Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin.
The stoichiometry of antithrombin III (AT) inhibition of alpha-thrombin (T) has been investigated in the presence and absence of heparin as a function of ionic strength by quantitative titration of enzyme active sites. In contrast to the ionic strength-independent stoichiometry of 1.0 mol of AT/mol of T observed in the absence of heparin, the presence of high-affinity heparin (HAH) resulted in an ionic strength-dependent increase in the apparent stoichiometry of inhibition from a molar ratio of 1.1 AT/T at an ionic strength of 0.3 to 9.8 mol of AT/T when the ionic strength was lowered to 0.01. Reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reaction products revealed that the increased AT/T stoichiometry was due to preferential formation of a specific proteolytically cleaved form of AT that was indistinguishable from the previously characterized reactive site-cleaved AT (ATM). Using high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to quantitate ATM, the cleaved inhibitor was shown to be formed rapidly and concomitant with the stable thrombin-antithrombin complex (TAT) and quantitatively accounted for the apparent increase in reaction stoichiometry at low ionic strength in the presence of HAH. The levels of HAH required to produce maximum ATM were catalytic at mu greater than or equal to 0.15, but became stoichiometric as the ionic strength decreased below 0.1. Substantially less ATM was produced in the presence of low-affinity heparin, while a low molecular weight HAH, virtually inactive in accelerating T inhibition by AT, was unable to promote significant ATM formation. These results indicate competition between substrate and inhibition reactions of AT with T which are affected by an ionic strength-dependent heparin interaction. A reaction mechanism accounting for these observations is proposed.
D000990:Antithrombin III / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009994:Osmolar Concentration; D011485:Protein Binding; D012995:Solubility; D013917:Thrombin / Q000378:metabolism*
D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin