Synthetic Heparin Pentasaccharide Depolymerization by Heparinase 1: Molecular and Biological Implications
- 1 January 2001
- journal article
- Published by SAGE Publications in Clinical and Applied Thrombosis/hemostasis
- Vol. 7 (1) , 58-64
- https://doi.org/10.1177/107602960100700112
Abstract
A synthetic pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 lU/mg> and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin pentasaccharides are resistant to the digestive effects of heparinase 1. To investigate the effect of heparinase I on the AXa activity of pentasaccharide SR90107/ORG31540. graded concentrations (1.25-100 μg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was eniployed to determine the potential breakdown products of the pentasaccharide. The experiment with the pentasaccharide included incubation (37°C) at mg/ml and exposure to graded concentrations of heparinase I (0.125-1 U/ml). After 30 min of incubation, the enzymatic activity was stopped by heat treatment and the mixture was analyzed using high performance size exclusion chromatography (HPSEC). Heparinase I concentration-dependent cleavage of the pentasaccharide was evident. The breakdown products exhibited a mass of 1,034 d and 743 d, respectively, suggesting the generation of a trisaccharide and a disaccharide moiety. The extinction of a disaccharide moiety in the UV region was high, indicating the presence of a double bond in this molecule. These data clearly, suggest that pentasaccharide SR90107/ORG31540 is digestible by heparinase I into its two components. Furthermore, these data support the hypothesis that heparinase I can be used as a neutralizing agent for pentasaccharide overdose. Additionally, a highly methylated analog of the previously mentioned synthetic pentasaccharide, SanOrg34006, which has also been subjected to similar experiments, has shown complete resistance to the depolymerizing function of heparinase I; therefore, its use may be appropriate in chronic situations as a long-acting form of the pentasaccharide.Keywords
This publication has 19 references indexed in Scilit:
- The Effects of Heparin, Protamine, and Heparinase 1 on Platelets in vitro Using Whole Blood Flow CytometryAnesthesia & Analgesia, 2000
- Allergy to protamine sulfateHeart & Lung, 1999
- Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic PentasaccharideBlood, 1998
- The Effects of Heparinase 1 and Protamine on Platelet ReactivityAnesthesiology, 1997
- Synthesis and Pharmacological Properties of a Close Analogue of an Antithrombotic Pentasaccharide (SR 90107A/ORG 31540)Journal of Medicinal Chemistry, 1997
- A Comparative Analysis of the Primary Sequences and Characteristics of Heparinases I, II, and III fromFlavobacterium heparinumBiochemical and Biophysical Research Communications, 1996
- Heparinase I from Flavobacterium heparinum: MAPPING AND CHARACTERIZATION OF THE HEPARIN BINDING DOMAINPublished by Elsevier ,1996
- Heparinase I from Flavobacterium heparinum. Identification of a Critical Histidine Residue Essential for Catalysis As Probed by Chemical Modification and Site-Directed MutagenesisBiochemistry, 1996
- Molecular Profiling and Weight Determination of Heparins and Depolymerized HeparinsJournal of Pharmaceutical Sciences, 1995
- Heparinase inhibitsneovascularization.Proceedings of the National Academy of Sciences, 1994