Effect of Pentosan Polysulphate Administration in Man on the Formation of Covalent Complexes Between Heparin Cofactor II and Thrombin Generated in Plasma by Contact Activation

Summary

An assay for the quantification of pentosan polysulphate (PPS) in plasma is described. As PPS has been shown to potentiate thrombin inhibition by the second heparin cofactor (HC II), the principle of this assay was to measure the formation of covalent complexes between HC II and the thrombin generated in plasma after contact activation and recalcification. The complexes were quantified by using purified ¹²⁵I-HC II added to the plasma as a tracer and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The assay was sensitive to low PPS concentrations (limit of detection: 0.1 μg/ml) and therefore suitable for the measurement of PPS in plasma after its administration to man.

The clearance of PPS was studied in 3 subjects receiving respectively 10, 50 and 100 mg intravenously (IV) and in 3 subjects receiving 35 mg subcutaneously (SC). PPS was still detectable 8 h after 50 and 100 mg IV and 6 h after 35 mg SC. The activated partial thromboplastin time (APTT) was, in comparison, relatively insensitive but for concentrations above 1 μg/ ml the values derived from the APTT and from the SDS-PAGE method fitted. The results were also in general agreement with those reported by McGregor et al. (5) who used a sensitive competitive binding assay. This indicates that low concentrations of PPS previously measured chemically are also pharmacologically active in plasma.

Keywords

Pentosan polysulphate - Heparin cofactor II - Anticoagulant

• References

• 1 Fischer AM, Barrowcliffe TW, Thomas DP. A comparison of pentosan polysulphate (SP 54) and heparin I: mechanism of action on blood coagulation. Thromb Haemostas 1982; 47: 104-108
• 2 Scully MF, Weerasinghe KM, Ellis V, Djazaeri B, Kakkar VV. Anticoagulant and antiheparin activities of a pentosan polysulphate. Thromb Res 1983; 31: 87-97
• 3 Scully MF, Kakkar VV. Identification of heparin cofactor II as the principal plasma cofactor for the antithrombin activity of pentosan polysulphate (SP 54). Thromb Res 1984; 36: 187-194
• 4 Tollefsen DM, Blank MK. Detection of a new heparin-dependent inhibitor of thrombin in human plasma. J Clin Invest 1981; 68: 589-596
• 5 MacGregor IR, Dawes J, Pepper DS, Prowse CV, Stocks J. Metabolism of sodium pentosan polysulphate in man measured by a new competitive binding assay for sulphated polysaccharides - comparison with effects upon anticoagulant activity, lipolysis and platelet alpha-granule proteins. Thromb Haemostas 1985; 53: 411-414
• 6 Sié P, Dupouy D, Pichon J, Boneu B. Turnover study of heparin cofactor II in healthy man. Thromb Haemostas 1985; 54: 635-638
• 7 Sié P, Dupouy D, Pichon J, Boneu B. Constitutional heparin cofactor II deficiency associated with recurrent thrombosis. Lancet 1985; 2: 414-416
• 8 Fischer AM, Merton RE, Marsh NA, Williams S, Gaffney PJ, Barrowcliffe TW, Thomas DP. A comparison of pentosan polysulphate and heparin II: effects of subcutaneous injection. Thromb Haemostas 1982; 47: 109-113
• 9 Vinazzer H, Haas S, Stemberger A. Influence on the clotting mechanism of sodium pentosan polysulphate (SP 54) in comparison to commercial beef lung sodium heparin. Thromb Res 1980; 20: 57-68
• 10 Harpel PC, Rosenberg RD. Alpha-2 macroglobulin and antithrom-bin-heparin cofactor: modulators of hemostatic and inflammatory reactions. In: Progress in Haemostasis and Thrombosis vol. 3 Spaet ThH. (ed) New York: 1976: 145-189
• 11 Tollefsen DM, Majerus DW, Blank MK. Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J Biol Chem 1982; 257: 2162-2169
• 12 Andersson TR, Larsen ML, Handeland GF, Abildgaard U. Heparin cofactor II activity in plasma: application of an automated assay method to the study of a normal adult population. Scand J Haematol 1986; 36: 96-102
• 13 Kakkar VV. Pentosan polysulfate in prevention of postoperative deep vein thrombosis. 1st International Symposium on Pentosan Polysulfate. Paris, June 8 1985
• 14 Sié P, Ofosu F, Fernandez F, Buchanan MR, Petitou M, Boneu B. Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulphated polysaccharides. Brit J Haematol(in press).
• 15 Wagenvoord R, Hendrix H, Soria C, Hemker HC. Determination of the non-antithrombin III dependent inhibitors sites of pentosane polysulphate in the blood coagulation. 1st International Symposium on Pentosan Polysulfate. Paris, June 8 1985