The glycosylation sites and structural characteristics of oligosaccharides on recombinant human thrombomodulin.

Thrombomodulin (TM) is an anticoagulant glycoprotein on the surface of endothelial cell that directly inhibits the procoagulant activities of thrombin, and the TM-thrombin complex accelerates thrombin-catalyzed activation of protein C. Soluble TM in urine has no glycosaminoglycan (GAG) chain which accelerates the anticoagulant activities. Therefore, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C127 cells. The glycosylation sites were determined by amino acid sequence analysis of peptides digested with trypsin after S-carboxymethylation. The structures of N-linked oligosaccharides were estimated by two-dimensional sugar mapping of pyridylaminated oligosaccharides that were treated with exoglycosidase. The disaccharide composition analysis of the GAG chain was performed by HPLC using digestion with chondroitinase ABC, ACII and B. Consequently, it was revealed that the N-linked oligosaccharides were assigned to Asn29, Asn98, Asn364, Asn391; those structures were estimated biantennary, 2-6 branched triantennary and 2-4 branched triantennary complex type oligosaccharides that were linked by fucose at the ratio of 1.0:0.5:0.1, respectively. Moreover, the attachment site of the GAG chain was assigned to Ser472. It was then estimated that the GAG chain contained chondroitin-4-sulfate and dermatan sulfate, which were repeated approximately 30 times. In this paper, the GAG attachment site and structural characteristics of GAG-UTM, were confirmed. Moreover, structures of the N-linked oligosaccharides of GAG-UTM are described for the first time.

Increased anticoagulant activity of recombinant thrombomodulin modified with glycosaminoglycan.

Thrombomodulin (TM) is a thrombin receptor on the endothelial cell surface, effective as an anticoagulant by changing procoagulant thrombin to an anticoagulant one. As rabbit TM with glycosaminoglycan (GAG) has a more potent anticoagulant activity than that without GAG, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C-127 cells. The effect of an additional GAG chain on anticoagulant activity was investigated in comparison with unmodified recombinant UTM (r-UTM). In vitro, the activity of cleavage of fibrinogen by thrombin or prothrombinase activity was more potently depressed by GAG-UTM than by r-UTM, and the generation of activated protein C by TM-thrombin complex was accelerated by GAG modification. The acceleration of antithrombin III-dependent anticoagulant activity was shown only by GAG-UTM. Parameters like thrombin time, prothrombin time and activated partial thromboplastin time in human plasma were prolonged by GAG-UTM more than by r-UTM. In vivo, the effect of GAG-UTM and r-UTM in endotoxin-induced disseminated intravascular coagulation (DIC) rats was investigated using hematological parameters. GAG-UTM and r-UTM significantly reduced the decrease in fibrinogen and platelet number induced by endotoxin at the dosage of 0.1 and 1.0 mg/kg/h, respectively, suggesting that the antithrombotic effect of GAG-UTM in endotoxin-induced DIC rats was 10-fold as potent as that of r-UTM. GAG-UTM reduced the prolongation of the bleeding time induced by endotoxin, while r-UTM accelerated it. These results suggest that the addition of a GAG chain may increase availability as an anticoagulant.

Importance of sialic acid in recombinant thrombomodulin in terms of pharmacokinetics and separation of desialyzed glycoprotein.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM) expressed in mouse C-127 cells has potent antithrombotic activity available as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with alpha-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo- and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each: RCA-120-Agarose column for separation and ELISA using anti-thrombomodulin antibody and RCA lectin for analysis.

Protein C activation by recombinant thrombomodulin in plasma.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM), which partially improved the amino acid sequence of human urinary thrombomodulin (UTM), was expressed in C127 cells. GAG-UTM accelerates protein C activation by thrombin and also thrombin inhibition by antithrombin III (ATIII) in the buffer system. Both accelerating activities of GAG-UTM are more potent than those of unmodified recombinant UTM (r-UTM) without a GAG chain. As ATIII in plasma also inhibits protein C activation by a thrombin-thrombomodulin complex, we studied whether GAG-UTM accelerates protein C activation in plasma. GAG-UTM suppressed the generation of thrombin in activating plasma protein C stronger than r-UTM. By Western blot analysis using anti-protein C antibody, activated protein C was generated by GAG-UTM more than by r-UTM. From these results, the acceleration of activated protein C formation by GAG-UTM was confirmed in plasma too.

Digestion of endothelin-1 on cultured vascular smooth muscle cells.

The degradation of 125I-endothelin-1 (125I-ET-1) was examined on cultured porcine aortic endothelial cell (EC) and rat vascular smooth muscle cell (SMC) by HPLC analysis. The degradation of ET-1 was observed on SMC and was slightly observed on EC. Membrane fractions of SMC had a strong potency for the degradation of ET-1 and retained activity even in plasma. This activity was inhibited with some of the noble inhibitors which were known as enkephalinase inhibitors. These results suggest that endothelin degradation enzyme on SMC is related to enkephalinase and that this enzyme plays a significant role in the degradation of ET-1 in vivo.

Purification and characterization of endothelin-1 degradation activity from porcine kidney.

In order to identify the membrane-bound peptidase that is responsible for the degradation of endothelin (ET), an endothelin-1 (ET-1) degradation enzyme was solubilized from membrane fractions of porcine kidney with 1% Triton X-100, and subsequently purified by column chromatographies, i.e., diethylamino-Sepharose ion exchange, gel permeation, Con A Sepharose and hydroxyapatite chromatography. On DEAE-Toyopearl ion exchange column chromatography, the ET degradation enzyme and aminopeptidase were separated, but ET degradation enkephalinase activities were not separable. In order to separate ET degradation enzyme and enkephalinase, the active fractions were loaded on each of the column chromatographies: sephacryl S-200, Con A Sepharose or hydroxyapatite. The ET degradation activities were co-migrated with enkephalinase activities on all of the three chromatographies. In addition, the ET degradation activities were inhibited by thiorphan, phosphoramidon and EDTA, which are known to inhibit enkephalinase. These results suggest that ET degradation activity in the membrane fractions of the kidney is related to enkephalinase and may be involved in the degradation of ET-1 in vivo.

Pyrogenic action of endothelin in conscious rabbit.

Injection of 0.3 nmol/kg endothelin(ET)-1 into the ear vein of conscious rabbits induced a significant increase in body temperature. ETB receptor specific agonist, namely 4-Ala-ET-1, also caused an elevation of the body temperature in a dose-dependent manner by injection into the ear vein of rabbit. These results suggest that ET play important roles in regulation of body temperature through selective stimulation of ETB receptor.

Characterization of immunoreactive endothelin in human urine.

We developed three antibodies, specific and sensitive to endothelin-1 (ET-1), and established two sandwich and three competitive enzyme immunoassays (EIAs). By using these EIAs, large immunoreactive ET (IR-ET) of molecular weight 10 k Da was identified as a main component of IR-ETs in human urine. This large IR-ET, which reacted with two antibodies specific for N-terminal region of ET-1 but not with the antibody against C-terminal peptide of ET-1, was partially purified by six-step procedure and examined by Western blotting after SDS polyacrylamide gel electrophoresis. The large IR-ET was detected as a single band at molecular weight of 10 k Da both in reduced and non-reduced conditions. From these results, the large IR-ET was thought to consist of a single polypeptide chain and possess the steric restricted N-terminal region of ET-1.

Inhibition of endothelin (ET)-1-and ET-2-induced vasoconstriction by anti-ET-1 monoclonal antibody.

We produced a monoclonal antibody to endothelin (ET)-1, tested cross-reactivities with the related peptides by enzyme immunoassay, and investigated the effects of the antibody on ET-1- or ET-2-induced vasoconstriction of rat isolated thoracic aorta. The antibody recognized ET-1, ET-2 and ET-3, and the immunoreactive site proved to be the N-terminal region but not the C-terminal region of ET-1. Moreover, at an approximate molar-equivalent concentration, the antibody absorbed ET-1 and ET-2, and significantly inhibited ET-1- and ET-2-induced vasoconstriction notwithstanding the presence of the endothelin receptor.

Protective effect of chromium(III) on acute lethal toxicity of carbon tetrachloride in rats and mice.

Trivalent chromium (Cr(III)) preadministered intraperitoneally (5 mg Cr/kg body weight) to rats and mice protected these animals from acute lethal toxicity of carbon tetrachloride (CCl4). Some other metals, Cr(VI), Cu(II), and Zn(II), had no effect on CCl4 lethal toxicity. DL-alpha-tocopherol, one of the antioxidative agents, showed similar protective effects to Cr(III). Activities of serum GOT and GPT in mice were increased sharply by the administration of CCl4, but these elevations were depressed by Cr(III) preadministration. Serum glucose levels of mice increased transiently after CCl4 administration and then in the control group fell to hypoglycemic levels after 6 hr, whereas the Cr(III)-pretreated group kept to homeostatic levels. Lipid peroxidation of microsomes in mice 24 hr after Cr(III) administration was lower than that of the control. These results suggest that Cr(III) preadministered to mice might act as a radical scavenger to CCl4 to form trichloromethyl radicals which are a major initial product of CCl4 in liver cells.