Abstract
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.
Highlights
high-affinity hep- (HAH) required to produce maximum ATM were cata- yield reactive site-cleaved antithrombin I11 (AT) when dissociated [17]
Homogeneous with high affnity; Low-affinity heparin (LAH), heparin species that lacks the specific pen- human antithrombin I11 was prepared from expired plasma by hepatasaccharide and binds antithrombin with low affinity; ATM,reactive rin-Sepharose affinity chromatography (Pharmacia) as describedpresite-cleaved antithrombin III; PEG, polyethylene glycol; Hepes, 4-(2- viously [23]
Reduced SDS-PAGE of reaction products formed in the absence of heparin revealed that the covalent thrombin-antithrombin complex (TAT*)was the sole product both at high (0.5) and low (0.025) ionic strength (Fig. IC), consistent with
Summary
Heparin-Porcine intestinal mucosal heparin (Diosynth) was purified by cetylpyridinium chloride (Sigma) precipitation [25], fractionated twice on Sephadex G-100 (Pharmacia) toobtain a narrow molecular weight distribution, and thenaffinity-fractionated with AT coupled to Affi-Gel 15 (Bio-Rad) as described previously [23], except that the NaCl concentration used to elute the high-affinity heparin was 3 M. A constant excess level of AT several times greater than the K d for HAH binding was used Under these conditions, product formation is exponential [27], and the observed rate constant varies linearly with heparin concentration [28]. HAH4m exhibited a bimolecular rate constant of 4.0 X 10' M" s-' while fluorescence titration of AT with HAH4mconfirmed a single high-affinity binding site/molecule [11] This represents a 5-fold enhancement of the inhibition rate constant from that measured in the absence of heparin under these conditions (7.60.5 X lo M" s-I [23])and is consistent with recent data of. Thrombin Inhibition Titrations-Experiments were performed in either 10 mM Hepes or 20 mM NaPi buffers containing 0.1 mM EDTA and 0.1% PEG 6000 (Baker) and varying NaCl concentrations at a final pH of 7.4 and 25 "C. Peak areas were converted to AT and ATMconcentrations using the purified proteins as standards and corrected for an uncomplexed AT contribution no greater than 17% which varied by less than 2% over an AT concentration range between 0.05 and 1 p ~ T.he accuracy of this method was confirmed using mixtures of AT and ATMof known composition
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