The Action of a Synthetic Pentasaccharide on Thrombin Generation in Whole Plasma

Summary

We investigated the effect on thrombin generation in plasma of the pentasaccharide that represent the AT II/binding site in heparin. This compound has no effect on the breakdown of thrombin in plasma. It dose-dependently inhibits the formation of thrombin in both the intrinsic and the extrinsic pathway. If coagulation is triggered by the complete prothrombinase complex (phospholipid – factor V_a – factor X_a) under conditions in which the large majority of factor X_a is bound to the complex, the inhibition of prothrombinase activity is only minor. If no factor V_a is present or if the prothrombinase activity is triggered by adding complete tenase (PL-FVIII_a-FIX_a) or incomplete tenase (PLFIX_a) to the plasma the inhibition by pentasaccharide is of the same magnitude as that in the intrinsic or extrinsic system.

We conclude that the pentasaccharide inhibits blood coagulation by katalysing the inactivation of free factor X_a. In contrast to classical heparin it does inhibit the peak of thrombin formation in platelet rich plasma, probably because it is less subject to inactivation by heparin binding proteins from platelets than classical heparin is.

Keywords

Heparin - Pentasaccharide - Thrombin generation - Factor X_a

• References

• 1 Barrowcliffe TW, Johnson EA, Eggleton CA, Kemball-Cook G, Thomas DP. Anticoagulant activities of high and low molecular weight heparin fractions. Br J Haematol 1979; 41: 573-583
• 2 Andersson LO, Barrowcliffe TW, Holmer E, Johnson EA, Söderstrom G. Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma Thromb Res 1979; 15: 531-541
• 3 Holmer E, Kurachi K, Söderstrom G. The molecular-weight heparin dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor X_a, factor IX_a, factor XI_a, factor XII_a and kallikrein by antithrombin. Biochem J 1981; 193: 395-400
• 4 Björk I, Lindahl U. Mechanism of the anticoagulant action of heparin. Mol Cell Biochem 1982; 48: 161-162
• 5 Lane A, Denton J, Flynn AM, Thunberg L, Lindahl U. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. Biochem J 1984; 218: 725-732
• 6 Casu B, Oreste P, Torri G, Zoppetti G, Choay J, Lormeau JC, Petitou M, Sinay P. The structure of heparin oligosaccharide fragments with high anti-(factor X_a) activity containing the minimal antithrombin Ill-binding sequence. Biochem J 1981; 197: 599-609
• 7 Choay J, Petitou M, Lormeau JC, Sinay P, Casu BJ, Gatti G. Structure-activity of relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high antifactor X_a activity. Biochem Biophys Res Comm 1983; 116: 492-499
• 8 Hemker HC, Willems GM, Béguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemostas 1986; 56: 9-17
• 9 Hemker HC. The mode of action of heparin in plasma. XIth Congress Thrombosis Haemostasis, Brussels. Verstraete M, Vermylen J, Lijnen HR, Amout J. (eds) Leuven University Press; Leuven: 1987. pp 17-36
• 10 Béguin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemostas 1988; 60: 457-462
• 11 Béguin S, Lindhout T, Hemker HC. The effect of trace amounts of tissue factor on thrombin generation in platelet rich plasma, its inhibition by heparin. Thromb Haemostas 1989; 61: 25-29
• 12 Hendriks H, Lindhout T, Mertens K, Engels W, Hemker HC. Activation of human prothrombin by stoichiometric levels of staphylocoagulase. J Biol Chem 1983; 258: 3637-3644
• 13 Rosing J, Tans G, Govers-Riemslag JW P, Zwaal RF A, Hemker HC. The role of phospholipids and factor V_a in the prothrombinase complex. J Biol Chem 1986; 255: 274-283
• 14 Owren PA, Aas K. The control dicoumarol therapy and the quantitative determination of prothrombin and proconvertin. Scand J Clin Lab Invest 1951; 3: 201-218
• 15 Petitou M, Duchaussoy P, Lederman I, Choay J, Jacquinet J-C, Sinaï P, Torri G. Synthesis of heparin fragments: A methyl-pentaoside with high affinity for antithrombin III. Carbohydrate Res 1987; 167: 67-75
• 16 Ma Xi, Béguin S, Hemker HC. The importance of factor IX activation in thromboplastin-dependent coagulation (the Josso pathway) in plasma. Haemostasis. 1989 accepted for publication
• 17 Pletcher CH, Nelsestuen GI. The rate determining step of the heparin-catalyzed antithrombin-thrombin reaction is independent of thrombin. J Biol Chem 1982; 257: 5342-5345
• 18 Lindhout T, Govers-Riemslag JW P, van der Waart P, Hemker HC, Rosing J. Factor V_a, factor X_a interaction. Effects of phospholipid vesicles of varying composition. Biochemistry 1982; 21: 5494-5502
• 19 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII. Biochemistry 1980; 19: 401-410
• 20 van Dieijen G, van Rijn JL M L, Govers-Riemslag JW P, Hemker HC, Rosing J. Assembly of the intrinsic factor X activating complex; interactions between factor IX_a, factor VIII_a and phospholipid. Thromb Haemostas 1985; 53: 396-400
• 21 Fujikawa K, Thompson AR, Legaz ME, Meyer RG, Davie EW. Isolation and characterization of bovine factor IX (Christmas Factor). Biochemistry 1973; 12: 4938-4945
• 22 Fujikawa K, Legaz ME, Kato H, Davie EW. The mechanism of activation of bovine IX (Christmas factor) by bovine factor XI_a(activated plasma thromboplastin antecedent). Biochemistry 1974; 13: 4508-4516
• 23 Barrowcliffe TW, Havercroft SJ, Kemball-Cook G, Lindahl U. The effect of Ca²⁺, phospholipid and factor V on the anti-(factor X_a) activity of heparin and its high oligosaccharides. Biochem J 1987; 243: 31-37
• 24 Walenga JM, Fareed J, Petitou M, Samama M, Lormeau JC, Choay J. Intravenous antithrombotic activity of a synthetic heparin polysaccharide in a human serum induced stasis thrombosis model. Thromb Res 1986; 43: 243-248