In murine Neuro-2a cells, 10–50 µM nafamostat mesylate reduced migration into a scratch wound over 24 hours. At 50 µM, the matched WST-8 assay showed no significant viability difference from vehicle (P=0.239), limiting a general cytotoxicity interpretation. [2]
Nafamostat mesylate reduced secreted VEGF in Neuro-2a cultures at 50 µM for 24 hours (P=0.042). NF-κB expression was unchanged in separate 10/100 µM immunoblot conditions. The study does not establish that VEGF reduction is caused by NF-κB inhibition. [2]
Purified serine-protease inhibition and hydrolysate comparison
Biochemical assay · Purified human uPA, FXIa, FXIIa and matriptase; murine uPA serine-protease domains [1]
NM inhibited multiple purified serine proteases in the nanomolar range. Human uPA inhibition was more potent than inhibition by either isolated hydrolysate. These assay IC50 values are not binding constants or cellular doses.
Literature-Reported Treatment Conditions
- Treatment concentration
- Various concentrations; exact range differs by panel (see Fig. 3)
- Exposure duration
- 15 min preincubation at 37 °C before substrate addition
- Method
- 5 nM enzyme; chromogenic substrates near each enzyme’s KM; initial rates at 405 nm in 20 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.1% BSA
- Reported limits
- Drug stock solvent not specified in the read assay methods. Fig. 3 caption specifies two independent experiments, each with “triplicate duplicate samples”; that wording is retained rather than reinterpreted.
Methods and source locations
Compound reported in the paper: Nafamostat mesylate; Compound label in the paper: NM
Evidence concerns the material and model reported by the authors; it does not validate A10617 lot performance.
Fig. 3A–G image/caption; Materials; inhibitory-effect methods; Statistics; Results first subsection
Protease activity relative to uninhibited baseline: Sigmoidal fits to initial substrate-cleavage rates; Fig. 3 caption: average of two independent experiments, each with triplicate duplicate samples. — Fig. 3A,D–G, printed fit annotations; inhibitory-effect methods; Statistics
Human uPA inhibition by isolated hydrolysis products GBA and 6A2N: Same chromogenic uPA assay; hydrolysates are comparator materials, not forms of the catalog product. — Fig. 3B–C printed annotations; Statistics
Structural evidence for a covalent uPA acyl-enzyme complex
Biochemical assay · Human uPA catalytic domain and inactive uPA-S195A; NM and isolated GBA/6A2N comparison complexes [1]
The published uPA:NM structure contains the GBA fragment covalently linked to Ser195. Isolated GBA and 6A2N bind noncovalently; inactive S195A retains intact NM in an opposite orientation. This supports a uPA-specific structural mechanism, not irreversible inhibition of every serine protease.
Literature-Reported Treatment Conditions
- Treatment concentration
- 500 µM NM or hydrolysate in crystallization mixtures
- Exposure duration
- Crystals appeared in about 3 days
- Method
- X-ray crystallography; NM-treated active uPA compared with isolated hydrolysates and inactive uPA-S195A
- Reported limits
- No washout/recovery kinetics adopted. SI Table S1 and omit maps were not retrieved; no refinement-quality or resolution claim is made.
Methods and source locations
Compound reported in the paper: Nafamostat mesylate; Compound label in the paper: NM
Evidence concerns the material and model reported by the authors; it does not validate A10617 lot performance.
Fig. 4A–D and caption; crystallization/structure-analysis methods; Results structural subsection; Data deposition
Ligand state in the S1 pocket: Published 2Fo−Fc maps displayed at 1.5 σ; four comparison structures. PDB identifiers in Data deposition: 7VM4, 7VM5, 7VM6, 7VM7. — Fig. 4A–D electron-density images/caption and structural Results
Neuro-2a migration with a matched viability check
Cell-based study · Murine Neuro-2a neuroblastoma cells (ATCC CCL-131), RPMI-1640 with 10% FBS [2]
Scratch-wound images showed reduced migration after NM treatment, while WST-8 absorbance at 50 µM for 24 h did not differ significantly from vehicle. The migration assay does not independently establish invasion inhibition.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 and 50 µM for scratch assay; 50 µM for WST-8
- Exposure duration
- 24 h exposure
- Method
- Scratch-wound imaging; WST-8 metabolic viability assay at 450 nm
- Vehicle
- Distilled water
- Reported limits
- No quantified wound-closure percentage reported in the adopted figure. Methods specify three independent experiments for the WST-8 result; no scratch-assay replicate count is separately specified.
Methods and source locations
Compound reported in the paper: Nafamostat mesylate; Compound label in the paper: Nafamostat
Evidence concerns the material and model reported by the authors; it does not validate A10617 lot performance.
Fig. 1A–B images/caption; Reagents; Cell culture; proliferation and wound-healing methods; first Results subsection
Migration into a scratch wound: Microscopy at 0 and 24 h; vehicle control. — Fig. 1A; wound-healing methods; first Results subsection
WST-8 absorbance relative to vehicle: Unpaired Student’s t-test; methods state mean ± SD from three independent experiments. No equivalence or absence-of-toxicity conclusion. — Fig. 1B printed P value; proliferation methods; Statistical analysis
Reduced secreted VEGF without decreased measured NF-κB expression
Cell-based study · Murine Neuro-2a cells; culture-supernatant ELISA and cytoplasmic-extract immunoblot [2]
NM reduced VEGF in culture supernatant at 50 µM for 24 h. NF-κB expression was not reduced in the separate 10/100 µM immunoblot conditions. VEGF causality and nuclear NF-κB activity were not established by these assays.
Literature-Reported Treatment Conditions
- Treatment concentration
- 50 µM for VEGF ELISA; 0, 10 and 100 µM for NF-κB immunoblot
- Exposure duration
- 24 h
- Method
- ELISA for secreted VEGF; NF-κB immunoblot with actin control
- Vehicle
- Distilled water (NM stock); 0 µM comparator in Fig. 3
- Reported limits
- No exact VEGF concentrations transcribed from bar heights. NF-κB assay measures expression in cytoplasmic extracts, not a nuclear DNA-binding endpoint.
Methods and source locations
Compound reported in the paper: Nafamostat mesylate; Compound label in the paper: Nafamostat
Evidence concerns the material and model reported by the authors; it does not validate A10617 lot performance.
Fig. 3A–B images/caption; ELISA and Western blotting methods; Results final subsection; Discussion
VEGF concentration in supernatant: ELISA; unpaired Student’s t-test; methods state experiments at least three times, data mean ± SD. — Fig. 3A printed P value; cytokine-secretion methods; Statistical analysis
NF-κB protein expression: Cytoplasmic extracts; actin loading control. No inference that all NF-κB signaling is unaffected. — Fig. 3B and caption; Western blotting methods; Results final subsection
