An improved microtiter plate method to measure the potency of plasminogen activators

THROMBOSIS RESEARCH 36; 467-474, 1984 0049-3848/84 $3.00 t .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.

BRIEF

COMMUNICATION

AN IMPROVED MICROTITER

PLATE METHOD To MEASURE THE

POTENCY OF PLASMINOGEN

ACTIVATORS

Wai-Pan Chan+and Virginia Mosher Coagulation Research, Hyland Therapeutics 1710 Flower Avenue, Duarte, California 91010, U.S.A. (Received 4.5.1984; Accepted in original form 23.8.1984 by Editor P.J. Gaffney)

Introduction

Several synthetic tripeptide substrates have been developed to measure the coagulation enzyme activities (1). The synthetic substrate H-D-Val-Leu-Lys-pNA (S-22511, Kabi) was used in an indirect method to determine plasminogen activator activity. The system contains plasminogen activator, plasminogen, the synthetic substrate and a mixture of soluble fibrinlogen) fragments prepared by cyanogen bromide treated fibrinogen. The plasminogen is converted to plasmin in the presence of plasminogen activator. The formed plasmin then catalyzes the hydrolysis of the tripeptide anilide to release the yellow dye: this paranitroanilide (~NA) release is detected by spectrophotometric absorption at 40s nm. Drapier, et al., (2) described a system for plasminogen activator determination and Wallen, et a1.,(3) later modified this assay to give a high sensitivity and lower detection limit in the presence of fibrin monomer. The insoluble fibrin monomer, however, was found to interfere with the assay. Recently, Verheijen, et al., (4) reported a method of increasing the activation rate of plasminogen by tissue plasminogen activator (t-PA) in the presence of fibrintogen) fragments using a polystyrene microtiter plate. The mechanism of enhancement of the activity in the presence of the fibrintogen) fragments is still not clear. The effect of various polyelectrolytes was studied by Allen (5). He found that poly-D-lysine, especially, mimicks the cofactor function of fibrin in the stimulation of the activation activity of plasminogen by human uterine plasminogen activator. Ericksson, et al., (6, 7) found that poly-D-lysine (MW=144,000) increased the t-PA activity by 20-fold at an optimal concentration of l-5 ug/ml of poly-D-lysine. They also found

Key Words: Tissue plasminogen activator, fibrintogen) fragments, poly-D-lysine, S-2251 +Present address : Syntex Medical Diagnostic, 3221, Porter Drive, Palo Alto, California 94303, U.S.A. Reprints available from above address. 467

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that lys-plasminogen is 10 times more active than the gluplasminogen. The t-PA from melanoma cells, pig heart, human uterus and urokinase were tested in their assay system. In our present study, a new formulation of the substrate buffer, containing both the cyanogen bromide treated fibrinogen and polyD-lysine, was found to have an enhancing effect in accelerating the activity of the plasminogen activation. With the use of microtiter plate, minimal amounts of reagents and samples are used and larger numbers of samples can be tested than in the manual spectrophotometric method. In the absence of the fihrintogen) fragments, the t-PA activity is minimal even though However, the urokinase activity is the poly-D-lysine is present. enhanced in the presence of the poly-D-lysine regardless of whether the fibrin(ogen) fragments are present in the system or not.

MATERIALS

AND METHODS

The plasminogen obtained from Sigma does not have a definition on the amount of lys-plasminogen or the glu-plasminogen present in each bottle. The plasminogen was reconstituted in a dilution buffer: 0.1 M Tris-HCl buffered at pH 7.45 containing 0.4% (v/v) Similarly, the poly-D-lysine of Tween-80 (obtained from Sigma). (MW=l40,000 obtained from Sigma) and the tripeptide substrate S2251 (obtained from Kabi) were reconstituted in the same buffer. The fibrintogen) fragments were made according to Rlomhack's procedure (8) using cyanogen bromide on the human This mixture of reagents for the fibrinogen obtained from Kabi. substrate buffer was made immediately prior to use. The substrate buffer contains 0.15 mg/ml of fibrintogen) fragments, 0.45 u mole of poly-D-lysine, 0.87 m mole of S-2251 and 0.38 u/ml of plasminogen in 0.1 M Tris-HC1/0.4% (v/v) Tween-80 buffered at pH 7.45. Drokinase obtained from Leo Pharmaceuticals (Denmark) was used as a reference standard by diluting the labelled activity to 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2.0, and 4.0 IV/ml. IJrokinase obtained from Winthrope (Winkinasee) and streptokinase from Calbiochem were diluted to the same concentrations according to the labelled activity units. Polystyrene microtiter plates were obtained from Flow Labs, Inc. (Linbroe Plates). Multichannel pipettors obtained from Flow Labs, Inc. were used The optical density of the plates was read by a for dispensing. Titerteke Multi-skan microtiter plate reader (Flow Labs, Inc.). were pipetted into each well first. Then, Samples of 40 160 ul substra r e buffer (made immediately prior to use) was put One into each well to make a total test volume of 200 ~1. hundred sixty$l of the substrate buffer and 40 ~1 of the dilution buffer was used as a blank and put into the wells in the first column in the plate (column 1: rows A to H). The parafilm covered plates were incubated at room temperature to develop the After 75 minutes of incubation at room yellow color. temperature, the plate was blanked against the first column with The optical density of the wells the microtiter plate reader.

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was read at 405 nm. The reading of the microtiter plate was finished in 2 minutes and the reference concentrations were A standard curve of plotted against the optical density. urokinase activity was established and a regression of the data was calculated with a Hewlett-Packard calculator HP4lC where the II R2 II (coefficient of determination), a (the intercept), and B (the slope of the curve) were determined. The t-PA samples obtained from melanoma cell culture conditioned media purified according to Collen's procedure (9) were also assayed with and without fibrintogen) fragments.

UROKINASE

ACTIVITY

( I Ulllll

I

FIG. 1 Standard Curve of Urokinase Reference Urokinase obtained from Leo Pharmaceutical (Denmark) was used. The labelled activity was converted to International Units (IU) from Ploug JJnits (1 IU=O.7 Ploug Units) and further diluted to 0.2, 0.4, in 0.1 M Tris-HC1/0.4% 0.8, 1.2, 1.6, 2.0 and 4.0 N/ml Tween-80, pH 7.45. The plate was read after incubating at room temperature for 75 minutes. The optical density at 405 nm was plotted against the activity in IV/ml.

TISSUE PLASMINOGEN ACTIVATORS

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1.5

0

1

I

I

0

t

I

I

UROKlNASE

ACT1

VlTY

r

I

2.0

1.0 ( IU/ml)

FIG. 2 Change of the Urokinase Reference Curves when the plate was rear? at different incubation times l/4 hr. 0 , 3/4 hr. ?? ,. The absorbance at 1 l/2 hr. A and2 hr. A . 1 hr. 0 405 nm wai plotted against the activity in IIJ/ml.

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RESULTS AND DISCUSSION The system used in this assay gave a linear regression between 0.2 ID/ml to 4.0 IU/ml of urokinase activity (see Fig. 1). The coefficient of correlation is between 0.985 to 0.999. The assay was optimized at 75 minutes of incubation at room temperature, as shown in Fig. 2. The urokinase obtained from Leo Pharmaceuticals gave a very similar standard curve as that of the Winkinasee. However, the streptokinase is more active than the urokinases (Fig. 3). The high activity in the streptokinase may be due to overestimated unitage labelled on the bottle or the streptokinase has a very different activity from the urokinases in this The results of the t-PA samples were found consistent system. with the results obtained from a manual method (1) as shown in Table I. The method has a formulation that requires only two pipetting steps and the time of incubation was decreased to less than two hours with a wide range of linearity, from 0.2 ID/ml to 4.n Ill/ml. This has been very helpful in the in-line monitoring of t-PA activity in t-PA purification processes. However, when t-PA was spiked into a normal pooled plasma, lower unitage was recovered. This may be due to the fact that plasmin inhibitors were present in the plasma and the activity was not fully expressed. The presence of fibrintogen) fragments is necessary to develop the t-PA activity (see Fig. 4).

I NCUBATION

TIME

(min.)

FIG. 3 Compare the Plasminogen Activation Activities between the urokinases, streptokinase and t-PA. The urokinases were obtained from Leo Pharmaceuticals (rt-i) and from Winthrope (o---o). The latter is used as a reference standard at the Office of Riologics. The streptokinase (e) and the t-PA (-1 were diluted with O.lM Tris HCl/fJ.4% Tween-80, pH 7.45 to 1.0 IIJ/ml equivalent to the urokinase activity according to the labelled value on the bottle. The absorbance at 405 nm was plotted against the incubation times in minutes.

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t-PA P with fragments

t-PA bout

t-PA bout

INCUBATION

TI ME

H fragments

P fragments

(min.)

FIG. 4 Effect of fihrin(ogen) fragments on t-PA samples obtained from melanoma cell culture. The urokinase with the fragments (0-O) had the same pattern as the urokinase without the fragments C-1. However, significant decrease in activity was found in the samples when the fragments were deleted. Fragments were put in the "H" (-1 and "P" (CL-Cl) samples as a comparison to the samples without fragments. The absorbance at 405 nm was plotted against time in minutes. The specificity of the t-PA for the fibrintogen) fragments made it possible to differentiate t-PA activity from non-t-PA activity by testing a sample both in the absence or in the presence of fibrintogen) fragments in this system. While most of the non-tPA fihrinolytic activity acts systemically in the body, only the t-PA acts on the "clot" where the fibrin fragments are located. This differential assay would therefore be useful to monitor t-PA therapy in the patients. More experiments are needed to confirm the validity of this test system as it could be applied to the determination of t-PA activity recovery in the human plasma samples.

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TABLE Comparison

I

of To

the Results of the Microtiter Plate Method the Results of the Manual Method t-PA Samples Were Obtained From Melanoma Cell Culture Purified by Collen's Procedure

Microtiter Plate Method Urokinase Standard Regression Data (n=lO) Range of Linearity

R2

Manual Method

= 0.9956 f 0.0041 a = 0.0357 f 0.0055 6 = 1.9873 f 0.5432

R2 = 0.9978 f 0.002 a = 0.3820 f 0.0426 B = 0.8015 f 0.1191

0.2 - 4.0 IV/ml

0.4 - 1.2 IU/ml

14.58 rt 1.18 IV/ml

14.70 f 0.38 IU/ml

244.98 rt 25.86 IV/ml

274.74 f 19.22 IU/ml

(n=10)

t-PA Sample: CR31213L (n=lO) C831213H (n=S)

R2

=

a

=

B

=

Coefficient of determination Intercept Slope of the line

ACKNOWLEDGEMENT We thank Mr. Chuck Weyand far his generosity in supplying us with the fibrin(ogen) fragments for this assay. Also, we thank Mr. Ray Rerkebile for his valuable suggestions and his time to Particularly, we like to thank Ray proofread this manuscript. for giving us freedom to pursue this problem on our own. We also thank Mrs. Jo Harris for her help in finalizing this manuscript in a very short time.

REFERENCES 1.

Fareed, J., Messmore, H.L., Walenga, J.M., and Hermes, Jr., Efficacy of Newer Synthetic-Substrate E.W. Diagnostic Methods for Assessing Coagulation Variables: A Critical Overview. Clin. Chem. 29, 225-236, 1983.

2.

Drapier, I.C., Tenue, I.P., Lemaine, G., and Petit, I.F. Regulation of Plasminogen Activator Secretion in Mouse Peritoneal Macrophages. Riochemie 61, 463-471, 1979.

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3.

Ranhy, M., Norrman, B. and Wallen, for tissue plasminogen activator. 749, 1983.

4.

Verheijen, J.H., Nieuwenhuizen, W., Traas, D.W., Chang, G.T.G., and Hoegee, E. Differences in effect of fihrin(ogen) fragments on the activation of 1-Gluplasminogen and 442-Val-plasminogen by tissue plasminogen activator. Throm. Res. 32, 87-92, 1983.

5.

Allen, R.A. The effect of poly-lysine on the activation of plasminogen. In: Progress in Fibrinolysis, Vol. V, J.F. Davidson, I.M. Nilsson and B. Astedt (Eds.) Churchill Livingstone, Edinghurgh, 1981, pp. 134-138.

6.

Eriksson, E., Gyzander, E. and Teger-Nilsson, A.C. Assay of tissue plasminogen activator utilizing plasminogen, poly-lysine and a chromogenic substrate. Haemostasis 11, Suppl. 1, 89, 1982.

7.

Eriksson, E., Rosen, S., Knos, M. and Friberger, P. Chromogenic substrate methods for the determination of Thrombosis FVIIIC, endotoxin and plasminogen activator. Haemostasis 46, 315, 1981.

P. A sentitive assay Thromb. Res. 27, 743-

&

8.

Rlomback, R., Blomback, M., Henschen, A., Hessel, B., N-Terminal Disulphide Knot of Iwanaga, S., and Woods, K.R. Nature (London) 218, 130-134, 1968. Human Fibrinogen.

9.

Rijken, D.C. and Collen, D. Purification and Characterization of the Plasminogen Activator Secreted by J. Biol. Chem. 256, 7035Human Melanoma Cells in Culture. 7042, 1981.