Haemostasis 5 : 295-305 (1976)
Differences in the Reactivities of Human Urokinase and the Porcine Tissue Plasminogen Activator Sixtus T horsen and T age Astrup Department of Clinical Chemistry, Hvidovre Hospital, Hvidovre, and Gaubius Institute, Netherlands Organization for Health Research (TNO), Leiden
Key Words. Fibrinolysis • Plasminogen activator ■Urokinase Abstract. It has previously been shown, that large differences exist between the effects of 6-aminohexanoic acid or ^-antitrypsin on fibrinolysis caused by a porcine tissue plas minogen activator or by human urokinase, while insignificant differences exist between the effects of a number of natural protease inhibitors on fibrinolysis caused by the two types of plasminogen activator. The present study shows that changes in substrate composition (pH, ionic strength, fibrinogen concentration, plasminogen concentration) may influence to different degrees the fibrinolytic activities of human urokinase and the porcine tissue plasminogen activator. It is suggested, that this finding is partly related to marked differences in affinity for fibrin of the two activators.
Introduction
Received: March 19, 1976; in revised form: June 6, 1976; accepted by editor H .S torJune 18, 1976.
morken :
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Plasminogen activators may originate from vascular endothelial cells, epithelial cells, mesothelial cells or leukocytes as well as from humoral precursors [5]. Highly purified activators have been prepared from human urine (urokinase) [28, 39], saline perfusion fluid from human cadavers (vascular activator) [3], animal tissues (tissue activator) [2, 6, 24, 31), or tissue culture supernatants [7, 17, 38]. Differences in molecular size, electro phoretic mobility, antigenic properties, or in reaction with inhibitors have been demonstrated between human activators of different origin [3, 4, 9, 26]
296
T horsen/A strup
or between human urokinase and activators from other species [18, 24-26, 32, 34]. Physicochemical, immunologic, or enzymatic similarities were re ported between human urokinase and plasminogen activator from human tissues (heart, peripheral veins, kidney cell cultures) [7, 10]. Particularly striking are the large differences in effect of 6-aminohexanoic acid or arantitrypsin on fibrinolysis caused by human urokinase or by porcine tissue plasminogen activator [18, 34], while insignificant differences exist in the effect of a number of natural protease inhibitors on fibrinolysis caused by the two activators [32]. The present report shows that differences between the reactivities of the two types of plasminogen activator can be demonstrated in the fibrinolytic assay system also in the absence of inhibitor. They respond differently to changes in pH, ionic strength, or substrate concentration.
Materials Buffers Barbital, Tris, or imidazole buffers: 0.05 m sodium diethylbarbiturate, 0.05 m Tris, HCI, or 0.05 m imidazole, HC1, respectively, in 0.10 m NaCI, with pH ranging from 6.50 to 8.25; ionic strength (I) 0.15. Gelatin buffers: buffer solution containing gelatin, 2.5 g/i.
Plasminogen Bovine plasminogen was prepared from a stock solution of bovine plasminogen-rich fibrinogen in gelatin barbital buffer, pH 7.75, by removing the fibrinogen by heat denaturation or by addition of thrombin [35].
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Fibrinogen, Thrombin and Activators Preparations of bovine plasminogen-rich fibrinogen (prepared by ammonium sulfate precipitation [13, 14], bovine plasminogen-free fibrinogen (prepared by adsorption of plas minogen to bentonite [12, 13] or obtained from Poviet Produkten, Amsterdam), bovine thrombin (Leo Pharmaceuticals, Copenhagen), human urokinase (Abbott Laboratories; Leo Pharmaceuticals and Sterling-Winthrop Laboratories) and porcine tissue plasminogen activator (product I and the ten times more purified product II prepared from pregnant hog ovaries [24]) were those previously reported [35]. Fibrinogen stock solutions were dia lyzed against 0.30 m NaCI, pH 7.40 (for pH experiments) or against buffer containing 0.02 m sodium diethylbarbiturate, pH 7.75 or 0.02 m Iris, HCI, pH 7.70 (20 C) in 0.28 m NaCI, 1 0.30. The concentration of fibrinogen in/(M in the dialyzed stock solutions was determined gravimetrically [13]. Prior to use the stock solutions were diluted with distilled water and adjusted with the appropriate buffer to the required fibrinogen concentration, pH and I. Solutions of thrombin and activators were prepared in gelatin buffer, or in 0.15 m NaCI containing gelatin, 2.5 g/l (for pH experiments).
Urokinase and Porcine Tissue Plasminogen Activator
297
Human plasminogen in gelatin-Tris-buffer, pH 7.70 or 8.00 (20 °C) was prepared from fresh serum euglobulin by gel filtration on Sephadex G-200 followed by ion exchange chromatography on DEAE Sephadex A-50 [1] or from fresh serum by affinity chromato graphy [20]. The concentration of plasminogen in micromolars was determined by active site titration with Trasylol® after conversion to plasmin [37]. It was assumed that the bovine and human preparations contained native plasminogen. They showed the expected increase in rate of activation by urokinase in the presence of 6-aminohexanoic acid (at concentrations from 0.1 to 3.3 mM) and the human plasminogen migrated with the /S2protein fraction in agarose gel electrophoresis at pH 8.6 [35, 37]. Dilutions were made in gelatin buffer.
Fibrinolytic activity was determined by a fibrin clot lysis time method. Solutions of fibrinogen and plasminogen were clotted in test tubes by mixtures of thrombin and acti vator. Air bubbles trapped in the fibrin network appeared during clot formation. The lysis time was recorded at 37 °C in seconds as the period of time between addition of the thrombin and until rising air bubbles passed midway through the liquifying clot. The reaction mixture, volume = 0.5 ml, was in buffer with pH ranging from 6.40 to 8.25 (37 °C) and with I ranging from 0.10 to 0.25. The thrombin concentration was 2 x I03 NIH U/l. An increase in the thrombin concentration from 103 NIH U/l (clotting time around 75 sec) to 8 x 103 NIH U/l (clotting time around 15 sec) did not change the lysis time with bovine plasminogen-rich fibrin (fibrinogen concentration, 4.4 // m , in buffer at pH 7.75 and I = 0.15), when lysis was induced by the tissue activator or urokinase within a 64-fold variation of activator concentration. Solutions of thrombin, plasminogen, and activators contained gelatin to protect against adsorption to glass [13]. The concen tration of gelatin varied from 0.5 to 1.25 g/1 in the reaction mixture. An increase in the gelatin concentration from 0 to 2.5 g/1 did not affect the lysis of the bovine plasminogenrich fibrin (fibrinogen concentration, 4 .4 /(M, in buffer at pH 7.75, I = 0.15) caused by gelatin-free solutions of tissue activator or urokinase within a 64-fold variation of acti vator concentration. The precision of the lysis time determination, mean ± SD for n = 10, at each of three concentrations of urokinase (covering a 32 times variation in activator concentration), using bovine plasminogen-rich fibrin (fibrinogen concentration, 4.4 /; m, in buffer at pH 7.75 and I = 0.15), was 349 ± 1.4, 729 ± 4, and 1,734 ± 8 sec. Interpolation on a reference curve of urokinase showed that SD was equivalent to a ± 0.5-1 % variation in activator concentration. Plotted double logarithmically with activator concentration as abscissa and lysis time as ordinate, dilutions of tissue activator or urokinase produced nearly linear dilution curves with slopes ranging from -0.45 to -0.70. Under similar assay conditions as above, the mean ± SD of the slopes of dilution curves obtained during a period of 1 years were, respectively, -0.48 ± 0.03 (n = 25) with tissue activator and -0.48 ± 0.02 (n = 36) with urokinase. The slopes were calculated from double logarithmic graphs with 5 points covering a 16-fold variation in activator concentration with lysis times between 300 and 1,800 sec.
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Method
298
T horsen/A strup
Results
Influence o f Changes in Ionic Strength An increase in I (from 0.10 to 0.25), caused by a change in the concen tration of sodium chloride in the final mixture at pH 7.35 (imidazole or Trisbuffer) or at pH 7.75 (barbital buffer), decreased the susceptibility of the fibrin to activator-induced lysis (fig. 2). The apparent decrease in activator activity was much more pronounced with the tissue activator than with urokinase. Similar patterns of results were obtained when the activator concentration was varied 32-fold. However, the decline in sensitivity of the substrate with increasing I, both at pH 7.35 and 7.75, became more pro-
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Influence o f Changes in pH The influence of a change in pH on activator-induced clot lysis in barbital and imidazole buffer is shown in figure 1. The apparent activity of the tissue activator is seen to decrease more rapidly than the activity of urokinase when pH is increased above 7.40. Similar differences in results with the two activators were obtained in Tris-buffer. The concentrations of the tissue activator at pH 8.00 in barbital, imidazole or Tris-buffer had to be in creased by a factor of 3.8, 2.7, and 2.0, respectively, in order to produce the same lysis time as that obtained at pH 7.40. In contrast, the concentrations of urokinase had to be increased by a factor of only 1.7, 1.5 and 1.4, respec tively. Each value is the mean of two experiments. The optimal pH range (defined as the range within which the concentrations of activator had to be increased by not more than 10% to yield the same lysis time as that at the pH optimum) was found with tissue activator to be from 7.05 to 7.55 in barbital or imidazole buffer and from 7.15 to 7.65 in the Tris-buffer. The optimal ranges were from 0.10 to 0.15 pH U higher with urokinase. Each pH range was obtained as the mean of two experiments. The relative in fluence of pH on fibrinolysis caused by the two activators between pH 6.50 and 8.00 remained the same when the activator concentrations were varied 32-fold. Variations in pH did not influence the slopes of the activator dilu tion curves. The pH curves obtained with preparations of tissue activator or urokinase of different purities (see Materials) were identical. Buffer concen trations and I (= 0 .1 5 ) in the final reaction mixtures were kept constant in all pH experiments. The pH was measured at 37 °C in solutions of the same composition as the final reaction mixtures except that thrombin and activator were absent. The pH was the same before and immediately after clot lysis.
Urokinase and Porcine Tissue Plasminogen Activator
299
2,0 0 0 -.
6.40 6.80 pH at 37 °C
7.20
7.60
800
8.40
Fig. 1. Influence of pH on bovine plasminogen-rich fibrin clot lysis caused by urokinase and tissue activator. Urokinase, 1.3 x 10‘ Ploug U/l, with barbital (•) and imidazole buffer (a ). Tissue activator, 2 x 105 A and A U/l, with barbital (O) and imidazole buffer ( a ). The fibrinogen concentration was 4.4 / im at I = 0.15. Component concentrations are in final reaction mixture.
1,000 -1
o
h -----------1-----
0.10
0.15
0.20
0.25
Fig. 2. Influence of I on bovine plasminogen-rich fibrin clot lysis caused by urokinase and tissue activator. Urokinase, 104 Ploug U/l (•), and tissue activator, 105 A and A U/l (O). The fibrinogen concentration was 4.4 /¿m in barbital buffer at pH 7.75. Compo nent concentrations are in final reaction mixture.
Downloaded by: King's College London 137.73.144.138 - 5/15/2018 10:51:25 AM
Ionic strength
300
T horsen /A strup
nounced at decreasing activator concentrations, because the slopes of the activator dilution curves decreased with increasing I. Thus, in an experiment at pH 7.35 the slope changed from -0.46 to -0.59 with urokinase and from -0.46 to -0.68 with tissue activator, when I was increased from 0.10 to 0.25. Tissue activator or urokinase preparations of different purity (see Mate rials) behaved similarly at varying I. Experiments with human plasminogen gave the same patterns as those obtained with the bovine plasminogen.
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Influence o f Changes in Substrate Concentration An increase in the concentration of the plasminogen-rich fibrinogen at pH 7.35 (imidazole buffer) or at pH 7.75 (barbital buffer) decreased the ap parent activity of the tissue activator. In contrast, the apparent activity of urokinase increased slightly (fig. 3). The tissue activator concentration had to be increased by factor of 3 to yield the same lysis time at a fibrinogen concentration of 17.6 /¿m as at a fibrinogen concentration of 1.10 /iM, while the concentration of urokinase had to be decreased by a factor of 0.75. The influence of the change in concentration of the plasminogen-rich fibrinogen remained the same when the activator concentration was varied 32-fold, since the slope of the activator dilution curves changed little within the concen tration range of fibrinogen studied. Identical results were obtained with tissue activator or urokinase preparations of different purity (see Materi als). When the concentrations of fibrinogen and human or bovine plasminogen were separately varied at pH 7.35 (Tris-buffer) or at pH 7.75 (barbital buffer) it was found that the differences observed with urokinase and the tissue activator at varying concentrations of the bovine plasminogen-rich fibrinogen (fig. 3) were caused mainly by the change in fibrinogen concen tration. The decrease in lysis time (fig. 3) resulting from a separate increase in the fibrinogen concentration from 1.15 to 18.5 /um corresponded to a 23-times change in tissue activator concentration and a 7-times change in urokinase concentration (both referred to a fibrinogen concentration of 4.4 /¿m). Similar patterns were obtained when the activator concentration was increased 8-fold or when the plasminogen concentration was decreased 4-fold. Figure 4 shows that an increase in the concentration of plasminogen at a constant concentration of fibrinogen produced a considerable increase in the apparent activity of both activators. The effect on urokinase was only slightly more pronounced than on the tissue activator. Similar results were obtained when the activator concentration was varied 5-fold.
Urokinase and Porcine Tissue Plasminogen Activator
301
4,000-i
■ 'H h ----------------- I---------------------- ,-----------------1-----------------1
1
2
5
10
20
Fibrinogen concentration, pM
Fig. 3. Fibrin clot lysis caused by urokinase and tissue activator at varying concentra tions of bovine plasminogen-rich fibrinogen or at varying concentrations of bovine plasminogen-free fibrinogen with a constant concentration of human plasminogen. Plasmin ogen-rich fibrinogen in imidazole buffer at pH 7.35 (37 °C) and I = 0.15. Urokinase, 8.3 X 103 Ploug U/l (•), and tissue activator, 105 A and A U/l (o ). Plasminogen-free fibrinogen with human plasminogen, 0.8 /
Differences in the Reactivities of Human Urokinase and the Porcine Tissue Plasminogen Activator Sixtus T horsen and T age Astrup Department of Clinical Chemistry, Hvidovre Hospital, Hvidovre, and Gaubius Institute, Netherlands Organization for Health Research (TNO), Leiden
Key Words. Fibrinolysis • Plasminogen activator ■Urokinase Abstract. It has previously been shown, that large differences exist between the effects of 6-aminohexanoic acid or ^-antitrypsin on fibrinolysis caused by a porcine tissue plas minogen activator or by human urokinase, while insignificant differences exist between the effects of a number of natural protease inhibitors on fibrinolysis caused by the two types of plasminogen activator. The present study shows that changes in substrate composition (pH, ionic strength, fibrinogen concentration, plasminogen concentration) may influence to different degrees the fibrinolytic activities of human urokinase and the porcine tissue plasminogen activator. It is suggested, that this finding is partly related to marked differences in affinity for fibrin of the two activators.
Introduction
Received: March 19, 1976; in revised form: June 6, 1976; accepted by editor H .S torJune 18, 1976.
morken :
Downloaded by: King's College London 137.73.144.138 - 5/15/2018 10:51:25 AM
Plasminogen activators may originate from vascular endothelial cells, epithelial cells, mesothelial cells or leukocytes as well as from humoral precursors [5]. Highly purified activators have been prepared from human urine (urokinase) [28, 39], saline perfusion fluid from human cadavers (vascular activator) [3], animal tissues (tissue activator) [2, 6, 24, 31), or tissue culture supernatants [7, 17, 38]. Differences in molecular size, electro phoretic mobility, antigenic properties, or in reaction with inhibitors have been demonstrated between human activators of different origin [3, 4, 9, 26]
296
T horsen/A strup
or between human urokinase and activators from other species [18, 24-26, 32, 34]. Physicochemical, immunologic, or enzymatic similarities were re ported between human urokinase and plasminogen activator from human tissues (heart, peripheral veins, kidney cell cultures) [7, 10]. Particularly striking are the large differences in effect of 6-aminohexanoic acid or arantitrypsin on fibrinolysis caused by human urokinase or by porcine tissue plasminogen activator [18, 34], while insignificant differences exist in the effect of a number of natural protease inhibitors on fibrinolysis caused by the two activators [32]. The present report shows that differences between the reactivities of the two types of plasminogen activator can be demonstrated in the fibrinolytic assay system also in the absence of inhibitor. They respond differently to changes in pH, ionic strength, or substrate concentration.
Materials Buffers Barbital, Tris, or imidazole buffers: 0.05 m sodium diethylbarbiturate, 0.05 m Tris, HCI, or 0.05 m imidazole, HC1, respectively, in 0.10 m NaCI, with pH ranging from 6.50 to 8.25; ionic strength (I) 0.15. Gelatin buffers: buffer solution containing gelatin, 2.5 g/i.
Plasminogen Bovine plasminogen was prepared from a stock solution of bovine plasminogen-rich fibrinogen in gelatin barbital buffer, pH 7.75, by removing the fibrinogen by heat denaturation or by addition of thrombin [35].
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Fibrinogen, Thrombin and Activators Preparations of bovine plasminogen-rich fibrinogen (prepared by ammonium sulfate precipitation [13, 14], bovine plasminogen-free fibrinogen (prepared by adsorption of plas minogen to bentonite [12, 13] or obtained from Poviet Produkten, Amsterdam), bovine thrombin (Leo Pharmaceuticals, Copenhagen), human urokinase (Abbott Laboratories; Leo Pharmaceuticals and Sterling-Winthrop Laboratories) and porcine tissue plasminogen activator (product I and the ten times more purified product II prepared from pregnant hog ovaries [24]) were those previously reported [35]. Fibrinogen stock solutions were dia lyzed against 0.30 m NaCI, pH 7.40 (for pH experiments) or against buffer containing 0.02 m sodium diethylbarbiturate, pH 7.75 or 0.02 m Iris, HCI, pH 7.70 (20 C) in 0.28 m NaCI, 1 0.30. The concentration of fibrinogen in/(M in the dialyzed stock solutions was determined gravimetrically [13]. Prior to use the stock solutions were diluted with distilled water and adjusted with the appropriate buffer to the required fibrinogen concentration, pH and I. Solutions of thrombin and activators were prepared in gelatin buffer, or in 0.15 m NaCI containing gelatin, 2.5 g/l (for pH experiments).
Urokinase and Porcine Tissue Plasminogen Activator
297
Human plasminogen in gelatin-Tris-buffer, pH 7.70 or 8.00 (20 °C) was prepared from fresh serum euglobulin by gel filtration on Sephadex G-200 followed by ion exchange chromatography on DEAE Sephadex A-50 [1] or from fresh serum by affinity chromato graphy [20]. The concentration of plasminogen in micromolars was determined by active site titration with Trasylol® after conversion to plasmin [37]. It was assumed that the bovine and human preparations contained native plasminogen. They showed the expected increase in rate of activation by urokinase in the presence of 6-aminohexanoic acid (at concentrations from 0.1 to 3.3 mM) and the human plasminogen migrated with the /S2protein fraction in agarose gel electrophoresis at pH 8.6 [35, 37]. Dilutions were made in gelatin buffer.
Fibrinolytic activity was determined by a fibrin clot lysis time method. Solutions of fibrinogen and plasminogen were clotted in test tubes by mixtures of thrombin and acti vator. Air bubbles trapped in the fibrin network appeared during clot formation. The lysis time was recorded at 37 °C in seconds as the period of time between addition of the thrombin and until rising air bubbles passed midway through the liquifying clot. The reaction mixture, volume = 0.5 ml, was in buffer with pH ranging from 6.40 to 8.25 (37 °C) and with I ranging from 0.10 to 0.25. The thrombin concentration was 2 x I03 NIH U/l. An increase in the thrombin concentration from 103 NIH U/l (clotting time around 75 sec) to 8 x 103 NIH U/l (clotting time around 15 sec) did not change the lysis time with bovine plasminogen-rich fibrin (fibrinogen concentration, 4.4 // m , in buffer at pH 7.75 and I = 0.15), when lysis was induced by the tissue activator or urokinase within a 64-fold variation of activator concentration. Solutions of thrombin, plasminogen, and activators contained gelatin to protect against adsorption to glass [13]. The concen tration of gelatin varied from 0.5 to 1.25 g/1 in the reaction mixture. An increase in the gelatin concentration from 0 to 2.5 g/1 did not affect the lysis of the bovine plasminogenrich fibrin (fibrinogen concentration, 4 .4 /(M, in buffer at pH 7.75, I = 0.15) caused by gelatin-free solutions of tissue activator or urokinase within a 64-fold variation of acti vator concentration. The precision of the lysis time determination, mean ± SD for n = 10, at each of three concentrations of urokinase (covering a 32 times variation in activator concentration), using bovine plasminogen-rich fibrin (fibrinogen concentration, 4.4 /; m, in buffer at pH 7.75 and I = 0.15), was 349 ± 1.4, 729 ± 4, and 1,734 ± 8 sec. Interpolation on a reference curve of urokinase showed that SD was equivalent to a ± 0.5-1 % variation in activator concentration. Plotted double logarithmically with activator concentration as abscissa and lysis time as ordinate, dilutions of tissue activator or urokinase produced nearly linear dilution curves with slopes ranging from -0.45 to -0.70. Under similar assay conditions as above, the mean ± SD of the slopes of dilution curves obtained during a period of 1 years were, respectively, -0.48 ± 0.03 (n = 25) with tissue activator and -0.48 ± 0.02 (n = 36) with urokinase. The slopes were calculated from double logarithmic graphs with 5 points covering a 16-fold variation in activator concentration with lysis times between 300 and 1,800 sec.
Downloaded by: King's College London 137.73.144.138 - 5/15/2018 10:51:25 AM
Method
298
T horsen/A strup
Results
Influence o f Changes in Ionic Strength An increase in I (from 0.10 to 0.25), caused by a change in the concen tration of sodium chloride in the final mixture at pH 7.35 (imidazole or Trisbuffer) or at pH 7.75 (barbital buffer), decreased the susceptibility of the fibrin to activator-induced lysis (fig. 2). The apparent decrease in activator activity was much more pronounced with the tissue activator than with urokinase. Similar patterns of results were obtained when the activator concentration was varied 32-fold. However, the decline in sensitivity of the substrate with increasing I, both at pH 7.35 and 7.75, became more pro-
Downloaded by: King's College London 137.73.144.138 - 5/15/2018 10:51:25 AM
Influence o f Changes in pH The influence of a change in pH on activator-induced clot lysis in barbital and imidazole buffer is shown in figure 1. The apparent activity of the tissue activator is seen to decrease more rapidly than the activity of urokinase when pH is increased above 7.40. Similar differences in results with the two activators were obtained in Tris-buffer. The concentrations of the tissue activator at pH 8.00 in barbital, imidazole or Tris-buffer had to be in creased by a factor of 3.8, 2.7, and 2.0, respectively, in order to produce the same lysis time as that obtained at pH 7.40. In contrast, the concentrations of urokinase had to be increased by a factor of only 1.7, 1.5 and 1.4, respec tively. Each value is the mean of two experiments. The optimal pH range (defined as the range within which the concentrations of activator had to be increased by not more than 10% to yield the same lysis time as that at the pH optimum) was found with tissue activator to be from 7.05 to 7.55 in barbital or imidazole buffer and from 7.15 to 7.65 in the Tris-buffer. The optimal ranges were from 0.10 to 0.15 pH U higher with urokinase. Each pH range was obtained as the mean of two experiments. The relative in fluence of pH on fibrinolysis caused by the two activators between pH 6.50 and 8.00 remained the same when the activator concentrations were varied 32-fold. Variations in pH did not influence the slopes of the activator dilu tion curves. The pH curves obtained with preparations of tissue activator or urokinase of different purities (see Materials) were identical. Buffer concen trations and I (= 0 .1 5 ) in the final reaction mixtures were kept constant in all pH experiments. The pH was measured at 37 °C in solutions of the same composition as the final reaction mixtures except that thrombin and activator were absent. The pH was the same before and immediately after clot lysis.
Urokinase and Porcine Tissue Plasminogen Activator
299
2,0 0 0 -.
6.40 6.80 pH at 37 °C
7.20
7.60
800
8.40
Fig. 1. Influence of pH on bovine plasminogen-rich fibrin clot lysis caused by urokinase and tissue activator. Urokinase, 1.3 x 10‘ Ploug U/l, with barbital (•) and imidazole buffer (a ). Tissue activator, 2 x 105 A and A U/l, with barbital (O) and imidazole buffer ( a ). The fibrinogen concentration was 4.4 / im at I = 0.15. Component concentrations are in final reaction mixture.
1,000 -1
o
h -----------1-----
0.10
0.15
0.20
0.25
Fig. 2. Influence of I on bovine plasminogen-rich fibrin clot lysis caused by urokinase and tissue activator. Urokinase, 104 Ploug U/l (•), and tissue activator, 105 A and A U/l (O). The fibrinogen concentration was 4.4 /¿m in barbital buffer at pH 7.75. Compo nent concentrations are in final reaction mixture.
Downloaded by: King's College London 137.73.144.138 - 5/15/2018 10:51:25 AM
Ionic strength
300
T horsen /A strup
nounced at decreasing activator concentrations, because the slopes of the activator dilution curves decreased with increasing I. Thus, in an experiment at pH 7.35 the slope changed from -0.46 to -0.59 with urokinase and from -0.46 to -0.68 with tissue activator, when I was increased from 0.10 to 0.25. Tissue activator or urokinase preparations of different purity (see Mate rials) behaved similarly at varying I. Experiments with human plasminogen gave the same patterns as those obtained with the bovine plasminogen.
Downloaded by: King's College London 137.73.144.138 - 5/15/2018 10:51:25 AM
Influence o f Changes in Substrate Concentration An increase in the concentration of the plasminogen-rich fibrinogen at pH 7.35 (imidazole buffer) or at pH 7.75 (barbital buffer) decreased the ap parent activity of the tissue activator. In contrast, the apparent activity of urokinase increased slightly (fig. 3). The tissue activator concentration had to be increased by factor of 3 to yield the same lysis time at a fibrinogen concentration of 17.6 /¿m as at a fibrinogen concentration of 1.10 /iM, while the concentration of urokinase had to be decreased by a factor of 0.75. The influence of the change in concentration of the plasminogen-rich fibrinogen remained the same when the activator concentration was varied 32-fold, since the slope of the activator dilution curves changed little within the concen tration range of fibrinogen studied. Identical results were obtained with tissue activator or urokinase preparations of different purity (see Materi als). When the concentrations of fibrinogen and human or bovine plasminogen were separately varied at pH 7.35 (Tris-buffer) or at pH 7.75 (barbital buffer) it was found that the differences observed with urokinase and the tissue activator at varying concentrations of the bovine plasminogen-rich fibrinogen (fig. 3) were caused mainly by the change in fibrinogen concen tration. The decrease in lysis time (fig. 3) resulting from a separate increase in the fibrinogen concentration from 1.15 to 18.5 /um corresponded to a 23-times change in tissue activator concentration and a 7-times change in urokinase concentration (both referred to a fibrinogen concentration of 4.4 /¿m). Similar patterns were obtained when the activator concentration was increased 8-fold or when the plasminogen concentration was decreased 4-fold. Figure 4 shows that an increase in the concentration of plasminogen at a constant concentration of fibrinogen produced a considerable increase in the apparent activity of both activators. The effect on urokinase was only slightly more pronounced than on the tissue activator. Similar results were obtained when the activator concentration was varied 5-fold.
Urokinase and Porcine Tissue Plasminogen Activator
301
4,000-i
■ 'H h ----------------- I---------------------- ,-----------------1-----------------1
1
2
5
10
20
Fibrinogen concentration, pM
Fig. 3. Fibrin clot lysis caused by urokinase and tissue activator at varying concentra tions of bovine plasminogen-rich fibrinogen or at varying concentrations of bovine plasminogen-free fibrinogen with a constant concentration of human plasminogen. Plasmin ogen-rich fibrinogen in imidazole buffer at pH 7.35 (37 °C) and I = 0.15. Urokinase, 8.3 X 103 Ploug U/l (•), and tissue activator, 105 A and A U/l (o ). Plasminogen-free fibrinogen with human plasminogen, 0.8 /
