The Binding of Low-Affinity and High-Affinity Heparin to Antithrombin. Fluorescence Studies

Abstract

The interaction between [human and bovine] antithrombin and 2 forms of heparin, differing in their affinity for the matrix-linked protein was studied by fluorescence. The binding of the high-affinity heparin fraction to antithrombin led to activation of the inhibitor, allowing it to react more rapidly with a number of serine proteases of the coagulation cascade. The interaction with the low-affinity heparin fraction had considerably less influence on this inhibition rate. The binding of either fraction to antithrombin resulted in an increase of the tryptophan fluorescence of the protein. This increase was much larger for high-affinity heparin than for low-affinity heparin, suggesting a different mode of binding of the 2 fractions to the protein. The fluorescence enhancement caused by high-affinity heparin is consistent with a conformational change of antithrombin related to its activation. Only the fluorescence enhancement observed on the binding of high-affinity heparin was of a sufficient magnitude to allow quantitative studies. These showed high-affinity heparin to bind to antithrombin with a stoichiometry of about 1 and with a binding constant at physiological ionic strength of about 8 .times. 107 M-1. At higher ionic strengths, the affinity decreased markedly.