Effect of dextran sulphates on thrombin activity.

Abstract

SUMMARY To clarify the action of dextran sulphate, a heparin analogue, in the clotting of fibrinogen by thrombin, determinations were carried out on the clotting activity, the release of fibrinopeptides from fibrinogen, and the hydrolytic activity of thrombin against a peptide chromogenic substrate in the absence or presence of antithrombin III (heparin cofactor). It was shown that dextran sulphate itself inhibited thrombin activity, and its inhibition was dependent on the molecular weight and the sulphur content of the dextran sulphate. Although heparin markedly enhanced the antithrombin activity of antithrombin HII, dextran sulphate did not activate antithrombin MI.Heparin is composed of amino sugar and uronic acid residues, and its molecular weight is roughly 10000-20000 (Jeanloz, 1975). As its synthesis has not yet been completely successful, a structural analogue of heparin (heparinoid), dextran sulphate, which is a synthetic dextrose polymer, has been investigated for its various biological and pharmacological actions. So far several influences of dextran sulphate on coagulation and the fibrinolytic system, along with its lipolytic, anticholesterolaemic, and antihyaluronidase activities, have been reported (Douglas, 1956;Yamada and Kuzuya, 1962). Of these actions, the anticoagulant activity is the most significant, but its mechanism seems to be very complicated and remains unclear.The present study was designed to clarify the anticoagulant mechanism of dextran sulphate on the fibrinogen-fibrin conversion process, fibrinopeptide release from fibrinogen by thrombin, and the hydrolytic activity of thrombin against a peptide chromogenic substrate in the absence or presence of antithrombin III. The relationships between molecular weight or sulphur content and the anticoagulant activity of dextran sulphate were also investigated. Material and methodsA 01M NaCl-0 05M Tris-HCI buffer (pH 7 5) was used throughout.Fibrinogen (97-98 % clottability) was purified from human plasma by the method of Blomback and Blomback, (1956 (1975) using barium sulphate adsorption, defibrination by heat, adsorption by aluminium hydroxide, fractionation with ammonium sulphate, gel filtration on Sephadex G-200 (Pharmacia Chem., Uppsala, Sweden), and chromatography of DEAEcellulose (Serva, Seikagaku Kogyo, Tokyo, Japan). The antithrombin III prepared by this method was concentrated 300-fold over plasma and showed essentially a single band on SDS-gel electrophoresis. This antithrombin III was dialysed against the Tris-HC1 buffer and then stored at -70°C until used.Quantitative determination of antithrombin III was carried out with immunodiffusion plates (M-Partigen, Behring Inst, West Germany) (Fahey and Mckelvey, 1965;Mancini et al., 1965).Solutions of sodium dextran sulphate (Kowa Pharm., Tokyo, Japan) of molecular weight (MW) 3500 (sulphur content; SY% = 5 3 or 18-0), MW 7500 (S% = 5 8 or 18-1), MW 10 000 (S% = 17-9), MW 50 000 (S% = 19-3), and MW 200 000 (S% = 19-0) were made at the specified concentrations in the TrisHCl buff...

The Molecular-Mass Dependence of Dextran Sulfate Enhancement of Inactivation of Thrombin and Fibrinogen and on Factor Xa Neutralization by Antithrombin III

“…The inhibition of fibrinopeptide release from fibrinogen by thrombin [5] and fibrin clotting reported by others [6] and shown here depended on the molecular mass of DS, as were the interactions of both the enzyme and the substrate protein. Therefore, it remains unclear whether DS interacted primarily with fibrinogen or thrombin in the three-component system.…”

Complexation between low‐molecular‐weight cationic ligands and negatively charged polymers as studied by capillary electrophoresis frontal analysis

“…Due to the features listed above, CE-FA as an analytical technique should be an ideal method for the study of interactions between low-molecular-weight ligands and polymers provided that the interacting species are soluble and at least one of them is charged. heparin [36,37]. Also the polymer has found use in drug delivery settings [3,5,38].…”

Treatment of Hyperlipidemia With a Modified Low Density Lipoprotein Apheresis System With Dextran Sulfate

“…Dextran sulfate was originally used as an anticoagulant agent (also called heparan) but was later replaced by heparin (with anticoagulation activity of 125 IU/mg), due to the low anticoagulation activity (10 IU/mg) and anaphylaxis in some clinical cases (13). In this study, we used DS as absorber of LDL‐C, and its low anticoagulation activity became advantageous in that the residual DS transfused back to patients was very low and would not cause spontaneous bleeding or require the removal of excess DS by absorption before transfusion.…”