Abstract
Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants.
Highlights
Thrombotic disorders, such as venous thromboembolism (VTE), stroke, myocardial infraction and other indications, constitute a major health burden for most countries
Which is located ~16–24 Å from the enzyme active site. This exosite bears no homology with thrombin’s exosite 2, which interacts with heparin (Fig 2C and 2D) [40], it may potentially serve as an allosteric site to which the sulfated GAGs and/or non-saccharide mimetics of GAGs may bind and inhibit Factor XIIIa (FXIIIa)
These sites differ in terms of their hydrophobic sub-sites [41,42,43], which may serve as recognition elements for sulfated nonsaccharide GAG mimetics (NSGMs), as demonstrated earlier [37, 38, 44,45,46]
Summary
Thrombotic disorders, such as venous thromboembolism (VTE), stroke, myocardial infraction and other indications, constitute a major health burden for most countries. Non-Saccharide Glycosaminoglycan Mimetics Inhibit Factor XIIIa current repertoire of antithrombotics used in the clinic, a large number of patients develop recurrent VTE and/or suffer from major long-term complications [1,2,3]. A range of anticoagulants is used in the clinic today to treat and prevent episodes of thrombosis including the antithrombin-based agents (heparins), vitamin K-based agents (coumarins), and the newer direct thrombin or factor Xa inhibitors [5]. Despite their success, each agent carries a number of drawbacks, of which a common risk is internal bleeding, which can be life-threatening [6]. Approaches that seek completely different routes of engineering anticoagulant activity, with a promise of reducing bleeding complications, are important to develop
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
