Dermatan Sulfate Is a More Potent Inhibitor of Clot-bound Thrombin than Unfractionated and Low Molecular Weight Heparins

Summary

Clot-bound thrombin proteolyses fibrinogen and amplifies the coagulation cascade at its close vicinity, thereby ensuring the growth of fibrin-rich thrombus. The present study compares the ability of various glycosaminoglycans (GAGs) to inhibit these 2 properties. Unfractionated heparin (UH), 3 low molecular weight heparins (LMWHs) with increasing antifactor Xa/antifactor Ha ratio, the synthetic pentasaccharide (PS), devoid of antifactor Ha activity, and dermatan sulfate (DS), a catalyst of thrombin inhibition by heparin cofactor II, were selected on the basis of their different properties. Proteolysis of fibrinogen by clot-bound thrombin was evaluated by measuring fibrinopeptide A (FPA) generation after an incubation of standardized washed clots in plasma for 120 min in absence or in presence of increasing concentrations of heparins or of DS. The results were compared to those obtained when free a-thrombin (0.4 nM) was added to plasma in the same experimental conditions. On the basis of equivalent antithrombin units, UH and LMWHs gave identical results. To inhibit by 70% fibrinogen proteolysis induced by clot-bound thrombin (IC 70), 5- to 9-fold higher concentrations of UH or of LMWHs were required in comparison with those required to inhibit free thrombin. For DS, only a 1.3 times higher concentration was required. PS (final concentration 1 anti Xa U • ml^-1) was devoid of any inhibitory effect. The amplification of the coagulation cascade induced by dot-bound thrombin was evaluated by measuring the shortening of whole blood clotting time (WBCT) resulting from the incubation of washed clots in native blood. In absence of GAG, clot-bound thrombin reduced WBCT from 18 ± 2 min to 9 ± 1 min. Each GAG prolonged WBCT in a dose-dependent manner but these prolongations were smaller in presence of washed clots. The most potent agent to suppress the shortening of WBCT was DS. LMWH and UH were less effective and PS (final concentration 1 anti Xa U/ml) was almost ineffective. Therefore, in these in vitro experiments, DS is a more potent inhibitor of clot-bound thrombin than heparin. Whether or not these observations are relevant for the treatment of established deep-vein thrombosis requires comparative clinical studies.

• References

• 1 Hogg PJ, Jackson CM. Fibrin monomers protects thrombin from inactivation by heparin-antihrombin III: implications for heparin efficacy. Proc Natl Acad Sci 1989; 86: 3619-3623
• 2 Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin Ill-independent inhibitors. J Clin Invest 1990; 86: 385-391
• 3 Okwusidi J, Anvari K, Kulczycky M, Blajchman MA, Buchanan MR, Ofosu FA. Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma. J Lab Clin Med 1991; 117: 359-64
• 4 Agnelli G, Renga C, Weitz J, Nenci G, Hirsh J. Sustained antithrombotic activity of hirudin after its plasma clearance: comparison with heparin. Blood 1992; 80: 960-965
• 5 Brill EP, Van Ryn-McKenna J, Cai L, Ofosu FA, Buchanan MR. Prevention of thrombus growth by antithrombin III dependent and two direct thrombin inhibitors in rabbits: implications for antithrombotic therapy. Thromb Haemost 1992; 68: 424-427
• 6 Van Ryn-McKenna J, Ofosu FA, Gray E, Hirsh J, Buchanan MR. Effects of dermatan sulfate and heparin on inhibition of thrombus growth in vivo. Ann NY Acad Sci 1989; 556: 304-312
• 7 Carrié D, Caranobe C, Gabaig AM, Larroche M, Boneu B. Effects of heparin dermatan sulfate and their association on the inhibition of venous thrombosis growth in the rabbit. Thromb Haemost 1992; 68: 637-641
• 8 Fernandez F, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. The haemorrhagic and antithrombotic effects of dermatan sulfate. Br J Haematol 1986; 64: 309-317
• 9 Petitou M, Duchaussoy P, Lederman I, Choay I, Jacquinet JC, Sinay P, Toni G. Synthesis of heparin fragments: a methyl alpha-pentaoside with high affinity to antithrombin III. Carbohydr Res 1987; 167: 67-75
• 10 Prins MH, Weitz JI. Heparin inhibits the amplification of coagulation mediated by clot-bound thrombin. Thromb Haemost 1991; 65: 759 (abst)
• 11 Sié P, Ofosu FA, Fernandez F, Buchanan MR, Petitou M, Boneu B. Respective role of AT III and HC II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharide. Br J Haematol 1986; 64: 704-714
• 12 Agnelli G, Cosmi B, Filippo PD, Ranucci V, Veschi F, Longetti M, Renga C, Barzi F, Gianese F, Lupattelli L, Rinonapoli E, Nenci GG. A randomised double blind placebo-controlled trial of dermatan sulphate for prevention of deep vein thrombosis in hip fracture. Thromb Haemost 1992; 67: 203-208
• 13 Lane DA, Ryan K, Ireland H, Curtis JR, Nurmohamed M, Krediet RT, Roggekamp M, Stevens P, Ten Cate JW. Dermatan sulphate in haemodialysis. Lancet 1992; 339: 334-335
• 14 Hemker HC, Choay J, Beguin S. Free factor Xa is on the main pathway of thrombin generation in clotting plasma. Bioch Bioph Acta 1989; 992: 409-411
• 15 Estivals M, Pelzer H, Sié P, Pichon J, Boccalon H, Boneu B. Prothrombin fragment 1 + 2, thromin-antithrombin III complexes and D-dimers in acute deep vein thrombosis: effects of heparin treatment. Br J Haematol 1991; 78: 421-424
• 16 Elias A, Bonfils S, Daoud-Elias M, Gauthier B, Sié P, Boccalon H, Boneu B. Influence of long term oral anticoagulants upon prothrombin fragment 1 + 2, thrombin-antithrombin III complex and D-Dimer levels in patients affected by proximal deep vein thrombosis. Thromb Haemost 1993; 69: 302-305
• 17 Cadroy Y, Hanson SR, Harker LA. Antithrombotic effects of synthetic pentasaccharide with high affinity for plasma antithrombin III in non-human primates. Thromb Haemost 1993; 70: 631-635
• 18 Carrié D, Caranobe C, Saivin S, Houin G, Petitou M, Lormeau HC, Van Boeckel C, Meuleman D, Boneu B. Pharmacokinetic and antithrombotic properties of two pentasaccharides with high affinity to antithrombin III in the rabbit: comparison with CY216. Blood. 1994. (in press)