heparitin-sulfate and isoleucyl-prolyl-arginine-4-nitroanilide

Inhibition of thrombin generation was studied in strongly diluted human plasma by continuously measuring the splitting of the thrombin-specific chromogenic substrate S2288 as a function of the inhibitor concentration. To avoid the activation of clotting cascade proenzymes other than prothrombin, the thrombin generation reaction was initiated by a mixture of calcium, phospholipids and (bovine) factor Xa. Using this assay we have estimated the relative potency of the following glycosaminoglycans: heparin; a fraction of heparin with high affinity for antithrombin III (high affinity heparin); the low molecular weight heparin Fragmin; the low molecular weight heparinoid Org 10172; a fraction of Org 10172 with high affinity for antithrombin III (high affinity Org 10172) and the O-methyl derivative of the pentasaccharide, representing the minimal structure required for binding to antithrombin III. Based on concentrations expressed in amidolytic anti-Xa units, the descending order of potency observed is: Heparin approximately High Affinity Heparin greater than Fragmin greater than Org 10172 greater than High Affinity Org 10172 greater than O-methyl pentasaccharide. The more potent the glycosaminoglycan the stronger the concentration dependence of its inhibitory effect. These findings could be due to the different, additional anti-thrombin activities of these glycosaminoglycans and/or to their different anti-prothrombinase activities. With the pentasaccharide a striking saturation of the inhibition is observed, due to saturation of the antithrombin III.

Topics: Antithrombins; Chondroitin Sulfates; Chromogenic Compounds; Dermatan Sulfate; Glycosaminoglycans; Heparin; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; In Vitro Techniques; Kinetics; Oligopeptides; Oligosaccharides; Thrombin