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.

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.

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.

Cobalt-nitrilotriacetic acid (Co(III)-NTA) chemistry is a recognized approach for oriented patterning of His6tagged bioreceptors. We have applied the matching strategy for the first time on an SPR platform, namely the commercialized 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 selfassembled monolayers (SAM), NTA allowed (1) more efficient FO-SPR surface coverage with bioreceptors compared to the former and (2) realization of thus far difficult-to-attain label-free bioassays on the FO-SPR platform in both buffer and 20fold diluted human plasma. Moreover, Co(III)-NTA surface proved to be compatible with traditional gold nanoparticlemediated 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 labelled formats.

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.