Plasmin

Infections were done by standard membrane feeding assay (SMFA) as previously described^{63 (link)}. Briefly, P. falciparum NF54 gametocyte cultures were diluted to 0.2% stage V gametocytemia with O + human RBC at 40% hematocrit in human plasma at 37 °C (Interstate Blood Bank Inc.). The infected blood was added to pre-heated (37 °C) glass membrane feeders and mosquitoes were allowed to feed for 30 min. Unfed and partially fed mosquitoes were removed from the experiment. Mosquitoes were kept with 10% corn syrup solution ad libitum until dissection. Mosquito midguts were dissected in 1× PBS 8–9 days post-infection and stained with 0.2% mercurochrome for 30 min. Mercurochrome-stained midguts were counted under the microscope and the infection was recorded.
Inhibition by huPAI-1 was assessed by supplementing the infectious blood meal with increasing concentrations of recombinant huPAI-1 (Sigma Aldrich, # A8111). For the plasmin rescue experiment, infected blood was supplemented with plasmin (Sigma-Aldrich) at a concentration of 100 or 200 µg/mL. The infected blood from control groups was supplemented with an equal volume of PBS. Mosquitoes were maintained and dissected for oocyst counts as described above.

TM5484 was developed, as a derivative of the PAI-1 inhibitor TM5441 [9 (link)], at the United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, Miyagi, Japan. Its PAI-1 inhibitory activity and specificity were assessed by a chromogenic assay as previously described [7 (link),8 (link)]. In brief, the reaction mixture includes 0.15 mol/L NaCl, 50 mmol/L Tris-HCl pH 8, 0.2mmol/L CHAPS, 0.1% PEG-6000, 1% dimethylsulfoxide, 5 nmol/L of either human active PAI-1 (Molecular Innovations, Southfield, MI), human antithrombin III (Sigma-Aldrich, Saint Louis, MO), or human α2-antiplasmin (Sigma-Aldrich), 2 nmol/L of either human 2-chain tPA (American Diagnostica Inc, Stanford, CT), thrombin (Sigma-Aldrich), or plasmin (Sigma-Aldrich), and 0.2 mmol/L of either Spectrozyme tPA (Chromogenix, Milano, Italy), chromogenic substrate S-2238 (Sekisui medical, Tokyo, Japan), or chromogenic substrate S-2251 (Sekisui medical) at a final concentration. Tested compounds were added at various concentrations and the half-maximal inhibition (IC50) was calculated by logit-log analysis. TM5484 inhibited PAI-1 activity with an IC50 value of 3.56 mM but did not inhibit α2-antiplasmin.

After incubation of 1 mg/ml unmodified and carbamylated fibrinogen (100 mM KOCN) with human thrombin (0.5 U/mg protein in the presence of 5 mM CaCl₂) for 1 h at 25°C, plasmin (12.5 µg/mg protein, Sigma Aldrich) was added to the samples and the fibrin clots digested at 37°C for 0, 3, 6 and 9 h, respectively. Reactions were terminated by addition of 5× Laemmli buffer (20 % DTT) and plasmin inactivated at 70°C for 20 min. Aliquots of the digests corresponding to 10 µg protein were separated by SDS gel electrophoresis under the same experimental conditions as described above. The gel was stained using Biosafe Coomassie G-250 Stain and plasmin catalysed fibrin clot lysis was evaluated based on the intensity of the β-chain band.

ELISA kits for BDNF, proBDNF and p75^NTR extracellular domain (p75^ECD) were purchased from Biosensis (Thebarton, Australia), those for sortilin and cortisol were from Abcam (Cambridge, MA, USA). Enzymatic kits were from the following providers: matrix metalloproteinase-3 (MMP-3) (AAT Bioquest, CA, USA), matrix metalloproteinase-7 (MMP-7), matrix metalloproteinase-9 (MMP-9) (Quickzyme, Leidan, The Netherlands), and plasmin (Sigma-Aldrich, Oakville, ON, Canada).

To determine the enzymatic specificity of FIB One, the probe was incubated at a concentration of 1 μM (unless otherwise stated) with the recombinant MMPs (Enzo Life Sciences) (active domains of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, and MMP-13, all at 30 nM), thrombin (Sigma Aldrich) (5 U/mL), factor Xa (Sigma Aldrich) (500 nM), plasmin (Sigma Aldrich) (30 nM), and human neutrophil elastase (30 nM). Where appropriate, enzymes were incubated with specific inhibitors for 30 min at 37°C prior to addition of FIB One. The pan-MMP inhibitor marimastat (Tocris Biosciences) was used at 20 μM, and the MMP inhibitor AZD1236 was used at 0-14 μM. Enzyme free reactions served as a control. Enzymatic reactions were performed in MMP buffer (10 mM CaCl₂, 6.1 g Tris-HCl, 8.6 g NaCl per litre, pH 7.5) in a final volume of 20 μL. Reactions were performed in duplicate in 384-well plates (Life Technologies) with optically clear plate seals (Thermo Scientific) and repeated thrice (independently). Fluorescence signal was measured for up to 60 min, ex/em 485/528 nm using microplate reader (BioTek Synergy H1 multimode reader). Data were normalised to buffer alone and are presented as fold-change in signal (relative fluorescent units, RFU) compared to enzyme-free controls. Data were plotted using Prism 7 (GraphPad Software Inc., La Jolla, CA, USA).

The screen was performed as previously described [58] (link), [85] (link). Except for the enzyme concentrations, all the rest was kept constant. The following assay concentrations were used. Thrombin (0.01 nM), α-chymotrypsin (0.03 nM), plasmin (0.8 nM), and chymase (0.45 nM) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Human skin β-tryptase (0.01 nM) was purchased from Promega (Madison, WI, USA), FXa (0.33 nM) from EMD Biosciences, Inc. (San Diego, CA, USA), FXIIa (0.1 nM) from Haematologic Technologies Inc. (Essex Junction, VT, USA), kallikrein (0.04 nM) from Fitzgerald Industries International (Concord, MA, USA), elastase (0.06 nM) from Elastin Products Company, Inc. (Owensville, MO, USA), and Cathepsin G (5.3 nM), FXIa (0.06 nM), urokinase plasminogen activator (uPA; 0.25 nM), and tissue plasminogen activator (t-PA; 0.02 nM) were purchased from Molecular Innovations (Southfield, MI, USA). Matriptase (0.03 nM) was obtained from R&D Systems (Minneapolis, MN, USA), proteinase 3 (11 nM) from Merck-Millipore (Billerica, MA, USA), and sequencing-grade trypsin (0.1 nM) was purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA).