PubMed · 8639413
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R W Carrell, D J Perry. 1996. Annotation. The unhinged antithrombins.. https://doi.org/10.1046/j.1365-2141.1996.458998.x
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Antithrombin (AT) circulates in two isoforms, alpha- (90-95%) and beta-AT (5-10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin's limitations, we prepared a covalent AT-heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% alpha-AT, and 45% beta-AT) was significantly different than that in the commercial AT starting material (80% alpha-AT and 20% beta-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the beta-variant reacted approximately 2-fold faster.
A series of potent and selective proline- and pyrazinone-based macrocyclic thrombin inhibitors is described. Detailed SAR studies led to the incorporation of specific functional groups in the tether that enhanced functional activity against thrombin and provided exquisite selectivity against trypsin and tPA. X-ray crystallography and molecular modeling studies revealed the inhibitor-enzyme interactions responsible for this selectivity.
Blood coagulation factor IXa has been presumed to be regulated by the serpin, antithrombin, and its polysaccharide activator, heparin, but it has not been clear whether factor IXa is inhibited by the serpin with a specificity comparable to that for thrombin and factor Xa or what determinants govern this specificity. Here we show that antithrombin is essentially unreactive with factor IXa in the absence of heparin (k(ass) approximately 10 M(-1) s(-1)) but undergoes a remarkable approximately 1 million-fold enhancement in reactivity with this proteinase to the physiologically relevant range (k(ass) approximately 10(7) M(-1) s(-1)) when activated by heparin in the presence of physiologic levels of calcium. This rate enhancement is shown to derive from three sources: (i) allosteric activation of antithrombin by a sequence-specific heparin pentasaccharide (300-500-fold), (ii) allosteric activation of factor IXa by calcium ions (4-8-fold), and (iii) heparin bridging of antithrombin and factor IXa augmented by calcium ions (130-1000-fold depending on heparin chain length). Mutagenesis of P6-P3' reactive loop residues of antithrombin further reveals that the reactivity of the unactivated inhibitor is principally determined by the P1 Arg residue, whereas exosites outside the loop which are present on the activated serpin and on heparin are responsible for heparin enhancement of this reactivity. These results together with our previous findings demonstrate that exosites are responsible for the unusual specificity of antithrombin and heparin for three clotting proteases with quite distinct substrate specificities.