Further Studies on the Mechanism for the Antithrombotic Effects of Naroparcil, an Orally Active Thioxyloside Compound

Summary

The antithrombotic β-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII.

The qualitative analysis of GAGs showed the presence of the ΔDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED₈₀ values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.

• References

• 1 Bellamy F, Horton D, Millet J, Picart F, Samreth S, Chazan JB. Glycosylated derivatives of benzophenone, benzhydrol, and benzhydril as potential venous antithrombotic agents. J Med Chem 1993; 36: 898-903.
• 2 Millet J, Theveniaux J, Brown NL. The venous antithrombotic profile of Naroparcil in the rabbit. Thromb Haemost 1994; 72: 874-9.
• 3 Martin N, Masson P, Sepulchre C, Theveniaux J, Millet J, Bellamy FD. Pharmacological and biochemical profiles of new venous antithrombotic β-D-xyloside derivatives: potential antiatherothrombotic drugs. Semin Thromb Hemost 1996; 22: 247-54.
• 4 Schwartz NB, Galligani L, Ho P, Dorfman A. Stimulation of synthesis of free chondroitin sulfate chains by β-D-xylosides in cultured cells. Proc Natl Acad Sci USA 1974; 71: 4047-51.
• 5 Masson PJ, Coup D, Millet J, Brown NL. The effect of the β-D-xyloside Naroparcil on circulating plasma glycosaminoglycans: an explanation for its known antithrombotic activity in the rabbit. J Biol Chem 1995; 270: 2662-8.
• 6 Sié P, Dupouy D, Caranobe C, Petitou M, Boneu B. Antithrombotic properties of a dermatan sulfate hexasaccharide fractionated by affinity for heparin cofactor II. Blood 1993; 81: 1771-7.
• 7 Dupouy D, Sié P, Dol F, Boneu B. A simple method to measure dermatan sulfate at sub-microgram concentrations in plasma. Thromb Haemost 1988; 60: 236-9.
• 8 Amar J, Caranobe C, Sié P, Boneu B.. Antithrombotic potencies of heparins Antithrombotic potencies of heparins in relation to their antifactor Xa and antithrombin activities: an experimental study in two models of thrombosis in the rabbit. Brit J Haematol 1990; 76:: 94-100.
• 9 Toulon P, Bardin JM, Blumenfeld N.. Increased heparin cofactor II levels in women taking oral contraceptives. Thromb Haemost 1990; 4:: 365-8.
• 10 Millet J, Theveniaux J, Brown NL. The venous antithrombotic effect of LF 1351 in the rat following oral administration. Thromb Haemost 1992; 67: 176-9.
• 11 Biggs RM, Mac Farlane RG. Blood coagulation and its disorders. In L’hémostase, méthodes d’exploration et diagnostic pratique. Caen J, Larrieu MJ, Samama M. eds Paris: L’Expansion Scientifique Française; 1968: 182-3.
• 12 Biggs R, Rizza C.. Human blood coagulation, haemostasis and thrombosis. vol 3. Oxford: Blackwell Scientific Publications; 1984
• 13 Teien AN, Lie M, Abildgaard U. Assay of heparin in plasma using a chromogenic substrate for activated factor X. Thromb Res 1976; 8: 413-6.
• 14 Teien AN, Lie M. Evaluation of an amidolytic heparin assay method. Increased sensitivity by adding purified antithrombin III. Thromb Res 1977; 10: 399-410.
• 15 Ofosu AF, Modi GJ, Smith LM, Cerskus AL, Hirsh J, Blajchman MA. Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways. Blood 1984; 64: 742-7.
• 16 Kumar R, Béguin S, Hemker HC.. The influence of fibrinogen and fibrin on thrombin generation – Evidence for feedback activation of the clotting system by clot bound thrombin. Thromb Haemost 1994; 72:: 713-21.
• 17 Maimone MM, Tollefsen DM. Structure of a dermatan sulfate hexasaccha-ride that binds to heparin cofactor II with high affinity. J Biol Chem 1990; 265: 18263-71.
• 18 Mascellani G, Liverani L, Prete A, Bergonzini G, Bianchini P, Torri G, Bisio A, Guerrini M, Casu B. Quantitation of dermatan sulfate active site for heparin cofactor II by 1H nuclear magnetic resonance spectroscopy. Anal Biochem 1994; 223: 135-41.
• 19 Denti A, Sini P, Tira ME, Balduini C. Structural heterogeneity of dermatan sulfate chains: correlation with heparin cofactor II activating properties. Thromb Res 1995; 79: 187-98.
• 20 Mascellani G, Liverani L, Prete A, Guppola PA, Bergonzini G, Bianchini P. Relative influence of different disulphate disaccharide clusters on the HCII-mediated inhibition of thrombin by dermatan sulphates of different origins. Thromb Res 1994; 74: 605-15.
• 21 Mascellani G, Liverani L, Parma B, Bergonzini G, Bianchini P. Active site for heparin cofactor II in low molecular mass dermatan sulfate. Contribution to the antithrombotic activity of fractions with high affinity for heparin cofactor II. Thromb Res 1996; 84: 21-32.
• 22 Bergonzini GL, Bianchini P, Hoppensteadt D, Parma B, Osima B, Walenga JM, Fareed J. Pharmacokinetics of native and low molecular weight derma-tans: preliminary studies in rats and primates. Semin Thromb Hemost 1991; 17: 235-9.