Anticoagulant and Antiprotease Profiles of a Novel Natural Heparinomimetic Mannopentaose Phosphate Sulfate (PI-88)

Abstract

Heparinomimetic mannopentaose phosphate sulfate (PI-88) (Progen Industries Ltd. Brisbane, Australia), currently developed as an anticoagulant and antiproliferative agent, mainly is composed of a pentomannan. However, tetrasaccharide and disaccharide components are also present. The molecular profile and the anticoagulant potency of PI-88 are investigated in this study. Gel permeation chromatography and polyacrylamide gel electrophoresis analyses were carried out to determine the molecular profile and separation of components of PI-88, respectively. Potentiation of antithrombin III (ATIII) and heparin cofactor-II (HC-11) activity were measured using chromogenic substrate assay. In order to determine anticoagulant and antiprcatease effects of PI-88, various global anticoagulant tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Hep-test@ (Haemachem Inc., St. Louis), ecarin clotting time (ECT), activated clotting time (ACT), and thromboelastography (TEG) were used. Anti-Xa and anti-IIa activities also were measured. The effect of PI-88 on the release of tissue factor pathway inhibitor (TFPI) was performed in nonhuman primates who received PI-88 and in endothelial cell culture systems. The relative susceptibility of PI-88 to heparinase 1, protamine sulfate (PS), and platelet factor 4 (PF4) also was evaluated. The high-performance liquid chromatography profiles of PI-88 showed that its average molecular weight is approximately 2300 Da. Separation and gradient electrophoretic . patterns of PI-88 showed that it is composed of five different fractions. This agent activates HC-II through inhibiting the thrombin generation but not inhibiting ATIII. Although PI-88 produced a concentration-dependent prolongation of all of the clotting tests, ECT gave the best correlation in the dose-response curve (ECT, r² = 0.94; TT, r² = 0.84; APTT, r² = 0.69). Heparinomimetic mannopentaose phosphate sulfate (PI-88) exhibited marked inhibition of FIIa, but not of FXa. Heparinase I failed to produce significant neutralization of PI-88 in all the assays used, whereas PS and PF4 partially neutralized the effects of this compound. Heparinomimetic mannopentaose phosphate sulfate (PI-88) produced fivefold increase in the TFPI levels at 15 minutes after intravenous (IV) injection to primates. The incubation of PI-88 in endothelial cell culture system also showed a strong effect on TFPI release. These results suggest that PI-88 exhibited strong antithrombotic and anticoagulant activity in addition to its known antiproliferative properties. Because of the molecular characteristics and the dual nature of the pharmacokgic action of PI-88., this agent represents an attractive pharmacotogic agent for the control of thrombotic and proliflerative disorders.

Keywords

Anticoagulant agent Antithrombotic agent Heparinase digestion Low-molecular-weight-heptrin Molecular profile Pentasaccharide PI-88 (heparinomimetic mannopentaose phosphate sulfate)Unfractionated heparin.

References

Fareed J. , Hoppensteadt DA , Bick RL An update on heparins at the beginning of the new millennium. Semin Thromb Hemost 2000;26(suppl 1):5.

Petitou M. , Herault JP , Bernat A. , et al. Synthesis of thrombin inhibiting heparin mimetics without side effects. Nature 1999;398: 417.

Choay J. , Lormeau JC , Petitou M. , et al. Structural study on a biologically active hexasaccharide obtained from heparin. Ann N Y Acad Sci 1981,370: 640.

Pierre S. Sugars slide into heparin activity. Nature 1999;398:378.

Walenga JM , Jeske WP , Bara L. , et al, Biochemical and pharmacological rationale for the development of a synthetic heparin pentasaccharide. Thromb Res 1997 86:1.

Turpie AG A dose ranging study of a novel synthetic pentasaccharide (Org3)540/SR90t07A) in the prevention of venous thromboembolism . after total hip replacement . hit J Hematol 2000; 72(suppl 1):54.

Parolis Las , Duus JQ , Parolis H. , et al. The extracellular polysaccharide of Pichia (Hansentila) holstii NRRL Y-2448: the structure of the phosphomannan backbone. Carbohydr Res 1996:293: 10 1.

McGarry M. The effect of the heparanase inhibitor PI-88 on neointimal formation following acute arterial injury [Ph.D. thesis]: Center for Research in Vascular Biology . Brisbane: University of Queensland ; 1999.

Parish CR Freeman C. , Brown KJ , et al. Identification of sulfated oligosaccharide-based inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity. Cancer Res 1999;59:3433.

10.

Daud AN Ahsan A. , Iqbal O. , et al. Synthetic heparin pentasaccharide depotymerization by heparinase I: molecular and biological implications. Clin Appl Thromb Hemost 2001;7:58--64.

11.

Yu G. , Le Brun L. , Guny NS , et al. Purification of synthetic pentasaccharide-derived oligosaccharide and its kinetics study on Heparinase I. Thromb Res 2000;100:549-556.

12.

Kaiser B. , Fareed J. , Hoppensteadt D. , et al. of recombinant hirudin and unfractionated heparin on thrombin and factor Xa generation in extrinsic and intrinsic activated systems. Thromb Res 1992; 65:157.

13.

Jeske WP , Fareed J. Antithrombin III- and heparin cofactor IImediated anticoagulant and antiprotease actions of heparin and its synthetic analogues. Semin Thromb Hemost 1993; 19(suppl 1):241.

14.

Iqbal O. , Daud AN , Kirschmaier B. , et al. Hepainase neutralization of low molecular weight heparins. Turk J Haematol 2000; 17(suppl): 102.

15.

Ahmad S. , Yang LH , Ahsan A. , et al. Pharmacokinetics of argatroban in primates: evidence on endogenous uptake. Int Angiol 2000; 19:126.

16.

Fareed J. , Kumar A. , Rock A. , et al. A primate model (Macaca mulatta) to study the pharmacokinetics of heparin and its fractions. Semin Thromb Hemost 1985; 11: 138.

17.

Fareed J. , Hoppensteadt DA Tissue factor pathway inhibitor as an endogenous modulator of thrombogenesis . Nova Acta Leopoldina NF 1 999;80: 105 .

18.

Van Deerlin V. , Tollefsen DM Molecular interactions between heparin cofactor II and thrombin. Semin Thromb Hemost 1992;18: 341.

19.

Brown KJ Maynes SF Bexos A. , et al. A novel in vitro assay for human arigiogenesis. Lab Invest 1996;75:539.

20.

Guimond S. , Maccarana M. , Olwin BB , et al. Activating and inhibitory heparin sequences for FGF-2 (Basic FGF): distinct requirements for FGF-1, FGF-2 and FGF-4. J Biol Chem 1993:268: 23906.

21.

Broze G. The role of tissue factor pathway inhibitor in a revised coagulation cascade . Semin Hematol 1992:29:159.

22.

Hoppensteadt DA Walenga JM , Fasanella A. , et al. TFPI antigen levels in normal human volunteers after IV and SC administration of heparin and low molecular weight heparin. Thromb Res 1994; 77:175.

23.

Kaiser B. , Hoppensteadt D. , Fareed J.

Tissue factor pathway inhibitor for cardiovascular disorders. Emerging

Drugs 2000:5:73.