Download PDF
Thromb Haemost 1992; 67(05): 556-562
DOI: 10.1055/s-0038-1648493
Original Articles
Schattauer GmbH Stuttgart

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

Ana-Victoria Bendetowicz
1   The Department of Biochemistry, Rijksuniversiteit Limburg, Maastricht, The Netherlands
,
Elisabeth Pacaud
2   Centre de Recherches de Vitry, Rhône-Poulenc Rorer, France
,
Suzette Béguin
1   The Department of Biochemistry, Rijksuniversiteit Limburg, Maastricht, The Netherlands
,
André Uzan
2   Centre de Recherches de Vitry, Rhône-Poulenc Rorer, France
,
H Coenraad Hemker
1   The Department of Biochemistry, Rijksuniversiteit Limburg, Maastricht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 04 June 1991

Accepted after revision 17 December 1991

Publication Date:
03 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 5 Barrowcliffe TW, Merton RE, Havercroft SJ, Thunberg L, Lindahl U, Thomas DP. Anticoagulant activities of heparin oligosaccharides. Thromb Res 1984; 34: 125-133
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 10 Jesty J. The kinetics of inhibition of a-thrombin in human plasma. J Biol Chem 1986; 261: 10313-10318
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 12 Beguin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemostas 1988; 60: 457-462
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    Google Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 21 Miller-Andersson M, Borg H, Andersson LO. Purification of antithrombin III by affinity chromatography. Thromb Res 1974; 5: 439-452
    CrossrefPubMedGoogle Scholar
  • 22 Smith RL. Titration of activated bovine factor X. J Biol Chem 1973; 248: 2418-2423
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 37 Beguin S, Dol F, Hemker HC. Factor IXa inhibition contributes to the heparin effect. Thromb Haemostas 1991; 66: 306-309
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 39 Hemker HC, Esnouf MP, Hemker PW, Swart ACW, Macfarlane RG. Formation of prothrombin converting activity. Nature 1968; 215: 246-251
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar