Tranexamic acid is an active site inhibitor of urokinase plasminogen activator

Abstract

1. TXA is an active-site inhibitor of uPA.

2. TXA attenuates MDA-MB-231 BAG cell migration and inhibits endogenous uPA activity.

Figure 1.

TXA is an active site inhibitor of uPA. (A) The residual enzyme activity of tPA (Actilyse; Boehringer Ingelheim), Plm (Haematologic Technologies), plasma kallikrein (KLK; residue 381-638 recombinantly expressed and purified from Expi293 cells), and uPA (urokinase medac) was measured in the presence of TXA and EACA (0-125 mM) in 50 mM Tris-HCl pH 7.4, 100 mM NaCl, and 0.01% Tween-80 at 450 nm, as previously described. IC₅₀ derived from the linear absorbance range and K_i are summarized in supplemental Table 1. The Michaelis-Menten constant (K_m) and the apparent K_m values for uPA in the presence of 0 to 15 mM TXA were also determined with 0 to 0.5 mM S-2444, and data were plotted using GraphPad Prism 7.0 to calculate K_i (supplemental Figure 1; supplemental Table 1). (B) The residual clot lysis activity (reciprocal plot of the normalized 50% clot lysis time in supplemental Figure 4) was recorded in the presence of 1 μM Plm, 1 μM Plg plus tPA, or 1 μM Plg plus uPA at the concentrations indicated. Each reaction consists of a 100-μL fibrin clot preformed by incubating human fibrinogen (12.5 μM; Enzyme Research Laboratories) and thrombin (55 nM; Diagnostic Reagents) in a lysis buffer (40 mM Tris, 75 mM NaCl, 3 mM CaCl₂, 0.01% Tween-20) at 37°C for 3 hours. An additional 100 μL of lysis buffer containing Plm or Plg plus tPA/uPA in the presence of 0 to 25 mM TXA was added to start the lysis; the progress of clot lysis was monitored using a nephelometer (BMG LABTECH), which records every minute for 5.5 hours at 37°C. Fifty percent clot lysis time was determined using an online application (https://drclongstaff.shinyapps.io/clotlysisCL/). (C) During fibrin clot lysis, Plm and uPA enzyme activities were also measured in the presence of TXA (0-50 mM), Plm substrate (H-Ala-Phe-Lys-AMC; Bachem), or uPA substrate (Glutaryl-Gly-Arg-AMC; Sekisui Diagnostics) in 100 μL of lysis buffer, as described in panel B. Enzyme activity was recorded using a FLUOstar Omega (BMG) at 37°C; excitation and emission were 355 nm and 460 nm, respectively. All data points represent the mean ± standard deviation of a minimum of 3 independent experiments. (D) Cocrystal structure of uPA and TXA. uPASP (serine protease domain of human uPA, residue 164-431) was expressed and purified from Expi293 cells, as previously described. uPASP was activated to the 2-chain form (tc-uPASP) by Plm (Haematologic Technologies), concentrated to 10 mg/mL, and mixed with 20 mM TXA for crystallization trials. Crystals were obtained in the presence of 0.03 M NaNO₃, 0.03 M Na₂HPO₄, 0.03 M (NH₄)₂SO₄, 0.1 M Tris-bicine pH 8.5, 25% (weight-to-volume ratio) polyethylene glycol monomethyl ether 500, and 10% (weight-to-volume ratio) polyethylene glycol 20 000 at 20°C. The electrostatic surface representation of uPA (basic, blue; acidic, red) is shown, with key binding residues (cyan sticks) to TXA (green sticks) labeled. There are 4 binary complex molecules in the asymmetric unit, and the model of monomer A with TXA is shown. Data collection and processing were performed as previously described (supplemental Table 2). Further information is shown in supplemental Figure 5.

Figure 2.

TXA inhibits cell migration of MDA-MB-231 BAG breast cancer cells in vitro. Two cell lines were studied: the MDA-MB-231 BAG breast cancer cell line (A-B) and the OVMZ6 ovarian adenocarcinoma cell line (C-D). Inhibition of cell migration by TXA is observed in MDA-MB-231 BAG cells (A) but not in the OVMZ6 cell line (C). (B) Endogenous uPA and Plm activities were detected in MDA-MB-231 BAG cells (panel i is the same as panel B with a reduced scale on the y-axis for better illustration). (Bii) The endogenous uPA activity (250 000 cells per reaction) was inhibited by TXA (0-50 mM) in a dose-dependent manner. (D) No uPA activity was recorded in OVMZ6 cells, but a high level of Plm activity (∼30 times that of MDA-MB-231 BAG cells) was recorded. The Transwell cell-migration assay was performed by incubation of cells with TXA at 0, 0.2, 1, and 5 mM or with EACA at 5 mM for 30 minutes, after which migration was allowed to occur at 37°C for 3 hours. Cells were stained with a Quick Dip staining kit (Fronine) and imaged with an Olympus IX71 microscope at ×200 magnification. Migrated cell numbers were counted and averaged from 10 individual microscopic frames in duplicate conditions and plotted as a percentage of 0 mM TXA. Endogenous Plm and uPA activity was measured (as for Figure 1A) using fluorogenic substrates (as for Figure 1C) on a range of cell number inputs. All data points represent the mean ± standard deviation of ≥3 independent experiments. Both cell lines were maintained at 37°C with 5% CO₂ in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL). **P < .005, ***P < .0005, 1-way analysis of variance using GraphPad Prism. AFU, arbitrary fluorescence units.