslated region of 661 nucleotides that concludes with a poly A tract. The open reading frame is subdivided into a secretory signal sequence of 19 amino acids, and a mature protein of 461 amino acids. Within the region comprising the mature protein, 87% of the amino acid residues are identical to those seen in human HCII. Expression of an appropriately modified form of the rabbit HCII clone in an in vitro reticulocyte expression system yielded two major polypeptides, of 60 and 56 kD respectively, both of which were able to form SDS-stable complexes with human alpha-thrombin, in a reaction accelerated by dermatan sulphate. The remarkable degree of homology observed between rabbit HCII and its human counterpart, indicating a high degree of conservation of structure through evolution, suggests an important function of HCII in hemostasis.

The production of heparin cofactor II is not regulated by inflammatory cytokines in human hepatoma cells: comparison with plasminogen activator inhibitor type-1.

Using the Northern blot technique, we screened 6 human hepatoma cell lines to investigate the regulation mechanism of heparin cofactor II (HC II) biosynthesis. We found that HuH-7 and Hep G2 cells constitutively expressed the HC II gene. In conditioned medium, HuH-7 cells constantly produced HC II that was functionally active and formed a complex with thrombin in the presence of dermatan sulfate. HC II is thought be an acute phase reactant, and, therefore, we examined the effects of the major inflammatory cytokines, IL-6, IL-1 beta, and TNF-alpha, on the regulation of HC II production in HuH-7 and Hep G2 cells. In HuH-7 cells, the antigen and mRNA levels of plasminogen activator inhibitor type-1 (PAI-1), an acute phase protein produced by hepatocytes, were increased in response to stimulation with either IL-6 or IL-1 beta or both, but HC II antigen and mRNA levels were not changed by the same stimulation. Even when Hep G2 cells were treated with a combination of three cytokines, IL-6, IL-1 beta, and TNF-alpha, HC II antigen and mRNA levels were not changed; however, PAI-1 antigen and mRNA levels were clearly increased. These results suggest that the production of HC II in hepatoma cells is not regulated by the major inflammatory mediators, IL-6, IL-1 beta, and TNF-alpha.

Regulation of thrombosis and hemostasis by antithrombin.

In this article, three cases, with antithrombin (AT) abnormality "Toyama" (type IIb), AT abnormality "Aomori" (type IIa), and congenital AT deficiency (type I) with pregnancy and delivery managed with administration of both AT concentrates and low-molecular-weight heparin, are described. Additionally, a case of AT-producing hepatocellular carcinoma, the first case in the world literature, is reported. Following these clinical reports, the development of AT studies on heparin cofactor II, characteristics of the vessel wall related to coagulation-fibrinolysis, and the development of the treatment of thrombosis with low-molecular-weight heparin and herbal drugs are discussed.