Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1

Abstract

Plasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.

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

“…By structural analysis of the PAI‐1/Nb64 complex we could confirm the crucial involvement of the latter loop. Importantly, Nb64 redirects the PAI‐1 mechanism toward substrate reaction, exceeding 90%, which correlates with previously reported biochemical data . Our structural data suggest that the key residues of the Nb64 epitope should be localized very close to the position of the PA after translocation to the opposite side of PAI‐1.…”

“…A Nb library was previously constructed, and a panel of Nbs with distinct inhibitory properties toward PAI‐1 activity were characterized and reported . The selected Nbs, wild‐type PAI‐1 (PAI‐1‐wt), PAI‐1‐W175F, and PAI‐1‐stab were expressed in Escherichia coli as His‐tagged small ubiquitin‐like modifier (SUMO) fusion proteins using auto‐induction ZYP‐5052 media .…”

“…The functional properties of Nb42 and Nb64 were determined by assessing their ability to inhibit active PAI‐1. PAI‐1 activity was determined by a chromogenic plasminogen‐coupled assay described by Verheijen et al and performed as previously described with minor modifications. PAI‐1‐wt (168 ng mL −1 with an activity of ~60% of the theoretical maximum value) was incubated with buffer or a 10‐fold molar excess of Vn, followed by incubation with serial two‐fold dilutions of Nb, resulting in a molar ratio between 0.5‐ and 256‐fold molar excess of Nb over PAI‐1.…”

“…Nbs (~15 kDa) are recombinant antigen‐binding variable domains derived from heavy‐chain‐only antibodies found in sera of Camelidae. Within a panel of Nbs, VHH‐2g‐42 (Nb42) and VHH‐2w‐64 (Nb64) exhibit distinct inhibitory properties toward PAI‐1 . According to biochemical characterization, Nb64 switched the PAI‐1/PA interaction to the substrate pathway, whereas Nb42 appeared to interfere with the initial steps of the PAI‐1/PA complex formation …”

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

“…In a mouse model of transient middle cerebral artery occlusion, inhibition of TAFI or PAI-1 significantly decreased fibrin(ogen) deposition in the ischaemic brain, thereby improving reperfusion, but the combined inhibition had an additive beneficial effect [158]. PAI-1 inhibition is also possible with nanobodies, which was demonstrated by Zhou et al who used nanobodies against PAI-1 to inhibit profibrinolytic activity in an in vitro clot lysis assay [159]. Clearly there are some promising benefits in the inhibition of PAI-1 with antibodies, which merit further in vitro and in vivo analysis.…”