The hallmark of serpins is the ability to undergo the so-called "stressed-to-relaxed" switch during which the surface-exposed reactive center loop (RCL) becomes incorporated as strand 4 in central beta-sheet A. RCL insertion drives not only the inhibitory reaction of serpins with their target serine proteases but also the conversion to the inactive latent state. RCL insertion is coupled to conformational changes in the flexible joint region flanking beta-sheet A. One interesting serpin is plasminogen activator inhibitor-1 (PAI-1), a fast and specific inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activator. Via its flexible joints' region, native PAI-1 binds vitronectin and relaxed, protease-complexed PAI-1 certain endocytosis receptors. From a library of 35-nucleotides long 2'-fluoropyrimidine-containing RNA oligonucleotides, we have isolated two aptamers binding PAI-1 by the flexible joint region with low nanomolar K(D) values. One of the aptamers exhibited measurable binding to native PAI-1 only, while the other also bound relaxed PAI-1. While none of the aptamers inhibited the antiproteolytic effect of PAI-1, both aptamers inhibited vitronectin binding and the relaxed PAI-1-binding aptamer also endocytosis receptor binding. The aptamer binding exclusively to native PAI-1 increased the half-life for the latency transition to more than 6 h, manyfold more than vitronectin. Contact with Lys124 in the flexible joint region was critical for strong inhibition of the latency transition and the lack of binding to relaxed PAI-1. We conclude that aptamers yield important information about the serpin conformational switch and, because they can compete with high-affinity protein-protein interactions, may provide leads for pharmacological intervention.

Summary. Background: Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co-administration of t-PA and a TAFIa inhibitor enhances t-PA treatment. Objective: We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t-PA induced clot lysis. Results: Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A ), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16-fold molar excess of nanobody over TAFIa, IC 50 Õs range between 0.38-and > 16-fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t-PA concentration. The nanobodies were found to bind in the active-site region of TAFIa and their timedependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition. Conclusion: These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.

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.

Background/Objectives This study aimed to study one-month recovery profile and to identify predictors of Quality of Recovery (QOR) after painful day surgery and investigate the influence of pain therapy on QOR. Methods/Design This is a secondary analysis of a single-centre, randomised controlled trial of 200 patients undergoing ambulatory haemorrhoid surgery, arthroscopic shoulder or knee surgery, or inguinal hernia repair between January 2016 and March 2017. Primary endpoints were one-month recovery profile and prevalence of poor/good QOR measured by the Functional Recovery Index (FRI), the Global Surgical Recovery index and the EuroQol questionnaire at postoperative day (POD) 1 to 4, 7, 14 and 28. Multiple logistic regression analysis was performed to determine predictors of QOR at POD 7, 14, and 28. Differences in QOR between pain treatment groups were analysed using the Mann-Whitney U test. Results Four weeks after haemorrhoid surgery, inguinal hernia repair, arthroscopic knee and arthroscopic shoulder surgery, good QOR was present in 71%, 76%, 57% and 24% respectively. Poor QOR was present in 5%, 0%, 7% and 29%, respectively. At POD 7 and POD 28, predictors for poor/intermediate QOR were type of surgery and a high postoperative pain level at POD 4. Male gender was another predictor at POD 7. Female gender and having a paid job were also predictors at POD 28. Type of surgery and long term fear of surgery were predictors at POD 14. No significant differences in total FRI scores were found between the two different pain treatment groups. Conclusions The present study shows a procedure-specific variation in recovery profile in the 4-week period after painful day surgery. The best predictors for short-term (POD 7) and long-term (POD 28) poor/intermediate QOR were a high postoperative pain level at POD 4 and type of surgery. Different pain treatment regimens did not result in differences in recovery profile. Trial registration European Union Clinical Trials Register 2015-003987-35.