On the Relationship Between Molecular Mass and Anticoagulant Activity in a Low Molecular Weight Heparin (Enoxaparin)

Summary

A low molecular weight heparin (enoxaparin, mean molecular weight ~ 4,400) was separated by gel chromatography into eight different fractions with a narrow distribution around the following mean molecular weights: 1,800, 2,400, 2,900, 4,200, 6,200, 8,600, 9,800 and 11,000. We compared the influence of enoxaparin on the generation of thrombin in plasma to that of the eight fractions.

We determined: a) the % of material with high affinity to antithrombin III (HAM) and the % of HAM above the critical chainlength necessary to allow for thrombin inhibition (ACLM), b) the specific catalytic activity on the decay of endogenous thrombin, and c) the inhibition of over-all thrombin formation in the extrinsic and the intrinsic pathway. From b and c we calculated the inhibition of prothrombin conversion in these pathways.

We found that a) there is a gradual decrease of the HAM fraction with decreasing molecular weight; b) the specific catalytic activity for the inactivation of thrombin does not vary significantly between the fractions when expressed in terms of ACLM; c) the potency to inhibit prothrombin conversion does not vary significantly between the fractions when expressed in terms of HAM.

• References

• 1 Chesebro JH, Zoldheyi P, Badimon L, Fuster V. Role of thrombin in arterial thrombogenesis: Implication for therapy. Thromb Haemostas 1991; 66: 1-4
• 2 Thunberg L, Lindahl U, Tengblad A, Laurent TC, Jackson CM. On the molecular-weight-dependence of the anticoagulant activity of heparin. Biochem J 1979; 181: 241-243
• 3 Andersson LO, Barrowcliffe TW, Holmer E, Johnson EA, Soden-strom 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
• 4 Lane DA, 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
• 5 Barrowcliffe TW, Merton RE, Havercroft SJ, Thunberg L, Lindahl U, Thomas DP. Anticoagulant activities of heparin oligosaccharides. Thromb Res 1984; 34: 125-133
• 6 Thomas DP, Merton RE, Barrowcliffe TW, Thunberg L, Lindahl U. Effects of heparin oligosaccharides with high affinity for antithrombin III in experimental venous thrombosis. Thromb Haemostas 1982; 47: 244-248
• 7 Bratt G, Tornebohm E, Lockner D, Bergstrom K. A human pharmacological study comparing conventional heparin and a low molecular heparin fragment. Thromb Haemostas 1985; 53: 208-211
• 8 Hemker HC, Willems GM, Beguin 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 Jesty J. Analysis of the generation and inhibition of activated coagulation factor X in pure systems and in human plasma. J Biol Chem 1986; 261: 8695-8702
• 10 Jesty J. The kinetics of inhibition of a-thrombin in human plasma. J Biol Chem 1986; 261: 10313-10318
• 11 Bendetowicz AV, Bara L, Samama M. The inhibition of intrinsic prothrombinase and its generation by heparin and four derivatives in prothrombin poor plasma. Thromb Res 1989; 58: 445-454
• 12 Beguin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemostas 1988; 60: 457-462
• 13 Choay J, Petitou M, Lormeau JC, Sinay P, Casu B, Gatti G. Structure-activity relationship in heparin: A synthetic pentasaccharide with high affinity for antithrombin III and eliciting high antifactor Xa activity. Biochem Biophys Res Commun 1983; 116: 492-499
• 14 Beguin S, Choay J, Hemker HC. The action of a synthetic pentasaccharide on thrombin generation in whole plasma. Thromb Haemostas 1989; 61: 397-401
• 15 Walenga JM, Bara L, Petitou M, Fareed J, Samama M, Choay J. The inhibition of the generation of thrombin and the antithrombotic effect of a pentasaccharide with sole anti-factor Xa activity. Thromb Res 1988; 51: 23-33
• 16 Hemker HC. The mode of action of heparin in plasma. Xlth Congress Thrombosis Haemostasis, Brussels. Verstraete M, Vermylen J, Lij-nen HR, Arnout J. (eds) Leuven University Press; Leuven: 1987. pp 17-36
• 17 Beguin S, Mardiguian J, Lindhout T, Hemker HC. The mode of action of low molecular weight heparin preparation (PK 10169) and two of its major components of thrombin generation in plasma. Thromb Haemostas 1989; 61: 30-34
• 18 Teien AN, Lie M. Evaluation of an amidolytic heparin assay method. Increased sensitivity by adding purified antithrombin III. Thromb Res 1977; 10: 399-410
• 19 Bara L, Combe-Tamzali S, Conard J, Horellou MH, Samama M. Modifications biologiques induites par trois heparines de bas poids moleculaire: PK 10169, Kabi 2165 et CY 216, comparees a l’heparine non fractionnee, injectees par voie sous-coutanee chez le volontaire sain en chirurgie generate et chez le sujet age en medecine. J Mai Vase 1987; 12: 78-84
• 20 Thaler E, Schmer G. A simple two-step isolation procedure for human and bovine antithrombin II/III (heparin cofactor): a comparison of two methods. Br J Haematol 1975; 31: 233-243
• 21 Miller-Andersson M, Borg H, Andersson LO. Purification of antithrombin III by affinity chromatography. Thromb Res 1974; 5: 439-452
• 22 Smith RL. Titration of activated bovine factor X. J Biol Chem 1973; 248: 2418-2423
• 23 Owren PA, Aas K. The control of dicumarol therapy and the quantitative determination of prothrombin and proconvertin. Scand J Clin Lab Invest 1951; 3: 201-218
• 24 Casu B, Oreste P, Torri G, Zopetti G, Choay J, Lormeau J-C, Petitou M. The structure of heparin oligosaccharide fragments with high antifactor Xa) activity containing the minimal antithrombin-III binding sequence. Biochem J 1981; 197: 599-609
• 25 Schoen P, Wielders S, Petitou M, Lindhout T. The effect of sulfation on the anticoagulant and antithrombin Ill-binding properties of a heparin fraction with low affinity for antithrombin III. Thromb Res 1990; 57: 415-423
• 26 Nordenman B, Danielsson A, Bjork I. The binding of low affinity and high affinity heparin to antithrombin. Fluorescence Studies. Eur J Biochem 1978; 90: 1-6
• 27 O’Neall-Speight M, Griffith MJ. Calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction by decreasing the apparent binding afinity of heparin for thrombin. Arch Biochem Biophys 1983; 225: 958-963
• 28 Barrowcliffe TW, Shirle Le Y. The effect of calcium chloride on anti-Xa activity of heparin and its molecular weight fractions. Thromb Haemostas 1989; 62: 95-954
• 29 Schoen P, Lindhout P, Franssen J, Hemker HC. Low molecular weight heparin-catalyzed inactivation of factor Xa and thrombin by antithrombin III. Effect of platelet factor 4. Thromb Haemostas 1991; 66: 435-441
• 30 Beguin S, Wielders S, Lormeau JC, Hemker HC. The mode of action of CY216 and CY222 in plasma. Thromb Haemostas 1992; 67: 33-41
• 31 Ellis V, Scully MF, Kakkar VV. The relative molecular mass dependence of the anti-factor Xa properties of heparin. Biochem J 1986; 238: 329-333
• 32 Barrowcliffe TW, Havercroft SJ, Kemball-Cook G, Lindhal U. The effect of Ca⁺², phospholipid and factor V on the anti(-factor Xa) activity of heparin and its high-affinity oligosaccharides. Biochem J 198 (243) 31-37
• 33 Danielsson A, Raub E, Lindahl U, Bjork I. Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the accelaration of the reactions between antithrombin and thrombin or factor Xa. J Biol Chem 1986; 261: 15467-15473
• 34 Ofosu FA, Sie P, Modi GJ, Fernandez F, Buchanan MR, Blajchman MA, Boneu B, Hirsh J. The inhibition of thrombin-dependent positive feedback reactions is critical to the expression of the anticoagulant effect of heparin. Biochem J 1987; 243: 579-588
• 35 Ofosu FA, Smith LM, Anvari N, Blajchman MA. An approach to assigning in vitro potency to unfractionated heparin and low molecular weight heparins based on the inhibition of prothrombin activation and catalysis of thrombin inhibition. Thromb Haemostas 1988; 60: 193-198
• 36 Pieters J, Lindhout T, Hemker HC. In situ generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin activated plasma. Blood 1989; 74: 1021-1024
• 37 Beguin S, Dol F, Hemker HC. Factor IXa inhibition contributes to the heparin effect. Thromb Haemostas 1991; 66: 306-309
• 38 Yin ET, Wessler S, Stoll PJ. Biological properties of the naturally occuring inhibitor to activated factor X. J Biol Chem 1971; 246: 3703-3711
• 39 Hemker HC, Esnouf MP, Hemker PW, Swart ACW, Macfarlane RG. Formation of prothrombin converting activity. Nature 1968; 215: 246-251
• 40 Rosing J, Tans G, Govers-Riemslag JWP, Zwaal RAF, Hemker HC. The role of phospholipid and factor Va in the prothrombinase complex. J Biol Chem 1980; 249: 7798-7807
• 41 Pieters J, Lindhout T. The limited importance of factor Xa inhibition to the anticoagulant property of heparin in thromboplastin-activated plasma. Blood 1988; 72: 2048-2052