The Activity of Unfractionated Heparin and Low Molecular Weight Heparins in Rabbit Plasma – The Need for Using Absolute Anti-factor Xa and Antithrombin Activities

V Peyrou; S Béguin; B Boneu; H C Hemker

doi:10.1055/s-0038-1655960

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1997; 77(02): 312-316
DOI: 10.1055/s-0038-1655960

Original Article

Schattauer GmbH Stuttgart

The Activity of Unfractionated Heparin and Low Molecular Weight Heparins in Rabbit Plasma – The Need for Using Absolute Anti-factor Xa and Antithrombin Activities

V Peyrou

¹Department of Biochemistry, Cardiovascular Research Institute and University of Limburg, Maastricht, The Netherlands

²Laboratoire de Recherche sur I’Hémostase et la Thrombose, Pavilion Charles Lefèbvre, Hôpital Purpan, Toulouse, France

,

S Béguin

¹Department of Biochemistry, Cardiovascular Research Institute and University of Limburg, Maastricht, The Netherlands

,

B Boneu

²Laboratoire de Recherche sur I’Hémostase et la Thrombose, Pavilion Charles Lefèbvre, Hôpital Purpan, Toulouse, France

,

H C Hemker

¹Department of Biochemistry, Cardiovascular Research Institute and University of Limburg, Maastricht, The Netherlands

› Institutsangaben

Abstract

als PDF herunterladen

Summary

Rabbit being a common animal model to evaluate the antithrombotic effect of heparins, our purpose was to apply the heparin Standard Independent Unit (SIU) approach to rabbit plasma. To take into account the specificities of the enzymes we have measured the decay constants of factor Xa and of thrombin from autologous and heterologous origins, in presence and in absence of heparin. Different heparins or heparin fractions with a mean molecular weight from 1.7 to 10.5 kDa were used.

We found that: a) the decay constants varied strongly between species and between enzymes; b) the decay constants of thrombin were always higher than those of factor Xa; c) the specific anti-factor Xa activity of heparins increased with the molecular weight and was 1.33 times higher when determined with bovine factor Xa than with rabbit factor Xa; d) the specific antithrombin activity of heparins also increased with the molecular weight but was similar when determined with rabbit and human thrombin; e) the specific anti-factor Xa activity was always lower than the specific antithrombin activity; f) the calibration of the heparins and heparin fractions against the 4th International Standard of Heparin expressed in International Units (IU) lead to a systematic overestimation of the anti-factor Xa activity and to an underestimation of the antithrombin activity.

These observations indicate that it may be very important to use the SIU approach and to know the accurate activities to better understand the mechanism of the antithrombotic activity of heparins in experimental models.

PDF (375 kb)

Referenzen

References
1 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
2 Rosenberg RD. Actions and interactions of antithrombin and heparin. N Engl J Med 1975; 292: 146-151
3 Choay J, Petitou M, Lormeau JC, Synaï P, Casu B, Gatti G. Structure activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. Biochem Biophys Res Comm 1983; 116: 492-499
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 Hemker HC, Béguin S. Standard and method independent units for heparin anticoagulant activities. Thromb Haemost 1993; 70: 724-728
6 Barrowcliffe TW, Le ShirleyY. The effect of calcium chloride on anti-Xa activity of heparin and its molecular weight fractions. Thromb Haemost 1989; 62: 950-954
7 Béguin S, Wielders S, Lormeau JC, Hemker HC. The mode of action of CY 216 and CY 222 in plasma. Thromb Haemost 1992; 67: 33-41
8 Peyrou V, Lormeau JC, Caranobe C, Gabaig AM, Crepon B, Saivin S, Houin G, Sié P, Boneu B. Pharmacological properties of CY 216 and of its ACLM and BCLM components in the rabbit. Thromb Haemost 1994; 72: 268-274
9 Briant L, Caranobe C, Saivin S, Sié P, Bayrou B, Houin G, Boneu B. Unfractionated heparin and CY 216: pharmacokinetics and bioavailabilities of the antifactor Xa and Ha effects after intravenous and subcutaneous injection in the rabbit. Thromb Haemost 1989; 61: 348-353
10 Josso F, Prou-Wartelle O. Exploration de l’hémostase. In: Techniques en hématologie. Alagille D. et al., Paris: Flammarion; eds. 1972. pp 101-108
11 Esnouf MP, Lloyd PH, Jesty J. A method for the simultaneous isolation of factor Xa and prothrombin from bovine plasma. Biochem J 1973; 131: 781-789
12 Weinstein RE, Ricklos FR, Walker FJ. Purification and preliminary characterization of rabbit vitamin K-dependent coagulation proteins. Thromb Res 1990; 59: 759-772
13 Béguin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemost 1988; 60: 457-462
14 Béguin S, Kessels H, Dol F, Hemker HC. The consumption of antithrombin III during coagulation, its consequences for the calculation of prothrombinase activity and the standardisation of heparin activity. Thromb Haemost 1992; 68: 136-142
15 Carrié D, Caranobe C, Boneu B. A comparison of the antithrombotic effects of heparin and of low molecular weight heparins with increasing anti-factor Xa/anti-factor Ha ratio in the rabbit. Br J Haematol 1993; 83: 622-626