Abstract
The role of the sequence-specific pentasaccharide region of high affinity heparin (HAH) in heparin acceleration of antithrombin-proteinase reactions was elucidated by determining the accelerating mechanism of low affinity heparin (LAH) lacking this sequence. LAH was shown to be free of HAH (< 0.001%) from the lack of exchange of added fluorescein-labeled HAH into LAH after separating the polysaccharides by antithrombin-agarose chromatography. Fluorescence titrations showed that LAH bound to antithrombin with a 1000-fold weaker affinity (KD 19 +/- 6 microM) and 5-6-fold smaller fluorescence enhancement (8 +/- 3%) than HAH. LAH accelerated the antithrombin-thrombin reaction with a bell-shaped dependence on heparin concentration resembling that of HAH, but with the bell-shaped curve shifted to approximately 100-fold higher polysaccharide concentrations and with a approximately 100-fold reduced maximal accelerating effect. Rapid kinetic studies indicated these differences arose from a reverse order of assembly of an intermediate heparin-thrombin-antithrombin ternary complex and diminished ability of LAH to bridge antithrombin and thrombin in this complex, as compared to HAH. By contrast, LAH and HAH both accelerated the antithrombin-factor Xa reaction with a simple saturable dependence on heparin or inhibitor concentrations which paralleled the formation of an antithrombin-heparin binary complex. The maximal accelerations of the two heparins in this case correlated with the inhibitor fluorescence enhancements induced by the polysaccharides, consistent with the accelerations arising from conformational activation of antithrombin. 1H NMR difference spectroscopy of antithrombin complexes with LAH and HAH and competitive binding studies were consistent with LAH accelerating activity being mediated by binding to the same site on the inhibitor as HAH. These results demonstrate that LAH accelerates antithrombin-proteinase reactions by bridging and conformational activation mechanisms similar to those of HAH, with the reduced magnitude of LAH accelerations resulting both from a decreased antithrombin affinity and the inability to induce a full activating conformational change in the inhibitor.
Highlights
From the HCenter for Molecular Biology of Oral Diseases and IlDepartment of Biochemistry, University of IllinoisChicago, Chicago, Illinois 60612, Ulenry Ford Hospital, Division of Biochemical Research, Detroit, Michigan 48202, Wepartment of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsola, Sweden, and **Sanofi Recherche, Gentilly Cedex, France
To determine whether this activity was an intrinsic property oflow affinity heparin or was due to small amounts of residual high affinity heparin, 30 /Lg of fluoresceinlabeled high affinity heparin were added to 3 mg of low affinity heparin, and the exchange oflabeled polysaccharide into unlabeled polysaccharide was measured after separating the polysaccharide mixture by antithrombin affinity chromatography
Several pieces of evidence argue that the acceleration of antithrombin-proteinase reactions by low affinity heparin is an intrinsic property of the polysaccharide and not due to the presence of a small amount of high affinity polysaccharide
Summary
From the HCenter for Molecular Biology of Oral Diseases and IlDepartment of Biochemistry, University of IllinoisChicago, Chicago, Illinois 60612, Ulenry Ford Hospital, Division of Biochemical Research, Detroit, Michigan 48202, Wepartment of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsola, Sweden, and **Sanofi Recherche, Gentilly Cedex, France. Heparin is a naturally occurring glycosaminoglycan which acts as a potent antagonist of blood coagulation This anticoagulant effect of heparin is primarily due to the polysaccharide accelerating the inactivation of blood clotting proteinases by their principal protein inhibitor, antithrombin, several thousandfold 1 and 2) The accelerating activity results from a sequence-specific pentasaccharide region, present in about one-third of heparin polysaccharide chains [3,4,5], which binds antithrombin with high affinity and induces an activating conformational change in the inhibitor [6,7,8,9,10,11]. Heparin rate enhancement of thrombin inhibition appears to be mostly due to the polysaccharide bridging antithrombin and thrombin in a ternary complex in which both the inhibitor and proteinase are bound to the same polysaccharide chain [12, 14,15,16,17,18,19,20,21]
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