Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors (original) (raw)
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Circulation, 1998
Background-Thrombolytic therapy induces a procoagulant state characterized by elevated plasma levels of fibrinopeptide A (FPA), but the responsible mechanism is uncertain. Methods and Results-Washed plasma clots were incubated in citrated plasma in the presence or absence of tissue plasminogen activator (t-PA), and FPA generation was monitored as an index of unopposed thrombin activity. FPA levels are almost twofold higher in the presence of t-PA than in its absence. This primarily reflects the action of thrombin bound to soluble fibrin degradation products because (a) there is progressive FPA generation even after clots are removed from t-PA-containing plasma, and (b) clot lysates produce concentration-dependent FPA generation when incubated in citrated plasma. Using thrombin-agarose affinity chromatography, (DD)E and fragment E but not D-dimer were identified as the thrombin-binding fibrin fragments, indicating that the thrombin-binding site is located within the E domain. Heparin inhibits thrombin bound to fibrin degradation products less effectively than free thrombin. In contrast, D-Phe-Pro-ArgCH 2 Cl, hirudin and hirugen inhibit free thrombin and thrombin bound to fibrin degradation products equally well. Conclusions-Thrombin bound to soluble fibrin degradation products is primarily responsible for the increase in FPA levels that occurs when a clot undergoes t-PA-induced lysis. Like clot-bound thrombin, thrombin bound to fibrin derivatives is protected from inhibition by heparin but susceptible to inactivation by direct thrombin inhibitors. These findings help to explain the superiority of direct thrombin inhibitors over heparin as adjuncts to thrombolytic therapy. (Circulation.
Thrombin generation and inactivation in the presence of antithrombin III and heparin
Biochemistry, 1986
W e have determined the rate constants of inactivation of factor X, and thrombin by antithrombin "heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor X, alone by antithrombin 111 as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X lo6 M-' min-'. Factor X, in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor V, or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 pM), was inhibited by antithrombin I11 with a second-order rate constant that was essentially the same: 1.2 X lo6 M-I min-'. When both factor V, and phospholipid were present during prothrombin activation, factor X, inhibition by antithrombin 111 was reduced about 10-fold, with a second-order rate constant of 1.3 X lo5 M-' min-'. Factor X, in the prothrombin activation mixture that contained both factor V, and phospholipid was even more protected from inhibition by the antithrombin 111-heparin complex. The first-order rate constants of these reactions a t 200 n M antithrombin 111 and normalized to heparin at 1 pg/mL were 0.33 and 9.5 min-' in the presence and absence of factor V, and phospholipid, respectively. When the prothrombin concentration was varied widely around the K,,, for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor X, when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin 111 in the absence as well as in the presence of heparin. The second-order rate constant (4.5 X lo5 M-I min-') of inhibition of thrombin activity generated during prothrombin activation was found to be (1) constant during the time course of prothrombin activation, (2) independent of the composition of the prothrombin converting complex, and (3) about 4-fold lower than the second-order rate constant of inhibition of purified a-thrombin (1.9 X lo6 M-' m i d ) . Surprisingly, heparin hardly enhanced the antithrombin 111 dependent
Mechanism of thrombin inhibition by antithrombin and heparin cofactor II in the presence of heparin
Biomaterials, 1997
The kinetics of thrombin inhibition by antithrombin (AT) and heparin cofactor II (HC II) were analysed as a function of the heparin concentration, from lo-' to 10e4M. The initial concentrations of inhibitor (I) and thrombin (E) were set at equimolar levels (C, = CE = lo-* M). The experimental data indicate that the reaction of thrombin inhibition was second-order both in the absence and in the presence of heparin, and that the apparent rate constant increased at heparin concentrations ranging from lo-' to 10-6~ and decreased at higher concentrations. The data fit with the kinetic model established by Jordan et al. [J. Biol. Chem. 1979, 254, 2902 for the catalysis of the thrombin-AT reaction by a low-molecular-weight heparin fraction. In this model, heparin (H) binds quickly to the inhibitor (I) and forms a heparin-inhibitor complex (HI), which is more reactive than the free inhibitor towards thrombin, leading to the formation of an inactive inhibitor-thrombin complex (I*E) and the release of free heparin, in a second step which is rate limiting. K ,,, ,, the dissociation constant of HI, and k, the second-order rate constant of free thrombin inhibition by HI, were found to be 3.7 x 1O-7 M and 1.3 x 10' M-' min-', respectively, for AT, compared to a K,,, , of 2.0 x 10m6 M and k of 6.4 x 10' M-' min-' for HC II. These data indicate that heparin-HC II complex reactivity is greater than that of the heparin-AT complex towards thrombin, whereas heparin affinity is stronger for AT. At heparin concentrations higher than 10e6 M, the decrease in the reaction rate was in keeping with the formation of a heparin-,thrombin complex (HE), whose inactivation by the heparin-inhibitor complex (HI) is slower than that of .the free protease. 0 1997 Elsevier Science Limited. All rights reserved
Biochemistry and Cell Biology, 1984
- Effects of heparin fractions of different affinities to antithrombin I11 and thrombin on the inactivation of thrombin and factor Xa by antithrombin 111. Cun. 3. Biochem. Cell Biol. 62, To investigate the relative contribution of heparin-binding thrombin and antithrombin I11 to the enhancement of the rate of inactivation of thrombin by antithrombin 111, standard heparin was fractionated on matrix-linked thrombin and (or) antithrombin 111. There was a good correlation between heparin affinity for antithrombin I11 and its ability to enhance the inactivation of thrombin and factor Xa. In addition, there was a good correlation between affinity of heparin for thrombin and its catalytic activity on the inactivation of thrombin by antithrombin 111. Thus fractions with high affinity to thrombin had similar rate-enhancing activity for thrombin inactivation to that of fractions with high affinity to antithrombin 111. Fractions with high affinity to both proteins were more potent than fractions with high affinity to either protein alone. No significant differences in mean molecular weight were observed among the various heparin fractions. A heparin fraction with very low affinity to thrombin and high affinity to antithrombin I11 was prepared by repeated fractionation of a low molecular weight heparin on the two affinity columns. This fraction had very weak rate-enhancing activity for the inactivation of thrombin by antithrombin 111, but retained substantial activity for the inactivation of factor Xa. The results of these studies support the concept that, for both standard and low molecular weight heparin, the enhancement of the inactivation of thrombin by antithrombin I11 requires the interaction of the heparin with both thrombin and antithrombin 111.
Action of heparin on thrombin-antithrombin reaction
Biochimica et biophysica acta, 1975
Thrombin partially purified from bovine plasma can be inactivated at 60 degress C. In the presence of 10 units of heparin the extent of inactivation decreases. When thrombin and heparin are mixed and incubated for 5 min at 0 degrees C before gel filtration on Sephadex G-200, thrombin with heparin is eluted prior to either thrombin or heparin laone. These data suggest a complex formation between thrombin and heparin. Immobilized heparin binds thrombin. The enzyme can be eluted with 0.05 M Tris-HCl buffer, pH 7.3, containing an ion mixture of Na+, K+ and Ca2+ at 73, 3 and 11 mM, respectively, at 0 degrees C and with 0.05 M Tris-HCl buffer, pH 7.3, containing 0.5 M NaCl at 20 degrees C. During the same chromatographic procedure, antithrombin-III (heparin cofactor) partially purified from human plasma is eluted with 0.05 M Tris-HCl buffer, pH 7.3, at 0 degrees C as well as 20 degrees C. Although, as described in the literature, heparin binds to antithrombin, our findings suggest another...
Haematologica, 2002
We evaluated a new test, based on prothrombin activation by Echis Carinatus snake venom in the absence/presence of unfractionated heparin, in patients with venous thromboembolism (VTE). The test (activated prothrombin heparin-inhibition test) was performed in 555 unselected, unrelated patients who had suffered from at least one objectively confirmed VTE and the results were compared with those obtained in 408 healthy controls. In 71 (12.8%) of the 555 patients the results of the test, expressed as a normalized ratio, were below the cut-off. This compared with 19 (5% by definition) results below the cut-off in the control group. The crude OR for VTE in subjects with altered vs those with normal results was 3.00 (95% CI: 1.78-5.07). ORs did not significantly change after adjustment for age (2.86, 95% CI: 1.68-4.85) and age/sex (2.80, 95% CI: 1.64-4.77) by logistic regression. After adjustment for antithrombin III, fibrinogen and prothrombin levels the risk associated with altered resu...