Turnover And Anticoagulant Properties Of Heparin-Antithrombin III Complexes, Stabilized By Covalent Bonds

High affinity heparin obtained by chromatography of clinical grade heparin on antithrombin III-Sepharose was covalently coupled to antithrombin III using a three step procedure: 1) introduction of free amino groups in the heparin molecule; 2) reaction of these amino groups with the difunctional reagent tolylene-2,4-diisothiocyanate; and 3) reaction of the remaining isothiocyanate group with amino groups of antithrombin III. Amino groups were introduced in the heparin molecule by limited N-desulfation or by reaction of carboxyl groups with hexamethylenediamine with the use of a water soluble carbodiimide. Between 1 and 4 moles of NH2 groups were introduced per 15,000 g heparin and the yield of the coupling procedures was 25 to 30 percent. The complexes could be separated from free active heparin by chromatography on antithrombin III-Sepharose and from unreacted antithrombin III by gel filtration on a high performance liquid chromatography column. Coupling occurred for 80 percent with an apparent 1:1 stoichiometry. The specific anticoagulant activity of the complexes (expressed per mg heparin) was approximately 75 percent of that of modified heparin and approximately 50 percent of the original high affinity heparin. The half-life of these complexes in blood following intravenous injection of about 100 heparin equivalent units in rabbits was 0.68 ± 0.08 hours for the N-desulfated heparin-antithrombin III complex and 0.99 ± 0.27 hours for the hexamethylenediamine substituted heparin-antithrombin III complex which is 2.4 and 3.5 times longer than the half-life of free heparin in the blood. This finding indicates that the main mechanism of disappearance of the anticoagulant activity following intravenous injection of heparin is by removal of free heparin and dissociation of the heparin-antithrombin III complex and not by clearing of the intact complex.