Colin Longstaff, Division of Haematology, Blanche Lane, South Mimms, Herts EN6 3QG, UK. Tel.: +44 1707 654753; fax: +44 1707 646730; e-mail: email@example.com
Summary. A method has been developed for accurately and precisely measuring the activity of a range of plasminogen activators (PAs) used as thrombolytic agents, including streptokinase, tissue plasminogen activator (tPA) and variants, and urokinase (uPA), both single and two chain forms. Plasminogen activation is monitored in a transparent, solid fibrin matrix but uses chromogenic substrate hydrolysis, rather than changes in fibrin, to quantitate the activity of PAs. The method has been tested in two recent international collaborative studies involving tPA and streptokinase where it has been shown to perform very well. Furthermore, the method is based on sound enzymological principles and once correction for the competitive inhibition of fibrin(ogen) is made, the generation of plasmin can be determined in molar terms and hence the activity of PAs can be expressed and compared in SI units (rate of increase in molar concentration of plasmin) as well as International Units. The assay is also arranged in such a way to reflect the behavior of PAs in vivo during thrombolytic therapy and it is shown that the specific activity of streptokinase and tPA in this system reflects plasmin generation capacity of these thrombolytics for doses given in infusions for treatment of myocardial infarction. The method would make a suitable reference method for PAs and provides a rigorous means of studying and modeling the enzymology of fibrinolysis and will be helpful in the rational design of third generation thrombolytic agents.
Recent initiatives by the IFCC and ISTH to promote standardization efforts have highlighted the need to co-ordinate the development of both reference materials and reference methods [1–3]. NIBSC traditionally has a role in the development of reference materials, but information on methods is also available from collaborative studies to calibrate new International Standards (IS). This information is helpful in the development of reference methods. It was apparent from the studies to calibrate the 3rd IS for tissue plasminogen activator (tPA)  and the 3rd IS for Streptokinase  that one new method performed very well and this method may also be an improvement on previous methods in fulfilling guidelines suggested for methods in the fibrinolytic system . Further development work has now been completed to demonstrate how this method can be used to express results for the potency of plasminogen activators (PAs) in SI units, as moles of plasmin produced per second, in this defined system, as well as International Units (IU). Thus, the method allows comparison of different PAs. Furthermore, the method has been field tested in two international collaborative studies where it has been shown to be both accurate and precise. This method is designed to use purified, well characterized reagents and appears to be reproducible and reflects the physiological situation encountered during thrombolytic therapy. The method would make a suitable reference method for the activity determination of many PAs and is now described in detail.
Materials and methods
The following ISs and other reference materials have been used in this study, all from the National Institute for Biological Standards and Control, Potters Bar, UK. Second IS for tPA (86/670), rec tPA (86/624), third IS for tPA (98/714), second IS for Streptokinase (88/826), streptokinase (88/824), third IS for streptokinase (00/464), 1st IS for high molecular weight uPA (87/594), low molecular weight uPA (90/642), glycosylated single chain uPA (scuPA, 95/564), non-glycosylated scuPA (95/668), Reteplase® (truncated tPA) (93/726), second IS (77/588) and third IS for Plasmin (97/536). Fibrinogen was fraction I, essentially plasminogen free, F-4883 from Sigma Chemical Co. S-2251 and Glu-plasminogen were from Chromogenix, Milan, Italy. Lys-plasminogen was from Immuno, Vienna, Austria. Plasminogen concentration was determined by OD at 280 nm using an extinction coefficient of 16.8 for a 1% solution. Thrombin was of bovine origin from Diagnostic Reagents, Thame, UK.
PA assay method
The principle of the method can be seen by reference to Figure 1, representing a single well of a microtitre plate.
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.
Data collection and analysis
OD was monitored as soon as possible to determine the initial rates for the generation of plasmin, using a plate reader fitted with a thermostat (e.g. Thermomax, using Softmax software, MDC, Stanford, CA, USA). The rate of plasmin generation was determined from the OD measurements at 405 nm by transforming raw data into plots of OD vs. s2, or data was fitted directly by non-linear regression analysis . The theoretical basis for this transformation has been discussed previously [10–13]. In practice, under these conditions, the rate of plasmin generation in mol L−1 s−1 is obtained by dividing the slope of the OD vs. s2 plot by 18 880 (calculated from the enzyme parameters and ε determined above).
Reaction conditions giving rise to maximum, stable conversion by plasmin of scuPA to uPA were investigated and found to be as follows. The optimum ratio of scuPA to plasmin was found to be 10 : 1 at concentrations of 300 nmol L−1 scuPA to 30 nmol L−1 plasmin. Activation was allowed to proceed for 10 min at 37 °C in Phosphate-buffered saline. Under these conditions maximum uPA activity was observed after 5 min and was stable for at least 30 min.
The proposed reference method has been used successfully at NIBSC in two international collaborative studies to establish the third IS for tPA (98/714)  in 1999 (laboratory number 14); and the study to establish the third IS for Streptokinase (00/464)  in 2001 (laboratory number 17). Generally speaking, linearization of OD vs. time plots using the time squared transformation was good using OD values up to 0.1, indicating the observed results conformed to theory (see below). Data from these two collaborative studies were reanalyzed to convert the original PA activity values expressed in IU to SI units, as the molar concentration of plasmin produced per second under conditions of the proposed reference method, as outlined in Materials and methods, above. A comparison of these units is shown in Table 1. Thus, it is possible to compare the specific activities of tPA and Streptokinase IS in this fibrin-based system and it can be seen that, on a molar basis, streptokinase is approximately sevenfold more active than tPA.
Table 1. Comparison of tPA and streptokinase IS activities expressed in IU or SI units1
Activity expressed as
1 Assuming contents of 1 ampoule of standard reconstituted in 1 mL of water.
a From references [4,5] using a range of methods as described. bValues recalculated from data collected in collaborative studies by NIBSC as laboratory 14 in  and laboratory 17 in . cIt is assumed that the initial activation reaction takes place at the interface between PA/S2251 solution and fibrin clot/plasminogen matrix. The solution phase above the clot consists of a 40 µL reaction volume. dThis assumes the molar concentrations for tPA and Streptokinase IS estimated in Materials and methods.
Rate of plasmin production nmol L–1 plasmin s–1 in reference method per ampouleb
Rate of plasmin production as picomoles plasmin produced/s in per ampoule in reference methodc
Specific activities for 1 mole of PA as µ micromoles of plasmin produced/s in reference methodd
Kinetic parameters for plasminogen activation in the proposed reference method
The results shown in Table 1 are derived from historical data from collaborative studies [4,5] and using the parameters for plasmin on S-2251 derived as described in Materials and methods. Representative data are shown in Fig. 1(A,B). An independent series of experiments was performed to determine apparent Km and kcat values over a range of plasminogen concentrations, using this method. Results from such a study are summarized in Table 2 where fixed concentrations of tPA (98/714), Streptokinase (00/464) and uPA (87/594) were all compared under identical conditions using the proposed reference method, and with a range of glu- and lys-plasminogen concentrations. PA concentrations were chosen so as to give a similar rate of S-2251 hydrolysis for all PAs. Curve fitting to the Michaelis-Menten equation by non-linear regression  generated values for apparent Km and Vmax for each PA with each plasminogen substrate form. The concentration of plasminogen used was the added final concentration in the fibrin clot, not in the whole reaction mixture, since it is expected that plasminogen will be remain associated with the fibrin matrix. The concentration of PA is arbitrarily stated as the concentration added in the 40 µL placed on the clot. Results were generally well fitted for tPA and streptokinase, but individual values for Km and Vmax (hence kcat) for uPA were more poorly estimated due to the relatively high Km. However, kcat/Km is valid for uPA, even where [S] < Km. kcat/Km is a measure of enzyme efficiency in this system and clearly follows the pattern streptokinase > tPA > uPA. Significantly, there is a sevenfold difference in kcat/Km values between streptokinase and tPA, exactly replicating the values obtained in the two previous collaborative studies shown in Table 1 from earlier data [4,5]. The results using Lys- and Glu-plasminogen as substrate show a very reproducible effect with only around twofold improvement in kcat/Km for Lys- compared to Glu-plasminogen. It is possible that this may be accounted for by the tighter binding of Lys-plasminogen to fibrin, as the effect seems to be independent of PA form.
Table 2. Kinetic parameters for plasminogen activation by streptokinase, tPA and uPA with Glu- or Lys-plasminogen in proposed reference method. Catalytic parameters were derived from curve fitting to the Michaelis-Menten equation by non-linear regression analysis
a The standard error of fitting was < 10% in all cases and < 5% in most cases. b The concentration of PA overlaid onto the clot in chromogenic substrate solution, which was 40 µL (the volume of the fibrin clot is not included), based on estimates of molar enzyme concentrations given in Materials and methods. cApp Km is calculated from the total added plasminogen concentration within the fibrin clot, not including the volume of PA + S-2251 solution overlaid. dEnzyme efficiency is given by kcat/Km.eThe kcat/Km for lys-plasminogen substrate/kcat/Km for glu-plasminogen substrate.fFor comparative purposes the kcat/Km of streptokinase, the most efficient PA, is set to 100 and the relative efficiencies of tPA and uPA are calculated. gIndicates not determined, due to high Km values. However, apparent kcat/Km values are valid.
Additional studies have been performed to investigate the applicability of the proposed reference method to the assay of other PAs. It is possible to perform the assay as outlined in Materials and methods and obtain valid data for Reteplase (truncated tPA) and for single chain uPA (scuPA or pro-uPA) and low molecular weight uPA. In the cases of scuPA and Reteplase there was some non-linearity of the time-squared transformations. This type of behavior has been noted elsewhere for Reteplase [14,15] in other assay systems. Non-linearity would also be expected for scuPA since an extra activation step is required in going from the zymogen scuPA to active uPA, which would be catalyzed initially by trace contaminating plasmin. However, slopes from transformed data of OD vs. s2 or a single endpoint method gave linear dose–response curves for both Reteplase and scuPA (data not shown), which could be used to assay these materials against an appropriate standard of the same material.
Investigations comparing glycosylated and non-glycosylated scuPA provide further evidence that the assay generates data in agreement with previous studies. Preparations of glycosylated scuPA (code 95/668) and non-glycosyated scuPA (95/564) were ampouled at NIBSC and were investigated both without and with prior activation to uPA by plasmin, as outlined in Materials and methods. In agreement with previous studies, the simple chromogenic activity of uPA on S-2444 is unaffected by glycosylation pattern. When glycosylated enzyme (uPA) or zymogen (scuPA) was compared with the non-glycosylated analog a consistent difference in specific activity due to glycosylation was found. Non-glycosylated scuPA was 1.27-fold more active (95% confidence interval 1.08–1.51) and non-glycosylated uPA was 1.52 fold more active (95% confidence interval 1.45–1.59), than the glycosylated partner. Thus, in agreement with previous data in a variety of assay systems , non-glycosylated (sc)uPA has an approximately 1.5-fold higher specific activity than glycosylated (sc)uPA.
The assay did not work where staphylokinase was the PA. The time-squared transformations were extremely non-linear, as were the dose–response curves. These observations highlight the very different molecular mechanisms that operate for streptokinase and staphylokinase.
There are many methods available for measuring activity of common PAs. Changes in clot physical structure may be used as the ability to support a glass bead  or retain trapped bubbles . Alternatively fibrin opacity may be measured as lysis zones on plates [19,20], or changes in OD in microtitre plate formats [21–23] or another automated device . The most common endpoint in these methods is the time to half lysis, but this may not be so easily automated . Alternative methods use radiochemical detection [25,26], or utilize modified fibrinogens with chromogenic detection . Methods using chromogenic substrates have been developed where fibrin is removed before OD measurement , or involving fibrin(ogen) chemically immobilized to microtitre plates , or in the presence of opaque fibrin . In some specific cases it has been possible to replace fibrin with soluble analogs to give a simplified chromogenic assay, for example for tPA , however, this may not give the same results as using fibrin  and cannot be used for other PAs.
At the simplest level, the proposed reference method has been demonstrated to give precise and accurate results when comparing a standard and test material that can be expressed in IU. The method has been tested in two international collaborative studies [4,5]. However, the format of the method, which relies on a chromogenic substrate, can be developed further to express results in SI units, according to the theory outlined in Materials and methods. Indeed, in some ways the use of SI units may be traced to the original standardized, ‘CTA’ units described for plasmin, plasminogen and urokinase , which was the forerunner of the IU unit for fibrinolysis proteins. The CTA unit was defined as the release of 0.1 microequivalent of tyrosine min−1 from a standard casein preparation.
IU permit a simple, direct comparison of standard and test materials but the option of SI units has several advantages. Absolute quantitation of the rate of plasmin generation is possible and hence different PAs can be compared in the same assay format. Such a comparison, shown in Table 2 for three common PAs, indicates that streptokinase has the highest specific activity and is around sevenfold more active than tPA. This difference is accounted for by the difference in apparent kcat values, the apparent Km values being very similar for these two PAs. The sevenfold difference in kcat/Km is duplicated in the specific activity results from the collaborative study data shown in Table 1, which are mean results from all labs in the study using a variety of methods. Interestingly, this difference is also reflected in the clinical doses of these two PAs. Normal doses of these thrombolytics when used to treat acute MI are infusion of 0.3 µmol of streptokinase  and 1.5 µmol of tPA . Thus the proposed reference method appears to give results broadly in line with clinical efficacy of these PAs. The proposed reference method may also be seen as replicating physiological conditions during thrombolytic therapy. Thrombolytics will be administered after a clot has established in an artery and will bind and initiate clot lysis from the external medium and progress through the clot, activating plasminogen and lysing fibrin along the way.
An advantage of SI units is that they can extend our understanding of the enzymology of fibrinolysis. In vitro studies and in silico modeling of coagulation have been very useful in furthering our knowledge or the normal operation and pathophysiology of the coagulation cascades [34,35]. In the future, the proposed reference method may be used in conjunction with computer modeling of fibrinolysis. Such studies will be valuable in understanding the normal and aberrant operation of fibrinolysis in conjunction with advances in functional genomics. Furthermore, computer modeling of fibrinolysis kinetics will assist in the rational design of new thrombolytic agents with optimized kinetic and binding constants leading to optimized clinical thrombolysis.