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),¹²  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.²¹  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/).²²  (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.¹²  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).¹²  Further information is shown in supplemental Figure 5.