PubMed · 8753177
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
N Sakuragawa. 1995. [Thrombin antithrombin III complex (TAT)].. https://pubmed.ncbi.nlm.nih.gov/8753177/
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A-beta-sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P1 Arg of alpha-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P1 His variant, antithrombin Glasgow, is unaffected, indicating that only the P1 Arg becomes accessible. Furthermore, the deimination of P1 Arg converts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P1 His variant, due to a lowered dissociation rate constant for the antithrombin-pentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this study by perturbation of the bonding of P1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibitory conformation with exposure of the P1 Arg.
Exosite I of the blood clotting proteinase, thrombin, mediates interactions of the enzyme with certain inhibitors, physiological substrates and regulatory proteins. Specific binding of a fluorescein-labeled derivative of the COOH-terminal dodecapeptide of hirudin ([5F] Hir54-65) to exosite I was used to probe changes in the function of the regulatory site accompanying inactivation of thrombin by its physiological serpin inhibitor, antithrombin. Fluorescence-monitored equilibrium binding studies showed that [5F]Hir54-65 and Hir54-65 bound to human alpha-thrombin with dissociation constants of 26 +/- 2 nM and 38 +/- 5 nM, respectively, while the affinity of the peptides for the stable thrombin-antithrombin complex was undetectable (>/=200-fold weaker). Kinetic studies showed that the loss of binding sites for [5F]Hir54-65 occurred with the same time-course as the loss of thrombin catalytic activity. Binding of [5F] Hir54-65 and Hir54-65 to thrombin was correlated quantitatively with partial inhibition of the rate of the thrombin-antithrombin reaction, maximally decreasing the bimolecular rate constants 1.7- and 2.1-fold, respectively. These results support a mechanism in which thrombin and the thrombin-Hir54-65 complex can associate with antithrombin and undergo formation of the covalent thrombin-antithrombin complex at modestly different rates, with inactivation of exosite I leading to dissociation of the peptide occurring subsequent to the rate-limiting inactivation of thrombin. This mechanism may function physiologically in localizing the activity of thrombin by allowing inactivation of thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the thrombin-antithrombin complex may be irreversibly released due to exosite I inactivation.
The beta-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated alpha-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of alpha- and beta-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for alpha-antithrombin than for beta-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrease in heparin binding affinity.