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A sensitive assay for tissue plasminogen activator
TH.ROMBOSISRESEARCH 27; 743-749, 1982 0049-3838/82/180743-07$03.00/O Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.
BRIEF
A SENSITIVE
COMMUNICATION
ASSAY FOR TISSUE PLASMINOGEN
Mats Rdnby, Bo Norrman
ACTIVATOR
and Per Wallen
Department of Physiological Chemistry, Umea University, S-901 87 Umea, Sweden
(Received 12.5.1982; in revised form 22.6.1982. Accepted by Editor G. Myllyla) INTRODUCTION Tissue plasminogen activator has been shown to be identical to the activator released into circulation upon physical stress or venous occlusion as judged by electrophoretic and immunological criteria (1). Strikingly, tissue plasminogen activator is a good plasminogen activator only in the presence of certain polypeptide structures (2). Fibrin (but not fibrinogen) contains such structures and fibrin can induce an increase in activator turn-over number from 0.0001 5-l to 0.1 s- 1 (3). This stimulating effect is utilized in the loyed activator assays e.g. fibrin plate (4), standard clot lysis i~~"~~$ ?h! I-fibrin radiometric assay (6). In these assays fibrin is utilized both as stimulator and secondary (plasmin) substrate which typically renders the assays non-linear dose-response characteristics since the fibrin stimulating effect is reduced as the fibrin is degraded. The present paper describes a parabolic rate (coupled) tissue plasminogen activator assay which utilizes the stimulating properties of des A fibrin (7). Compared to a previously published protocol (8), the present assay procedure is 10 fold more sensitive, less reagent consuming and much easier to use. Furthermore, it is demonstrated that the presence of tissue plasminogen activator antibodies quantitatively quenches the activator activity whereas normal immunoglobulins have no effect.
MATERIALS Tissue plasminogen Key Words:
activator
AND METHODS
of the one-chain
form was prepared
from the
Tissue plasminogen activator, enzymatic assays, D-Val-Leu-Lys-paranitroaniline, S-2251, Glu-plasminogen, Fibrin I
743
744
ACTIVATOR ASSAY
vo1.27, No.6
culture medium of a human melanoma cell line by immunosorbent chromatography, argininesepharose affinity chromatography, and G-150 gel filtration chromatography (9, 10). The purity of the preparation was shown to be over 98 % by analysis of S-carboxymethylated samples on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The specific activity was 218,000 IU/mg. Stock solutions (20,400 IU/ml, 1.3 pM, assuming M, = 72,000) in 1 M ammonium bicarbonate containing 0.1 g/l Tween 80 were stored at -90 oC. Two-chain tissue plasminogen activator was prepared by incubating one-chain activator with Sepharose-plasmin as previously described (9). Plasminogen was prepared from freshly frozen titrated human plasma by lysine-Sepharose, gel filtration, and ion exchange chromatography (11). It was shown to fulfill the criteria specified by acetic urea polyacrylamide gel electrophoresis, streptokinase complex active site titration and N-terminal analysis (3, 11). Stock solutions (33.6 PM, assuming Mr = 93,000) in 0.065 M NaCl, 0.06 M Tris-HCl pH 8.5 buffer were stored at -90 oC. Fibrinogen was prepared from freshly frozen titrated human plasma (12). Stock in 0.3 M NaCl, 0.02 M sodium phossolutions (60 pM, assuming M, = 340,000 phate pH 7.3 buffer were stored at -20 b C. Bathroxobin (from Bothrops Moojeni) was a gift from Dr. K. Stocker, Pentapharm. AG, Basel, Switzerland. Solubilized des A fibrin (fibrin I monomer) was prepared by adding 10 ~1 250 BU/ml bathroxobin to 5 ml 60 uM fibrinogen and incubating at room temperature (a22 oC) for 6 hours. To the formed gel 5 ml 7 M urea was added and the mixture gently stirred until the gel dissolved. The completion. of the bathroxobin reaction was checked by centrifugating crushed gels at 15,000 x g for 15 minutes, followed by measurement of the UV-absorbance at 280 nm. This was found to decrease as a function of reaction time and reach a level consistent with a protein content of 3 % after about 4 hours. Fibrin I monomer in 250 ul portions was stored at -90 oC. No reduction in the stimulating capacity of the frozen monomer has been observed (6 months). The same is true for thawed preparations (24 hours observation time). The immunoglobulin fractions of goat anti-human tissue plasminogen activator serum and serum from non-imnunized goats were isolated by ammonium sulphate precipitation and DEAE-ion exchange chromatography (13). D-Valyl-L-Leusyl-L-Lysine-paranitroaniline (D-Val-Leu-Lys-pNA) was obtained from AB KABI, Peptide Research, Milndal, Sweden, by courtesy of Dr. L. Melstam. Concentration of the sub trate was determined spectrophotometrically at 316 nm where E = 12,800 M-lcm- 7 . Triton X-100 was purchased from Sigma, St. Louis, MO. USA, and Tween 80 from Kebo AB, Stockholm, Sweden. Protein and amino acid composition determinations of tissue plasminogen activator, plasminogen and fibrinogen were made by amino acid analysis. The samples were hydrolysed in redistilled constant boiling 5.7 M HCl in vacua for 24 h at 110 OC, and the determinations made on a modified Beckman 120 C amino acid analyzer. Clot lysis time assay (14) was performed at 37 'C in systems composed of fibrinogen (final concentration 0.3 PM), plasminogen (final concentration 0.65 PM) and activator (final concentration range 2.5 - 50 IU/ml). The incubation was started by addition of thrombin (final concentration 10 NIHunits/ml). Clot lysis times were in the range 3-11 minutes. Standard curves were constructed by plotting reciprocal lysis time against activator con-
Vo1.27, No.6
centration,
ACTIVATOR ASSAY
using an urokinase*
745
standard.
Spectrophotometric determinations were performed on a Titertek'Multiscan spectrophotometer (Flow Laboratories, Cal. USA) using 8 x 12 well flat bottomed polystyrene microtitration plates. The volume of the systems analysed was 202 ul.
ASSAY PROCEDURE Activator reagent composed of 1 uM Glu-plasminogen and 0.6 mM D-Val-Leu-LyspNA in 10 mM EDTA, 0.1 g/l sodium azide, 0.1 g/l Triton X-100, 0.1 M sodium phosphate pH 7.3 buffer was prepared and stored in 2 ml portions at -90 oC. Tissue plasminogen activator samples were dissolved in the sodium phosphate buffer described above to an expected concentration in the range 0.0005 0.1 Ill/ml. In the wells of a microtiter plate 100 ul of sample, 100 ul activator reagent and 2 ul 30 uM fibrin I monomer solution were mixed. The fibrin I monomer was added with a disposable plastic pipette, with which the final stirring was done. After incubation for 3 to 28 hours at 25 'C in a high humidity box the absorbance at 405 nm was measured. Background absorbance (due to turbidity and variations among the wells) was corrected for by subtracting the absorbance at 492 nm. At this wave length the absorbance of the liberated dye (para-nitro-aniline) is negligable (~1 % of A405). The correction was typically in the range 0.01 - 0.03.
RESULTS AND DISCUSSION Standard curves for 3, 8, and 28 hours incubation time are shown in figure 1. The absorbance at 405 nm is plotted against activator concentration in the 100 ul sample. As shown the assay measures activator in the concentration range 0.0003 - 0.08 IU/ml (0.001 - 0.4 ng/ml). The standard curves although linear do not pass through origo. This could be due to the presence of inhibitors (e.g. o2-antiplasmin), conversion of onechain to two-chain activator or an effect associated with the fibrin I stimulation. To investigate this, preparations of urokinase, one-chain and twochain tissue plasminogen activator were tested. The urokinase but not the one- and two-chain activator standard curves passed through origo. It was concluded that the lag observed is an effect associated with the fibrin I stimulation. Under assay conditions the stimulating effect of fibrin I on tissue plasminogen activator was found to be 3,000-fold but on urokinase fibrin I had no effect. Figure Id shows an alternative manner of plotting standard curves. The absorbance is plotted against activator concentration x time square, since the activator induced rise in absorbance is reported to be proportional to
* WHO International Laboratory for Biological Hampstead, London (code no 66/46).
Standards,
Holly Hill,
746
ACTIVATOR ASSAY
Vo1.27, No.6
both activator concentration and time square (3, 7). This linear plot for parabolic rate assays is convenient since arbitrary incubation times can be used. When plotted in this manner, the standard curves are almost parallel, but the lag increases somewhat with the incubation time (Fig. Id). The reason for this is unclear.
@ os-
z f 8 5 e::
::
IXa1 -
402
0.04
Activator
activity
W/ml)
activity
(IU/mlI
0.06
0.08
Activator
activity
(IU/ml)
a
b
Activator
Activator
d
FIG.
activity
x hours
square
(lIU/mllxh21
1
Assay of tissue plasminogen activator samples using 3, 8 and 28 hours incubation (la, lb, and lc, respectively). The absorbance at 405 nm is plotted against the activator activity (1 IU/ml = 4.6 ng/ml = 64 PM). The mean (0) and the standard deviation (I) of three determinations are shown. Mean absorbances below 0.040 have been omitted. The lines represent linear least square regression. In figure Id, the same data as on figure la (......), lb (------), and lc (-) have been used but the absorbance at 405 nm is plotted against activator concentration x times square. Urokinase, one-chain and two-chain tissue plasminogen activator preparations of equal strength (as determined by clot-lysis) were prepared. After identical dilutions the three preparations were tested in the assay system described and were found to be of nearly equal strength (15 % deviation). The Glu-plasminogen used in these experiments was of good quality and contained very small amounts of plasmin (less than 10 ppm). The absorbance in-
Vo1.27, No.6
ACTIVATOR ASSAY
747
crease when analysing a blank was about 0.0005lhour and no corrections were made. For longer incubation times ( >28 h) or when more plasmin is present corrections using a blank or activator antibodies (see below) should be made. The effect of variations in the reagent component concentrations was studied. A 20 % variation in Glu-plasminogen, fibrin I monomer, D-Val-Leu-Lys-pNA or urea concentration, all caused less than 5 % variation in assay reproducibility. As shown in figure 2, when more gen activator immunoglobulin is activity is practically reduced goats have no effect. Urokinase globulins.
than 5 r.g/ml goat anti-human tissue plasminoincluded in the assay system, the activator to zero. Immunoglobulins from non-immunized is not affected by the anti-activator immuno-
Immunogkbulin
concentration
(pq/ml)
FIG. 2. Inhibition of activator activity by goat anti-human tissue plasminogen activator immunoglobulin (A,=) or normal goat immunoglobulins (A,Cl). Apparent activator activity is plotted against final immunoglobulin concentration. The activator concentration was 0.1 (A, A) or 0.01 (I,O) IU/ml.
For highly specific tissue plasminogen activator measurements the following procedure was devised. Two batches of reagent were prepared, one containing 10 ug/ml anti-human tissue plasminogen activator immunoglobulins and the other 10 pg/ml normal immunoglobulins. Both reagents contained 1 uM Glu-plasminogen and 0.6 mM D-Val-Leu-Lys-pNA. The samples are analysed as described above using both reagents. Since only human tissue plasminogen activator (not urokinase) is quenched by the antibodies the difference between the two analyses can be taken as a measure of this activator in the sample. Contributions to the total absorbance at 405 nm from other activators or amidolytic
748
ACTIVATOR ASSAY
Vo1.27, No.6
activity (from sample or reagents) cancel out. This procedure can measure human tissue plasminogen activator concentrations in the presence of comparable amounts of urokinase and it makes the assay resistant to amidolytic activity in the sample. The assay can also be made tissue plasminogen activator specific by assaying in the presence and absence of fibrin I. This works fine in purified systems, but when analysing biological samples which may contain fibrin (or fibrinlike structures) the method may lead to grave underestimations of the tissue plasminogen activator content.
ACKNOWLEDGEMENT This study was supported by the Swedish Medical Research Council (project no. 13X-03906) and Bergwall's Stiftelse. Skilful technical assistance by Ms. Ulla Uhman is gratefully acknowledged.
REFERENCES 1.
RIJKEN, D.C., WIJNGAARDS, G. and WELBERGEN, J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall Thrombosis Research I&, 815-830, 1980.
2.
CAMIOLO, S.M., THORSEN, S. and ASTRUP, T. Fibrinogenolysis and Fibrinolysis with Tissue Plasminogen Activator, Urokinase, Streptokinase-Activated Human Globulin and Plasmin. Proc. Sot. Exp. Biol. and Med. 138, 277-280, 1971.
3.
RANBY, M. Studies on the kinetics of plasminogen activator by tissue plasminogen activator. Biochim. Biophys. Acta (in press) 1982.
4.
HAVERKATE, F., BRAKMAN, P. Fibrin Plate Assay. In: Progress in Chemical Fibrinolysis and Thrombosis. Vol. 1. J.F. Davidsson, M.M. Samama, P.C. Desnoyers (Eds.), Raven Press, New York, 1975, pp. 151-159.
5.
FEARNLEY, C.R., BALMFORTH, G. and FEARNLEY, E. Evidence of a diurnal fibrinolytic rhythm; with a simple method of measuring natural fibrinolysis. Clin. Sci. -16, 645-650, 1957.
6.
TOBIA, A., OSSOWSKI, L., QUIGLEY, J.P., RIFKIN, D.B. and UNKELESS, J.C., REICH, E. An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. J. Exp. Med. 137, 85-111, 1973.
7.
WALLEN, P., RANBY, M. and BERGSDORF, N. On the Stimulating Effect of Bathroxobin Induced Fibrin (Fibrin I) or Tissue Activator Induced Fibrinolysis. VIIIth International Congress on Thrombosis and Haemostasis (Abstract) 1981.
8.
RANBY, M. and WALLEN, P. A sensitive parabolic rate assay for tissue plasminogen activator. In: Progress in Fibrinolysis Vol. 5, J.F. Davidsson, I.M. Nilsson and 8. Astedt (Eds.), Churchill-Livingstone, Edinburgh, 1981, pp. 233-235.
Vo1.27, No.6
9.
ACTIVATOR ASSAY
749
WALLEN, P., RANBY, M., BERGSDORF, N. and KOK, P. Purification and characterization of tissue plasminogen activator: on the occurrence of two different forms and their enzymatic properties. In: Progress in Fibrinolysis Vol. 5, J.F. Davidsson, I.M. Nilsson and B. AChurch1'11 Livingstone, Edinburgh, 1981, pp. 16-23.
10.
WALLEN, P., POHL, G., BERGSDORF, N., RANBY, M., NY, T. and JURNWALL, H. Purification and structural characterization of a melanoma cell plasminogen activator. (submitted for publication) 1982.
11.
WALLEN, P. and WIMAN, B. Characterization Biophys. Acta 257, 122-134, 1972.
12.
BLOMBACK, B. and BLOMBACK, M. Purificat ion of human and bovine fibrinogen. Arkiv for kemi -10, 415-443, 1956.
13.
HARBOE, N. and INGILD, A. Immunization, isolation of immunoglobulins, estimation of antibody titre. In: Quant tative Immunoelectrophoresis, N.H. Axelsen, J. Levi11 and B. Weem s.), Universitetsfdrlaget, Oslo, 1973, p. 161.
14.
WALLiN, P., RANBY, M. and BERGSDORF, N. Purification of tissue plasminogen activator by fibrin adsorption. Biochim. Biophys. Acta (submitted for publication) 1982.
of human plasminogen.
Biochim.
BRIEF
A SENSITIVE
COMMUNICATION
ASSAY FOR TISSUE PLASMINOGEN
Mats Rdnby, Bo Norrman
ACTIVATOR
and Per Wallen
Department of Physiological Chemistry, Umea University, S-901 87 Umea, Sweden
(Received 12.5.1982; in revised form 22.6.1982. Accepted by Editor G. Myllyla) INTRODUCTION Tissue plasminogen activator has been shown to be identical to the activator released into circulation upon physical stress or venous occlusion as judged by electrophoretic and immunological criteria (1). Strikingly, tissue plasminogen activator is a good plasminogen activator only in the presence of certain polypeptide structures (2). Fibrin (but not fibrinogen) contains such structures and fibrin can induce an increase in activator turn-over number from 0.0001 5-l to 0.1 s- 1 (3). This stimulating effect is utilized in the loyed activator assays e.g. fibrin plate (4), standard clot lysis i~~"~~$ ?h! I-fibrin radiometric assay (6). In these assays fibrin is utilized both as stimulator and secondary (plasmin) substrate which typically renders the assays non-linear dose-response characteristics since the fibrin stimulating effect is reduced as the fibrin is degraded. The present paper describes a parabolic rate (coupled) tissue plasminogen activator assay which utilizes the stimulating properties of des A fibrin (7). Compared to a previously published protocol (8), the present assay procedure is 10 fold more sensitive, less reagent consuming and much easier to use. Furthermore, it is demonstrated that the presence of tissue plasminogen activator antibodies quantitatively quenches the activator activity whereas normal immunoglobulins have no effect.
MATERIALS Tissue plasminogen Key Words:
activator
AND METHODS
of the one-chain
form was prepared
from the
Tissue plasminogen activator, enzymatic assays, D-Val-Leu-Lys-paranitroaniline, S-2251, Glu-plasminogen, Fibrin I
743
744
ACTIVATOR ASSAY
vo1.27, No.6
culture medium of a human melanoma cell line by immunosorbent chromatography, argininesepharose affinity chromatography, and G-150 gel filtration chromatography (9, 10). The purity of the preparation was shown to be over 98 % by analysis of S-carboxymethylated samples on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The specific activity was 218,000 IU/mg. Stock solutions (20,400 IU/ml, 1.3 pM, assuming M, = 72,000) in 1 M ammonium bicarbonate containing 0.1 g/l Tween 80 were stored at -90 oC. Two-chain tissue plasminogen activator was prepared by incubating one-chain activator with Sepharose-plasmin as previously described (9). Plasminogen was prepared from freshly frozen titrated human plasma by lysine-Sepharose, gel filtration, and ion exchange chromatography (11). It was shown to fulfill the criteria specified by acetic urea polyacrylamide gel electrophoresis, streptokinase complex active site titration and N-terminal analysis (3, 11). Stock solutions (33.6 PM, assuming Mr = 93,000) in 0.065 M NaCl, 0.06 M Tris-HCl pH 8.5 buffer were stored at -90 oC. Fibrinogen was prepared from freshly frozen titrated human plasma (12). Stock in 0.3 M NaCl, 0.02 M sodium phossolutions (60 pM, assuming M, = 340,000 phate pH 7.3 buffer were stored at -20 b C. Bathroxobin (from Bothrops Moojeni) was a gift from Dr. K. Stocker, Pentapharm. AG, Basel, Switzerland. Solubilized des A fibrin (fibrin I monomer) was prepared by adding 10 ~1 250 BU/ml bathroxobin to 5 ml 60 uM fibrinogen and incubating at room temperature (a22 oC) for 6 hours. To the formed gel 5 ml 7 M urea was added and the mixture gently stirred until the gel dissolved. The completion. of the bathroxobin reaction was checked by centrifugating crushed gels at 15,000 x g for 15 minutes, followed by measurement of the UV-absorbance at 280 nm. This was found to decrease as a function of reaction time and reach a level consistent with a protein content of 3 % after about 4 hours. Fibrin I monomer in 250 ul portions was stored at -90 oC. No reduction in the stimulating capacity of the frozen monomer has been observed (6 months). The same is true for thawed preparations (24 hours observation time). The immunoglobulin fractions of goat anti-human tissue plasminogen activator serum and serum from non-imnunized goats were isolated by ammonium sulphate precipitation and DEAE-ion exchange chromatography (13). D-Valyl-L-Leusyl-L-Lysine-paranitroaniline (D-Val-Leu-Lys-pNA) was obtained from AB KABI, Peptide Research, Milndal, Sweden, by courtesy of Dr. L. Melstam. Concentration of the sub trate was determined spectrophotometrically at 316 nm where E = 12,800 M-lcm- 7 . Triton X-100 was purchased from Sigma, St. Louis, MO. USA, and Tween 80 from Kebo AB, Stockholm, Sweden. Protein and amino acid composition determinations of tissue plasminogen activator, plasminogen and fibrinogen were made by amino acid analysis. The samples were hydrolysed in redistilled constant boiling 5.7 M HCl in vacua for 24 h at 110 OC, and the determinations made on a modified Beckman 120 C amino acid analyzer. Clot lysis time assay (14) was performed at 37 'C in systems composed of fibrinogen (final concentration 0.3 PM), plasminogen (final concentration 0.65 PM) and activator (final concentration range 2.5 - 50 IU/ml). The incubation was started by addition of thrombin (final concentration 10 NIHunits/ml). Clot lysis times were in the range 3-11 minutes. Standard curves were constructed by plotting reciprocal lysis time against activator con-
Vo1.27, No.6
centration,
ACTIVATOR ASSAY
using an urokinase*
745
standard.
Spectrophotometric determinations were performed on a Titertek'Multiscan spectrophotometer (Flow Laboratories, Cal. USA) using 8 x 12 well flat bottomed polystyrene microtitration plates. The volume of the systems analysed was 202 ul.
ASSAY PROCEDURE Activator reagent composed of 1 uM Glu-plasminogen and 0.6 mM D-Val-Leu-LyspNA in 10 mM EDTA, 0.1 g/l sodium azide, 0.1 g/l Triton X-100, 0.1 M sodium phosphate pH 7.3 buffer was prepared and stored in 2 ml portions at -90 oC. Tissue plasminogen activator samples were dissolved in the sodium phosphate buffer described above to an expected concentration in the range 0.0005 0.1 Ill/ml. In the wells of a microtiter plate 100 ul of sample, 100 ul activator reagent and 2 ul 30 uM fibrin I monomer solution were mixed. The fibrin I monomer was added with a disposable plastic pipette, with which the final stirring was done. After incubation for 3 to 28 hours at 25 'C in a high humidity box the absorbance at 405 nm was measured. Background absorbance (due to turbidity and variations among the wells) was corrected for by subtracting the absorbance at 492 nm. At this wave length the absorbance of the liberated dye (para-nitro-aniline) is negligable (~1 % of A405). The correction was typically in the range 0.01 - 0.03.
RESULTS AND DISCUSSION Standard curves for 3, 8, and 28 hours incubation time are shown in figure 1. The absorbance at 405 nm is plotted against activator concentration in the 100 ul sample. As shown the assay measures activator in the concentration range 0.0003 - 0.08 IU/ml (0.001 - 0.4 ng/ml). The standard curves although linear do not pass through origo. This could be due to the presence of inhibitors (e.g. o2-antiplasmin), conversion of onechain to two-chain activator or an effect associated with the fibrin I stimulation. To investigate this, preparations of urokinase, one-chain and twochain tissue plasminogen activator were tested. The urokinase but not the one- and two-chain activator standard curves passed through origo. It was concluded that the lag observed is an effect associated with the fibrin I stimulation. Under assay conditions the stimulating effect of fibrin I on tissue plasminogen activator was found to be 3,000-fold but on urokinase fibrin I had no effect. Figure Id shows an alternative manner of plotting standard curves. The absorbance is plotted against activator concentration x time square, since the activator induced rise in absorbance is reported to be proportional to
* WHO International Laboratory for Biological Hampstead, London (code no 66/46).
Standards,
Holly Hill,
746
ACTIVATOR ASSAY
Vo1.27, No.6
both activator concentration and time square (3, 7). This linear plot for parabolic rate assays is convenient since arbitrary incubation times can be used. When plotted in this manner, the standard curves are almost parallel, but the lag increases somewhat with the incubation time (Fig. Id). The reason for this is unclear.
@ os-
z f 8 5 e::
::
IXa1 -
402
0.04
Activator
activity
W/ml)
activity
(IU/mlI
0.06
0.08
Activator
activity
(IU/ml)
a
b
Activator
Activator
d
FIG.
activity
x hours
square
(lIU/mllxh21
1
Assay of tissue plasminogen activator samples using 3, 8 and 28 hours incubation (la, lb, and lc, respectively). The absorbance at 405 nm is plotted against the activator activity (1 IU/ml = 4.6 ng/ml = 64 PM). The mean (0) and the standard deviation (I) of three determinations are shown. Mean absorbances below 0.040 have been omitted. The lines represent linear least square regression. In figure Id, the same data as on figure la (......), lb (------), and lc (-) have been used but the absorbance at 405 nm is plotted against activator concentration x times square. Urokinase, one-chain and two-chain tissue plasminogen activator preparations of equal strength (as determined by clot-lysis) were prepared. After identical dilutions the three preparations were tested in the assay system described and were found to be of nearly equal strength (15 % deviation). The Glu-plasminogen used in these experiments was of good quality and contained very small amounts of plasmin (less than 10 ppm). The absorbance in-
Vo1.27, No.6
ACTIVATOR ASSAY
747
crease when analysing a blank was about 0.0005lhour and no corrections were made. For longer incubation times ( >28 h) or when more plasmin is present corrections using a blank or activator antibodies (see below) should be made. The effect of variations in the reagent component concentrations was studied. A 20 % variation in Glu-plasminogen, fibrin I monomer, D-Val-Leu-Lys-pNA or urea concentration, all caused less than 5 % variation in assay reproducibility. As shown in figure 2, when more gen activator immunoglobulin is activity is practically reduced goats have no effect. Urokinase globulins.
than 5 r.g/ml goat anti-human tissue plasminoincluded in the assay system, the activator to zero. Immunoglobulins from non-immunized is not affected by the anti-activator immuno-
Immunogkbulin
concentration
(pq/ml)
FIG. 2. Inhibition of activator activity by goat anti-human tissue plasminogen activator immunoglobulin (A,=) or normal goat immunoglobulins (A,Cl). Apparent activator activity is plotted against final immunoglobulin concentration. The activator concentration was 0.1 (A, A) or 0.01 (I,O) IU/ml.
For highly specific tissue plasminogen activator measurements the following procedure was devised. Two batches of reagent were prepared, one containing 10 ug/ml anti-human tissue plasminogen activator immunoglobulins and the other 10 pg/ml normal immunoglobulins. Both reagents contained 1 uM Glu-plasminogen and 0.6 mM D-Val-Leu-Lys-pNA. The samples are analysed as described above using both reagents. Since only human tissue plasminogen activator (not urokinase) is quenched by the antibodies the difference between the two analyses can be taken as a measure of this activator in the sample. Contributions to the total absorbance at 405 nm from other activators or amidolytic
748
ACTIVATOR ASSAY
Vo1.27, No.6
activity (from sample or reagents) cancel out. This procedure can measure human tissue plasminogen activator concentrations in the presence of comparable amounts of urokinase and it makes the assay resistant to amidolytic activity in the sample. The assay can also be made tissue plasminogen activator specific by assaying in the presence and absence of fibrin I. This works fine in purified systems, but when analysing biological samples which may contain fibrin (or fibrinlike structures) the method may lead to grave underestimations of the tissue plasminogen activator content.
ACKNOWLEDGEMENT This study was supported by the Swedish Medical Research Council (project no. 13X-03906) and Bergwall's Stiftelse. Skilful technical assistance by Ms. Ulla Uhman is gratefully acknowledged.
REFERENCES 1.
RIJKEN, D.C., WIJNGAARDS, G. and WELBERGEN, J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall Thrombosis Research I&, 815-830, 1980.
2.
CAMIOLO, S.M., THORSEN, S. and ASTRUP, T. Fibrinogenolysis and Fibrinolysis with Tissue Plasminogen Activator, Urokinase, Streptokinase-Activated Human Globulin and Plasmin. Proc. Sot. Exp. Biol. and Med. 138, 277-280, 1971.
3.
RANBY, M. Studies on the kinetics of plasminogen activator by tissue plasminogen activator. Biochim. Biophys. Acta (in press) 1982.
4.
HAVERKATE, F., BRAKMAN, P. Fibrin Plate Assay. In: Progress in Chemical Fibrinolysis and Thrombosis. Vol. 1. J.F. Davidsson, M.M. Samama, P.C. Desnoyers (Eds.), Raven Press, New York, 1975, pp. 151-159.
5.
FEARNLEY, C.R., BALMFORTH, G. and FEARNLEY, E. Evidence of a diurnal fibrinolytic rhythm; with a simple method of measuring natural fibrinolysis. Clin. Sci. -16, 645-650, 1957.
6.
TOBIA, A., OSSOWSKI, L., QUIGLEY, J.P., RIFKIN, D.B. and UNKELESS, J.C., REICH, E. An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. J. Exp. Med. 137, 85-111, 1973.
7.
WALLEN, P., RANBY, M. and BERGSDORF, N. On the Stimulating Effect of Bathroxobin Induced Fibrin (Fibrin I) or Tissue Activator Induced Fibrinolysis. VIIIth International Congress on Thrombosis and Haemostasis (Abstract) 1981.
8.
RANBY, M. and WALLEN, P. A sensitive parabolic rate assay for tissue plasminogen activator. In: Progress in Fibrinolysis Vol. 5, J.F. Davidsson, I.M. Nilsson and 8. Astedt (Eds.), Churchill-Livingstone, Edinburgh, 1981, pp. 233-235.
Vo1.27, No.6
9.
ACTIVATOR ASSAY
749
WALLEN, P., RANBY, M., BERGSDORF, N. and KOK, P. Purification and characterization of tissue plasminogen activator: on the occurrence of two different forms and their enzymatic properties. In: Progress in Fibrinolysis Vol. 5, J.F. Davidsson, I.M. Nilsson and B. AChurch1'11 Livingstone, Edinburgh, 1981, pp. 16-23.
10.
WALLEN, P., POHL, G., BERGSDORF, N., RANBY, M., NY, T. and JURNWALL, H. Purification and structural characterization of a melanoma cell plasminogen activator. (submitted for publication) 1982.
11.
WALLEN, P. and WIMAN, B. Characterization Biophys. Acta 257, 122-134, 1972.
12.
BLOMBACK, B. and BLOMBACK, M. Purificat ion of human and bovine fibrinogen. Arkiv for kemi -10, 415-443, 1956.
13.
HARBOE, N. and INGILD, A. Immunization, isolation of immunoglobulins, estimation of antibody titre. In: Quant tative Immunoelectrophoresis, N.H. Axelsen, J. Levi11 and B. Weem s.), Universitetsfdrlaget, Oslo, 1973, p. 161.
14.
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