The principal of the assay method is that in the first stage a fibrin clot is formed in the wells of a microtitre plate by mixing fibrinogen, plasminogen and thrombin. This is allowed to clot for 30 min. At NaCl concentrations above approximately 70 mmol L−1, the fibrin network is transparent. When clotting is complete, a mixture of PA and chromogenic substrate is added to the clot followed by mineral oil to prevent evaporation and clot shrinkage. Fibrinolysis takes place at the boundary of the solid fibrin matrix and solution phase as PA penetrates the clot and encounters plasminogen bound to fibrin. The plasmin generated is able to digest fibrin and also hydrolyzes the chromogenic substrate, S-2251, present as a reporter in the solution phase. The generation of p-nitroaniline is monitored by following the increase in OD at 405 nm.
Three buffers were used, buffer A, 0.5 mol L−1 Tris/HCl, pH 7.4 at 37 °C, buffer B, 40 mmol L−1 stock Tris/HCl (buffer A diluted 16 mL up to 200 mL) containing 0.01% Tween 20 and 75 mm NaCl. Buffer C is buffer B +1 mg mL−1 albumin. Microtitre plates (not high protein binding) were blocked using 0.1% Tween 20 in buffer B for 2 h at 37 °C. Fibrinogen was made up fresh to 2.65 mg mL−1 (total protein) in buffer B and maintained at 37 °C until required and 1.0 mg mL−1 plasminogen (50 µL to 4 mL fibrinogen solution) was added just prior to the clotting reaction. Clotting was initiated by addition of 60 µL fibrinogen-plasminogen solution to wells containing 20 µL of 4.0 IU mL−1 thrombin solution. PA dilutions were prepared separately in a microtitre plate using buffer C. Aliquots of 20 µL of these solutions were then dispensed into empty wells and 80 µL of 0.75 μmol L−1 S-2251 was added (a stock solution of 3 mmol L−1 made up in water and subsequently diluted fourfold using buffer B). Clot lysis was initiated by adding 40 µL of these PA/S-2251 solutions to the top of the preformed clots, followed by 60 µL of mineral oil. A suitable concentration range of PA added to the clot in this way was found to be from 12 to 1.5 IU mL−1 (doubling dilutions) for tPA, 0.4 to 0.05 IU mL−1 for streptokinase and 40 to 5 IU mL−1 for uPA.
To study the effect of substrate (plasminogen) concentration on activation rate, activator concentration was fixed and Glu- or Lys-plasminogen concentration added to the fibrin clot was varied from 0 to 1.2 µmol L−1, where stock plasminogen concentrations were determined from absorbance at 280 nm. Data were corrected for hydrolysis of S-2251 with no added plasminogen (due to contaminants in reagents and thrombin). Plasminogen solutions were dialyzed at 4 °C before use against a large volume of 20 mmol L−1 Na acetate buffer pH 4.5 containing 0.15 mol L−1 NaCl, followed by deionized water, adjusted to pH 4 with 1 mol L−1 HCl. This procedure removed traces of aminohexanoic acid remaining after the purification procedure. It is possible to calculate the apparent Vmax (and hence kcat) values and Km values for tPA, streptokinase and uPA on plasminogen in this system from the generation rate of plasmin measured using the rate of S-2251 hydrolysis, but a correction factor is needed for the competition of fibrin at the active site of plasmin. An initial series of experiments showed that plasmin activity over a range [S-2251] was indistinguishable in the presence of fibrinogen or fibrinogen + thrombin (forming fibrin). Thus, for practical purposes it was simpler to investigate the competition between fibrinogen and S-2251 for the plasmin active site, rather than fibrin and S-2251. To quantitate the competitive effect of fibrinogen, plasmin activity against 92–920 µmol L−1 S-2251 was measured in the presence of 3.8, 7.6, 11.4 and 15.2 µmol L−1 fibrinogen. A total of four sets of independent assays were performed and data for initial rates used to fit Ks, VmS and Ki using the program Dynafit, a program for performing numerical integration using a series of differential equations describing a system of chemical reactions , according to the following:
where E is plasmin, S is S-2251, P is p-nitroaniline and Fg is fibrinogen. Fibrinogen is treated as a competitive inhibitor . Fitting gave values of KS = 266 ± 28 µmol L−1, VmS =8.69 ± 0.31 mOD min−1 and Ki = 1.39 ± 0.11 µmol L−1 (mean ± SE). Therefore, apparent Ks accounting for the inhibition of fibrin(ogen) was, apparent Ks = Ks(1 + [Fg]/Ki) = 1.43 mmol L−1, using 2.0 mg mL−1 fibrinogen. The plasmin used was the second IS (77/588), the concentration of which had been determined previously by active site titration  and this produced a value of kcatS = 51.1 s−1. S-2251 was allowed to hydrolyze completely under the conditions in this method to derive an extinction coefficient (ε) for p-nitroaniline, which was found to be 2500 OD/mol L−1. These parameters were used in subsequent analyses.
In order to calculate apparent Vmax and kcat values for tPA, streptokinase and uPA action on plasminogen, it was necessary to estimate the molar concentrations of active enzyme. This was done using the protein concentration from records of the fills for each standard and resulted in enzyme concentrations of 300 nmol L−1 for tPA (98/714), 210 nm for streptokinase (00/464) and 550 nmol L−1 for uPA (87/594), when each ampoule was reconstituted in 1 mL deionized water as directed. These estimates gave specific activities (IU/mg) within the expected range using traditional clot lysis methods.