The Effect of the Synthetic Pentasaccharide SR 90107/ORG 31540 on Thrombin Generation Ex Vivo Is Uniquely due to ATIII-Mediated Neutralization of Factor Xa

Summary

The inhibition of thrombin generation (TG) was studied in plasma from human volunteers after single subcutaneous administrations of 4000, 8000 or 12,000 anti-Xa units (i.e., 6, 12 or 18 mg) of the synthetic pentasaccharide (SR 90107/ORG 31540) (SP).

SP impaired TG in plasma for up to 18 h after injection, and the time-courses of TG and factor Xa inhibitions were similar.

In untreated plasma supplemented in vitro with SP to obtain the same anti-Xa activity as in ex vivo samples, equivalent TG inhibitions were observed thus showing that no transformed SP molecules were involved in the TG inhibition ex vivo.

Functional as well as immunological assay of TFPI indicated that subcutaneous injection of 12,000 anti-Xa units of SP did not induce any TFPI release, whereas under the same conditions, 13,000 IU of Fraxiparine® produced a significant rise of TFPI in plasma.

The plotting of TG inhibition versus SP concentration could be fitted with a good correlation (r = 0,94) to the graphical representation linking [ATIII-SP] to [SP].

These results demonstrate that following subcutaneous administration to man, SP inhibits TG ex vivo and likely in vivo exclusively through the same selective ATIII-mediated inhibition of factor Xa as the one elicited in vitro.

• References

• 1 Barrowcliffe TW, Thomas DP. Heparin and Low Molecular Weight Heparin. In: Haemostasis and Thrombosis. Bloom AL, Forbes CD, Thomas DP, Tuddenham EG D. eds. Churchill Livingstone; Edinburgh, London, Madrid, Melbourne, New York and Tokyo: 1994. pp 1417-1438
• 2 Choay J, Petitou M, Lormeau JC, Sinay P, Casu BJ, 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
• 3 Béguin S, Choay J, Hemker HC. The action of a synthetic pentasaccharide on thrombin generation in whole plasma. Thromb Haemost 1989; 6: 397-401
• 4 Lormeau JC, Herault JP. Comparative inhibition of extrinsic and intrinsic thrombin generation by standard heparin, a low molecular weight heparin and the synthetic ATIII-binding pentasaccharide. Thromb Haemost 1993; 69: 152-156
• 5 Walenga JM, Fareed J, Petitou M, Samama M, Lormeau JC, Choay J. Intravenous antithrombotic activity of a synthetic heparin pentasaccharide in a human serum induced stasis thrombosis model. Thromb Res 1986; 43: 243-248
• 6 Walenga JM, Petitou M, Lormeau JC, Samama M, Fareed J, Choay J. Antithrombotic activity of a synthetic heparin pentasaccharide in a rabbit stasis thrombosis model using different thrombogenic challenges. Thromb Res 1987; 46: 187-198
• 7 Hobbelen PM J, Van Dinther TG, Vogel GM, Van Boeckcl CA A, Moelker HC T, Meuleman DG. Pharmacological profile of the chemically synthetized antithrombin III binding fragment of heparin (pentasaccharide) in rats. Thromb Haemost 1990; 63: 266-270
• 8 Amar J, Caranobe C, Sié P, Boneu B. Antithrombotic potencies of heparins in relation to their antifactor Xa and antithrombin activities: an experimental study in two models of thrombosis in the rabbit. Hr J Haematol 1990; 76: 94-100
• 9 Boneu B, Necciari J, Magnani H, Kieffer G, Mant T, Morrison P, Binger B, Lamond G, Dickinson J. Pharmacokinetics of the natural pentasaccharide (SR 90107A/ORG 31540) with high affinity for antithrombin III following subcutaneous administration in man. Thromb Haemost 1995; 69: 406 abstract
• 10 Boneu B, Necciari J, Cariou R, Gabaig AM, Kieffer G, Dickinson J, Lamond G, Moelker H, Mant T, Magnani H. Pharmacokinetics and tolerance of the natural pentasaccharide (SR 90107 A/ORG 31540) with high affinity to antithrombin III. (submitted for publication)
• 11 Hemker BC, Willems GM, Béguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemost 1986; 56: 9-17
• 12 Sandset PM, Abildgaard U, Pettersen M. A sensitive assay of extrinsic coagulation pathway inhibitor (EP1) in plasma and plasma fractions. Thromb Res 1987; 47: 389-400
• 13 Crepon B, Donat F, Barzu T, Hérault JP. Pharmacokinetic parameters of ATIII-binding pentasaccharides in three animal species: Predictive value for humans. Thromb Haemost 1993; 69: 403 abstract
• 14 Barrowcliffe TW, Johnson EA, Thomas D. Antithrombin III and heparin. Br Med Bull 1978; 34: 143-150
• 15 Valentin S, Ostergaard P, Kristensen H, Nordfang O. Synergism between full length TFP1 and heparin: evidence for TFPI as an important factor for the antithrombotic activity of heparin. Blood Coag Fibrinol 1992; 3: 221-222
• 16 Novotny WF, Brown S, Miletich JP, Rader DJ, Broze Jr GJ. Plasma antigen levels of the lipoprotein-associated coagulation inhibitor in patient samples. Blood 1991; 78: 387-393
• 17 Lojewski B, Bacher P, Jeske W, Fareed J. Molecular weight dependence on the release of tissue factor pathway inhibitor after subcutaneous and intravenous actions of heparins. Thromb Haemost 1993; 69: 2263 abstract
• 18 Novotny WF, Maffi T, Mehta RL, Milner PG. Identification of novel heparin-releasable proteins, as well as the cytokines midkine and pleiotrophin, in human postheparin plasma. Arterioscler Thromb 1993; 13: 1798-1805
• 19 Ellis V, Scully MF, Kakkar VV. The relative mass dependence of the anti-factor Xa properties of heparin. Biochem J 1986; 238: 329-333
• 20 Hemker HC, Choay J, Beguin S. Free factor Xa is on the main pathway of thrombin generation in clotting plasma. Biochem Biophys Acta 1989; 992: 409-411
• 21 Jesty J. Analysis of the generation and inhibition of activated factor X in pure system and in human plasma. J Biol Chem 1986; 261: 5695-5702
• 22 Messmore HL, Griffin B, Fareed J, Coyne E, Seghatchian J. In vitro studies of the interaction of heparin, low molecular weight heparin and heparinoids with platelets. Ann NY Acad Sci 1989; 556: 217-231