Arie Visser

DETERMINATION OF THE ANTI-FACTOR Xa ACTIVITY OF THE SYNTHETIC PENTASACCHARIDE SR 90107A/ORG 31540 AND OF TWO STRUCTURAL ANALOGUES

The anti-factor Xa activity of the synthetic pentasaccharide SR 90107A/ORG 31540 was assayed by a chromogenic method at pH 8.4 and pH 7.35, comparatively to the 4th International Heparin Standard (IHS) or to the Ist International Low Molecular Weight Heparin Standard (LMWHS). At pH 8.4, SR 90107A/ORG 31540 was found to have a specific anti-factor Xa activity of 639 +/- 14 and 659 +/- 19 IU/mg (mean +/- sem, n = 6) when assayed in comparison with the 4th ISH and the Ist LMWHS respectively. At pH 7.35, the corresponding figures were 864 +/- 6 and 1160 +/- 51 IU/mg (mean +/- sem, n = 6) respectively. The dissociation constants of the ATIII-pentasaccharide complex formed by SR 90107A/ORG 31540 and by two close analogues: SR 80327A and SR 80027A in the presence of purified human ATIII were found to be 41 +/- 8, 96 +/- 1 and 3 +/- 1.4 nM (mean +/- sem, n = 3) respectively. For the three compounds, the pseudo-first order molar catalytic constants for factor Xa inactivation by the ATIII-pentasaccharide complex were shown to be statistically comparable, in the range of 7-8 x 10(7) min-1 per mole. It is concluded that the differences in specific anti-factor Xa activities between SR 90107A/ORG 31540 and its synthetic chemical analogues can be attributed to variations of the dissociation constants whereas the catalytic constants for factor Xa inactivation remain unchanged.

Peptide-derived transition state analogue inhibitors of thrombin; Synthesis, activity and selectivity

In a study to combine the transition state analogue concept with the principle of catalytic site spanning, a series of peptide-derived transition state analogue (TSA) inhibitors of thrombin has been synthesized and tested. In the sequence H-D-Phe-Pro-Arg-Gly-OH (2) the Arg-Gly amide bond has been replaced by three classes of transition state analogues, being the ketomethylene, the hydroxyethylene and the hydroxymethylene amide bond replacements. Compound 12a, in which the amide bond has been replaced by the ketomethylene group, was found to be the most potent thrombin inhibitor of the series studied. Subsequently, penta- and hexapeptide sequences with good affinity for thrombin were developed, i.e. H-D-Phe-Pro-Arg-Gly-Phe-OH (16) and H-D-Phe-Pro-Arg-Gly-Phe-Lys-OH (26). In these sequences the Arg-Gly amide bond was then replaced by the ketomethylene group. The resulting compounds 43a and 47a, respectively, were evaluated in vitro as inhibitors of thrombin and factor Xa. Compound 47a was found to be the most potent thrombin inhibitor of the series studied (Ki = 29 nM). The combination of the transition state analogue concept and the principle of peptide elongation (tetrapeptide-->hexapeptide) yields thrombin inhibitors of high potency and selectivity. The effects of these two alterations reinforce each other indicating a synergistic effect. This might be rationalized by entropy factors.

Inhibition of the early stages of the thrombin generation reaction by various glycosaminoglycans

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.

Transition State Analogue Inhibitors of Thrombin: Synthesis, Activity and Molecular Modelling

Thrombin, an important enzyme in the blood coagulation system, cleaves four Arg-Gly peptide bonds in fibrinogen in order to form fibrin monomers, which form blood clots after polymerisation. In thrombosis the blood coagulation system is disturbed, resulting in an excess of blood clot formation. Thrombosis can be inhibited by inactivation of thrombin.