Electrostatic interactions in the heparin-enhanced reaction between human thrombin and antithrombin

Abstract

Binding of heparin to thrombin is monitored by means of an aqueous two-phase partition system, and binding of heparin to antithrombin is monitored by means of heparin induced enhancement of the intrinsic fluorescence of the protein. Both types of binding are studied at various electrolyte compositions of the medium. Heparin is displaced from thrombin at lower concentrations of electrolyte than those necessary for its displacement from antithrombin. K+ is more efficient than Na+, which is again more efficient than Li+ in displacing heparin from these proteins. The kinetics of the reaction between thrombin and antithrombin in the presence of heparin were studied by using an assay where synthetic peptide substrate is present in the reaction mixture during the reaction between proteinase and inhibitor. The kinetics are studied at various electrolyte compositions of the medium and the results are compared with those obtained from the binding studies performed under similar conditions. The results are consistent with a model where binding of heparin to antithrombin causes enhancement of the reaction rate, and where this enhancement is abolished again when additional binding of heparin to thrombin takes place on further addition of heparin.

CE frontal analysis based on simultaneous UV and contactless conductivity detection: A general setup for studying noncovalent interactions

“…It appears that the potassium ion, which also has the lowest hydrated radius, seems to have the strongest affinity for dextran sulfate. This finding is in accordance with literature results obtained using the negatively charged polymer heparin [30]. However, there does not seem to be any clear-cut correlation between the charge localization and the degree of interaction.…”

Mechanisms responsible for the failure of protamine to inactivate low‐molecular‐weight heparin

“…This result was not surprising as, unlike factor Xa, thrombin must bind to heparin in order to be neutralized (Andersson et al , 1979). Thus, anti‐IIa neutralization can be accomplished by displacement of thrombin from the heparin [wherethrombin binds heparin molecules with significantly reduced affinity to that of antithrombin (AT) (Petersen & Jorgensen, 1983)]. The sensitivity of UFH and LMWH antifactor Xa activities to protamine did not appear to be owing to the amount of protamine bound per mg of heparin (unfractionated or low molecular weight).…”

Covalent Antithrombin-Heparin Complexes with High Anticoagulant Activity

“…Unlike noncovalent mixtures of AT and heparin, there is no initial binding of serpin and GAG required, which is the rate-determining step (26). In addition, AT may have selected for heparins with higher average anionic density, which would enhance the interactions of thrombin with ATH since its binding is a charge-dependent phenomenon (27). All of the heparin molecules in ATH contain the high affinity AT binding site, and, since the average heparin chain length in the conjugate was greater than the starting heparin, thrombin would have a shorter mean path distance for contacting the inhibitor complex.…”