Experimental proof for the structure of a thrombin-inhibiting heparin molecule.

Kinetic studies of thrombin inhibition by antithrombin in the presence of heparin have shown that thrombin binds to heparin in a preformed heparin-antithrombin complex. To study the relative position of the thrombin binding domain and the antithrombin binding domain on a heparin molecule we have designed and synthesized heparin mimetics, which structurally are very similar to the genuine polysaccharide. Their inhibitory properties with respect to factor Xa and thrombin provide experimental evidence that in heparin the thrombin binding domain must be located at the nonreducing end of the antithrombin binding domain to observe thrombin inhibition. As expected, factor Xa inhibition is not affected by elongation of the antithrombin binding pentasaccharide sequence, regardless of the position in which this elongation takes place.

Identification of a hexasaccharide sequence able to inhibit thrombin and suitable for 'polymerisation'.

Three hexasaccharides, having from low to very high affinity for antithrombin, were synthesised from disaccharide building block precursors. One of them, methyl(sodium 2,3-di-O-methyl-4-O- sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]2-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside, obtainable from a single disaccharide building block precursor, constitutes a good starting point for obtaining simple oligosaccharidic heparin mimetics able to inhibit the two coagulation factors thrombin and Factor Xa.

Assessment through chemical synthesis of the size of the heparin sequence involved in thrombin inhibition.

Deca- to eicosasaccharides having the generic structure methyl(sodium 2,3-di-O-methyl-4-O-sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]n-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside have been synthesized from a single disaccharide precursor. All of them bind to and activate antithrombin. When n < or = 6 only Factor Xa inhibition is observed, whereas when n > 6 Factor Xa and thrombin are both inhibited in the presence of antithrombin. These results indicate that, in heparin, the sequence involved in antithrombin-catalyzed thrombin inhibition is a pentadeca- or a hexadecasaccharide.

Synthesis and pharmacological properties of a close analogue of an antithrombotic pentasaccharide (SR 90107A/ORG 31540).

The synthetic pentasaccharide (1) corresponding to the heparin sequence which binds to, and activates, antithrombin III (AT III) is a potent antithrombotic compound in several animal models of venous thrombosis. We describe here the preparation and the pharmacological properties of 34, an analogue of oligosaccharide 1 with the latter's N-sulfates being replaced by sulfate esters and hydroxyl groups being methylated. These structural modifications allow a simpler and more efficient synthesis of such anionic oligosaccharides. Affinity for human AT III, anti-factor Xa activity, ability to inhibit thrombin generation, antithrombotic activity in a rat model of venous thrombosis, and elimination half-life in the rat have been determined for 1 and 34. Surprisingly, introduction of O-sulfates in place of N-sulfates, and methylation of hydroxyl groups, contributes to reinforce the binding to AT III, resulting in an improved pharmacological profile.