Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin‐activatable fibrinolysis inhibitor activation and activated thrombin‐activatable fibrinolysis inhibitor activity

Abstract

Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-Å-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-Å-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-Å-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics.

Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk

“…One nanobody was shown to bind close to the TAFI activation site, and the other close to a possible thrombomodulin binding site. These findings explained the interference of the two nanobodies with TAFI activation, and thrombin-thrombomodulin-mediated activation, respectively [151]. Although these studies are promising and shed light on the mechanistic properties of TAFI-inhibitory compounds, in vivo data are lacking.…”

Structural Insights into the Mechanism of a Nanobody That Stabilizes PAI-1 and Modulates Its Activity

“…The crucial involvement of the RCL is also in agreement with the observed selectivity of Nb93 for the active conformation of PAI-1. Previous affinity determination using surface plasmon resonance showed a lack of binding to latent PAI-1 and two latent variants of PAI-1, PAI-1-H185A-R186A-H187A, or PAI-1-D89A-T94A [ 25 ]. The current structure reveals that these residues do not reside within the epitope of Nb93, with the closest residue His185 located approximately 17 Å away from the proposed epitope of Nb93.…”

Importance of Endogenous Fibrinolysis in Platelet Thrombus Formation

“…With the understanding of the importance of spontaneous, endogenous fibrinolysis and its role in modulating platelet thrombus formation, medication that impacts favourably on this pathway may be an alternative approach to reperfusion or to regulate thrombus formation. This could be in the form of inhibition of regulators of fibrinolysis (TAFI inhibitors [ 109 , 110 ], PAI-1 inhibitors [ 111 , 112 ] and α-2 plasmin inhibitors [ 113 , 114 ]).…”