A Narrative Review on Plasminogen Activator Inhibitor-1 and Its (Patho)Physiological Role: To Target or Not to Target?

Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of plasminogen activators (PAs) and is therefore an important inhibitor of the plasminogen/plasmin system. Being the fast-acting inhibitor of tissue-type PA (tPA), PAI-1 primarily attenuates fibrinolysis. Through inhibition of urokinase-type PA (uPA) and interaction with biological ligands such as vitronectin and cell-surface receptors, the function of PAI-1 extends to pericellular proteolysis, tissue remodeling and other processes including cell migration. This review aims at providing a general overview of the properties of PAI-1 and the role it plays in many biological processes and touches upon the possible use of PAI-1 inhibitors as therapeutics.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI): An Updated Narrative Review.

Thrombin activatable fibrinolysis inhibitor (TAFI), a proenzyme, is converted to a potent attenuator of the fibrinolytic system upon activation by thrombin, plasmin, or the thrombin/thrombomodulin complex. Since TAFI forms a molecular link between coagulation and fibrinolysis and plays a potential role in venous and arterial thrombotic diseases, much interest has been tied to the development of molecules that antagonize its function. This review aims at providing a general overview on the biochemical properties of TAFI, its (patho)physiologic function, and various strategies to stimulate the fibrinolytic system by interfering with (activated) TAFI functionality.

Structural Insight into the Two-Step Mechanism of PAI-1 Inhibition by Small Molecule TM5484.

Plasminogen activator inhibitor-1 (PAI-1), a key regulator of the fibrinolytic system, is the main physiological inhibitor of plasminogen activators. By interacting with matrix components, including vitronectin (Vn), PAI-1 plays a regulatory role in tissue remodeling, cell migration, and intracellular signaling. Emerging evidence points to a role for PAI-1 in various pathological conditions, including cardiovascular diseases, cancer, and fibrosis. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1-related pathologies. A class of small molecule inhibitors including TM5441 and TM5484, designed to bind the cleft in the central ß-sheet A of PAI-1, showed to be potent PAI-1 inhibitors in vivo. However, their binding site has not yet been confirmed. Here, we report two X-ray crystallographic structures of PAI-1 in complex with TM5484. The structures revealed a binding site at the flexible joint region, which is distinct from the presumed binding site. Based on the structural analysis and biochemical data we propose a mechanism for the observed dose-dependent two-step mechanism of PAI-1 inhibition. By binding to the flexible joint region in PAI-1, TM5484 might restrict the structural flexibility of this region, thereby inducing a substrate form of PAI-1 followed by a conversion to an inert form.

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

Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Apart from being critically involved in fibrinolysis and wound healing, emerging evidence indicates that PAI-1 plays an important role in many diseases, including cardiovascular disease, tissue fibrosis, and cancer. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1 related pathologies. Despite ongoing efforts no PAI-1 inhibitors were approved to date for therapeutic use in humans. A better understanding of the molecular mechanisms of PAI-1 inhibition is therefore necessary to guide the rational design of PAI-1 modulators. Here, we present a 1.9 Å crystal structure of PAI-1 in complex with an inhibitory nanobody VHH-s-a93 (Nb93). Structural analysis in combination with biochemical characterization reveals that Nb93 directly interferes with PAI-1/PA complex formation and stabilizes the active conformation of the PAI-1 molecule.

Expanding a Portfolio of (FO-) SPR Surface Chemistries with the Co(III)-NTA Oriented Immobilization of His6-Tagged Bioreceptors for Applications in Complex Matrices.

Cobalt-nitrilotriacetic acid (Co(III)-NTA) chemistry is a recognized approach for oriented patterning of His6-tagged bioreceptors. We have applied the matching strategy for the first time on a surface plasmon resonance (SPR) platform, namely, the commercialized fiber optic (FO)-SPR. To accomplish this, His6-tagged bioreceptor (scFv-33H1F7) and its target PAI-1 were used as a model system, after scrutinizing the specificity of their interaction. When benchmarked to traditional carboxyl-based self-assembled monolayers (SAM), NTA allowed (1) more efficient FO-SPR surface coverage with bioreceptors compared with the former and (2) realization of thus far difficult-to-attain label-free bioassays on the FO-SPR platform in both buffer and 20-fold diluted human plasma. Moreover, Co(III)-NTA surface proved to be compatible with traditional gold nanoparticle-mediated signal amplification in the buffer as well as in 10-fold diluted human plasma, thus expanding the dynamic detection range to low ng/mL. Both types of bioassays revealed that scFv-33H1F7 immobilized on the FO-SPR surface using different concentrations (20, 10, or 5 µg/mL) had no impact on the bioassay sensitivity, accuracy, or reproducibility despite the lowest concentration effectively resulting in close to 20% fewer bioreceptors. Collectively, these results highlight the importance of Co(III)-NTA promoting the oriented patterning of bioreceptors on the FO-SPR sensor surface for securing robust and sensitive bioassays in complex matrices, both in label-free and labeled formats.

Targeting PAI-1 in Cardiovascular Disease: Structural Insights Into PAI-1 Functionality and Inhibition.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily with antiprotease activity, is the main physiological inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Apart from being crucially involved in fibrinolysis and wound healing, PAI-1 plays a pivotal role in various acute and chronic pathophysiological processes, including cardiovascular disease, tissue fibrosis, cancer, and age-related diseases. In the prospect of treating the broad range of PAI-1-related pathologies, many efforts have been devoted to developing PAI-1 inhibitors. The use of these inhibitors, including low molecular weight molecules, peptides, antibodies, and antibody fragments, in various animal disease models has provided ample evidence of their beneficial effect in vivo and moved forward some of these inhibitors in clinical trials. However, none of these inhibitors is currently approved for therapeutic use in humans, mainly due to selectivity and toxicity issues. Furthermore, the conformational plasticity of PAI-1, which is unique among serpins, poses a real challenge in the identification and development of PAI-1 inhibitors. This review will provide an overview of the structural insights into PAI-1 functionality and modulation thereof and will highlight diverse approaches to inhibit PAI-1 activity.

Molecular mechanism of two nanobodies that inhibit PAI-1 activity reveals a modulation at distinct stages of the PAI-1/plasminogen activator interaction.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasminogen activators (PAs) tissue-type PA (tPA) and urokinase-type PA (uPA) plays a crucial role in many (patho)physiological processes (e.g., cardiovascular disease, tissue fibrosis) as well as in many age-related pathologies. Therefore, much effort has been put into the development of small molecule or antibody-based PAI-1 inhibitors. OBJECTIVE: To elucidate the molecular mechanism of nanobody-induced PAI-1 inhibition. METHODS AND RESULTS: Here we present the first crystal structures of PAI-1 in complex with two neutralizing nanobodies (Nbs). These structures, together with biochemical and biophysical characterization, reveal that Nb VHH-2g-42 (Nb42) interferes with the initial PAI-1/PA complex formation, whereas VHH-2w-64 (Nb64) redirects the PAI-1/PA interaction to PAI-1 deactivation and regeneration of active PA. Furthermore, whereas vitronectin does not have an impact on the inhibitory effect of Nb42, it strongly potentiates the inhibitory effect of Nb64, which may contribute to a strong inhibitory potential of Nb64 in vivo. CONCLUSIONS: These findings illuminate the molecular mechanisms of PAI-1 inhibition. Nb42 and Nb64 can be used as starting points to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions.