Comp. Biochem. Physiol. Vol. 102B, No. 1, pp. 159-162, 1992 Printed in Great Britain
0305-0491/92 $5.00+0.00 © 1992 Pergamon Press Ltd
E F F E C T OF V A R I O U S P O L Y A M I N O A C I D S A N D D- A N D L-AMINO A C I D S O N THE B L O O D F I B R I N O L Y T I C SYSTEM HIROYUKI SUMI,* KYOKO KAWABE~ and NOBUYOSHINAKAJIMA* *Department of Nutrition and Food Science, Okayama Prefectural College, Okayama 700; and ?Department of Physiology, Miyazaki Medical College, Miyazaki 889-16, Japan (Received 29 July 1991) Abstract--l. Comparative additional effects of 24 commercially obtained amino acids and their derivatives were studied by the whole blood clot lysis time (WBCLT) method, o-Arg and L-Lys (> 50 #g/ml) activated the fibrinolytic system, and poly-L-Glu (mol. wt 6000-90,000) (< 50/lg/ml) were less effective. 2. Poly-L-Arg, poly-L-Lys and poly-(Lys-Ala-Glu-Tyr) accelated the TPA inhibition in the presence of human plasma. 3. For in rico experiments, 5 mg of poly-L-Glu were given intravenously to 10 rats. 4. The shortening of WBCLT and elevation of EFA (P < 0.01) were found after 1 hr administration. 5. The main enzymes which increased in plasma were proved to be the endogenous plasminogen activators with mol. wt higher than 70,000, by zymography.
INTRODUCTION
(1941). Astrup and Miillertz's (1952) revised method was adopted for the preparation of a fibrin plate to see the effect of the sample on UK and with TPA. According to this method a plate was prepared by adding 1% of agar to 0.4% of fibrinogen (by Miles Laboratories, USA) to 0.17M borate of pH 7.8. With 30 #l of sample applied, a lysis area (square millimetres), created 18 hr after incubation at 37°C, was measured. A zymography was performed according to Tissot et aL's method (1982). To prepare a fibrin-agar plate for the zymography, regular plasminogen-rich fibrinogen (Type lS, Lot 58F-9468: Sigma Chemical Co.) was used. Plasminogen-free fibrinogen passed through the Lys-Sepharose column by the conventional method, was used in some tests.
Since the discovery of a strong fibrinolytic enzyme nattokinase (NK) in one of the Japanese traditional foodstuffs, natto, we have reported the effect of this enzyme, when administered orally, on the blood fibrinolytic system (Sumi et al., 1987, 1989, 1990). In the process of our experiment on the enzyme it has become clear that a peculiar viscous matter contained in the natto (amino acid polymer) apparently contributes to the stimulation of clot lysis. Inspired by this finding, we have compared the effects of 24 kinds of D- or L-amino acid polymer or m o n o m e r on the blood fibrinolytic system for this report. An in vivo test was also carried out on poly-L-Glu using rats. It was proved that poly-L-Glu reacts to produce an endogenous plasminogen activator.
RESULTS The effects of various kinds of D- and L-amino acid monomers and polymers on W B C L T were compared. The result indicated, as shown in Table 1, that D-Arg and D-Lys had the strongest clot lysis stimulation power when added in high density (500 #g/ml), but that when added in low density (12.5 or 50/tg/ml), among polyamino acids, poly-L-Lys and poly-L-Glu effected clot lysis stimulation, though only slightly, at different mol. wt. However, poly-Lys, especially those of high mol. wt were found to have a clot involuting effect at the same time, and in high density to introduce a strong clot lysis inhibiting effect. Meanwhile, little direct effect was found to be made on T P A by any amino acid m o n o m e r or polyamino acid in observations by the fibrin plate method (Table 2). Under the same conditions, polymers of some molecular types [poly-L-Arg, poly-L-lys and poly-(Lys-Ala-Giu-Tyr)] proved to augment a T P A inhibiting effect in the presence of the plasma (Table 2, upper right). Yet such a phenomenon could not be recognized when U K was used. With poly-L-Glu, the effect o f its administration to rats by i.v. injection (5 mg) was examined as well. Changes in W B C L T and fibrinolytic activity in plasma euglobulin showed that the administration of
MATERIALS AND METHODS Polyamino acids and D- and L-amino acids were purchased from Sigma Chemical Co., and urokinase (UK) from Green Cross Co., Osaka, Japan. A tissue plasminogen activator (TPA), extracted from the pig uterus with 0.3 M potassium acetate by Bachmann's method (1964), was refined by ammonium sulphate graduation to make a sample with a spec. act. of 37.3 IU/mg. Male Wistar strain rats, weighing 400-420 g, were fasted for 24 hr prior to use. To determine the effect of poly amino acid, it was injected i.v. in the tails of fasted rats at 5.0 mg 0.5 ml saline-i body wt. The control received 0.5 ml of saline without enzyme. Each group comprised 10 animals. Blood was drawn at intervals from each animal in 1/10 vol of 3.8% citrate and plasma obtained by centrifugation at 3000 rpm for 10 min. Blood fibrinolytic activity in the blood was measured by Chohan's (1975) whole blood clot lysis time (WBCLT) technique; 0.1 ml thrombin (50 U/ml by Mochida Pharmaceutical Co., Tokyo, Japan) was added to each 0.2 ml citrated blood in the system of reaction of 0.12 M sodium acetate in pH 7.4 (2.0 ml), which was incubated at 37°C to measure lysis time after clot formation. The preparation of plasma euglobulin was performed by Milstone's method 159
160
HIROYUKI SUMI et al. Table 1. The effect of the addition of various amino acid monomers and polymers on whole WBCLT blood clot lysis time (Approx. mol. wt) L-Arg D-Arg Poly-L-Arg L-Lys D-Lys Poly-L-Lys Poly-D-Lys
Poly-(Lys-Ala-Glu-Tyr) L-Glu D-GIu Poly-L-GIu Poly-D-Glu L-GIn D-GIn DL-Asp DL-Asn Gly
12.5 #g/ml
50 #g/ml
500 #g/ml
112
109 119 113 117 113 164" 109 117 --* --* ~
151 217 1733'* 149 219 173" 133" 274* --* ~* ~*
-
(100,000)
-
--
-
-
-
(14,000) (90,000) (12,000) (65,000) (100,000) (200,000)
--* 112 -131 --* 112
(540,000)
142
~
(52,000)
--
~
~*
(6000) (32,000) (60,000) (66,000)
ND ND ND 108 ND -ND ND
ll3 115 113
102 -102
-
-
ND ND
~*
--
--
117 115 ND ND 120 ND ND
----
-
113 --
--
Two tenths of a millilitre of normal human blood and various agents were added to 1.7 ml 0.12 M sodium acetate buffer of pH 7.4 (12.5-500 #g/ml blood), to which in turn 0.1 ml thrombin (50 U/ml) was added to let it react. Blood fibrinolytic activity is represented by I/clot lysis time (minutes), while figures represent relative values to the assumed control value of 100 (clot lysis time: 30 min) under the condition that no agent is added. *Signifies the swollen clot owing to deficient clot involution; **fallen clot owing to deficient clot involution; clot lysis strongly inhibited with the advance of clot involution; --blood clot remaining even after 530 min incubation at 37°C.
poly-L-Glu effected a slow process of clot lysis stimulation with its peak appearing about 1 hr after the administration, with WBCLT 2 3 2 _ 26 min before the administration, tending to diminish to 225 + 42, 190+ 15 and 211 __+23 min 20, 60 and 90min after the administration, respectively, and fibrin resolving power in plasma euglobulin, which had been
2 8 _+ 1 1 m m 2 b e f o r e t h e a d m i n i s t r a t i o n , c h a n g i n g t o 53_+16, 78_13 and 65_ ll mm 2(n=10;P
0305-0491/92 $5.00+0.00 © 1992 Pergamon Press Ltd
E F F E C T OF V A R I O U S P O L Y A M I N O A C I D S A N D D- A N D L-AMINO A C I D S O N THE B L O O D F I B R I N O L Y T I C SYSTEM HIROYUKI SUMI,* KYOKO KAWABE~ and NOBUYOSHINAKAJIMA* *Department of Nutrition and Food Science, Okayama Prefectural College, Okayama 700; and ?Department of Physiology, Miyazaki Medical College, Miyazaki 889-16, Japan (Received 29 July 1991) Abstract--l. Comparative additional effects of 24 commercially obtained amino acids and their derivatives were studied by the whole blood clot lysis time (WBCLT) method, o-Arg and L-Lys (> 50 #g/ml) activated the fibrinolytic system, and poly-L-Glu (mol. wt 6000-90,000) (< 50/lg/ml) were less effective. 2. Poly-L-Arg, poly-L-Lys and poly-(Lys-Ala-Glu-Tyr) accelated the TPA inhibition in the presence of human plasma. 3. For in rico experiments, 5 mg of poly-L-Glu were given intravenously to 10 rats. 4. The shortening of WBCLT and elevation of EFA (P < 0.01) were found after 1 hr administration. 5. The main enzymes which increased in plasma were proved to be the endogenous plasminogen activators with mol. wt higher than 70,000, by zymography.
INTRODUCTION
(1941). Astrup and Miillertz's (1952) revised method was adopted for the preparation of a fibrin plate to see the effect of the sample on UK and with TPA. According to this method a plate was prepared by adding 1% of agar to 0.4% of fibrinogen (by Miles Laboratories, USA) to 0.17M borate of pH 7.8. With 30 #l of sample applied, a lysis area (square millimetres), created 18 hr after incubation at 37°C, was measured. A zymography was performed according to Tissot et aL's method (1982). To prepare a fibrin-agar plate for the zymography, regular plasminogen-rich fibrinogen (Type lS, Lot 58F-9468: Sigma Chemical Co.) was used. Plasminogen-free fibrinogen passed through the Lys-Sepharose column by the conventional method, was used in some tests.
Since the discovery of a strong fibrinolytic enzyme nattokinase (NK) in one of the Japanese traditional foodstuffs, natto, we have reported the effect of this enzyme, when administered orally, on the blood fibrinolytic system (Sumi et al., 1987, 1989, 1990). In the process of our experiment on the enzyme it has become clear that a peculiar viscous matter contained in the natto (amino acid polymer) apparently contributes to the stimulation of clot lysis. Inspired by this finding, we have compared the effects of 24 kinds of D- or L-amino acid polymer or m o n o m e r on the blood fibrinolytic system for this report. An in vivo test was also carried out on poly-L-Glu using rats. It was proved that poly-L-Glu reacts to produce an endogenous plasminogen activator.
RESULTS The effects of various kinds of D- and L-amino acid monomers and polymers on W B C L T were compared. The result indicated, as shown in Table 1, that D-Arg and D-Lys had the strongest clot lysis stimulation power when added in high density (500 #g/ml), but that when added in low density (12.5 or 50/tg/ml), among polyamino acids, poly-L-Lys and poly-L-Glu effected clot lysis stimulation, though only slightly, at different mol. wt. However, poly-Lys, especially those of high mol. wt were found to have a clot involuting effect at the same time, and in high density to introduce a strong clot lysis inhibiting effect. Meanwhile, little direct effect was found to be made on T P A by any amino acid m o n o m e r or polyamino acid in observations by the fibrin plate method (Table 2). Under the same conditions, polymers of some molecular types [poly-L-Arg, poly-L-lys and poly-(Lys-Ala-Giu-Tyr)] proved to augment a T P A inhibiting effect in the presence of the plasma (Table 2, upper right). Yet such a phenomenon could not be recognized when U K was used. With poly-L-Glu, the effect o f its administration to rats by i.v. injection (5 mg) was examined as well. Changes in W B C L T and fibrinolytic activity in plasma euglobulin showed that the administration of
MATERIALS AND METHODS Polyamino acids and D- and L-amino acids were purchased from Sigma Chemical Co., and urokinase (UK) from Green Cross Co., Osaka, Japan. A tissue plasminogen activator (TPA), extracted from the pig uterus with 0.3 M potassium acetate by Bachmann's method (1964), was refined by ammonium sulphate graduation to make a sample with a spec. act. of 37.3 IU/mg. Male Wistar strain rats, weighing 400-420 g, were fasted for 24 hr prior to use. To determine the effect of poly amino acid, it was injected i.v. in the tails of fasted rats at 5.0 mg 0.5 ml saline-i body wt. The control received 0.5 ml of saline without enzyme. Each group comprised 10 animals. Blood was drawn at intervals from each animal in 1/10 vol of 3.8% citrate and plasma obtained by centrifugation at 3000 rpm for 10 min. Blood fibrinolytic activity in the blood was measured by Chohan's (1975) whole blood clot lysis time (WBCLT) technique; 0.1 ml thrombin (50 U/ml by Mochida Pharmaceutical Co., Tokyo, Japan) was added to each 0.2 ml citrated blood in the system of reaction of 0.12 M sodium acetate in pH 7.4 (2.0 ml), which was incubated at 37°C to measure lysis time after clot formation. The preparation of plasma euglobulin was performed by Milstone's method 159
160
HIROYUKI SUMI et al. Table 1. The effect of the addition of various amino acid monomers and polymers on whole WBCLT blood clot lysis time (Approx. mol. wt) L-Arg D-Arg Poly-L-Arg L-Lys D-Lys Poly-L-Lys Poly-D-Lys
Poly-(Lys-Ala-Glu-Tyr) L-Glu D-GIu Poly-L-GIu Poly-D-Glu L-GIn D-GIn DL-Asp DL-Asn Gly
12.5 #g/ml
50 #g/ml
500 #g/ml
112
109 119 113 117 113 164" 109 117 --* --* ~
151 217 1733'* 149 219 173" 133" 274* --* ~* ~*
-
(100,000)
-
--
-
-
-
(14,000) (90,000) (12,000) (65,000) (100,000) (200,000)
--* 112 -131 --* 112
(540,000)
142
~
(52,000)
--
~
~*
(6000) (32,000) (60,000) (66,000)
ND ND ND 108 ND -ND ND
ll3 115 113
102 -102
-
-
ND ND
~*
--
--
117 115 ND ND 120 ND ND
----
-
113 --
--
Two tenths of a millilitre of normal human blood and various agents were added to 1.7 ml 0.12 M sodium acetate buffer of pH 7.4 (12.5-500 #g/ml blood), to which in turn 0.1 ml thrombin (50 U/ml) was added to let it react. Blood fibrinolytic activity is represented by I/clot lysis time (minutes), while figures represent relative values to the assumed control value of 100 (clot lysis time: 30 min) under the condition that no agent is added. *Signifies the swollen clot owing to deficient clot involution; **fallen clot owing to deficient clot involution; clot lysis strongly inhibited with the advance of clot involution; --blood clot remaining even after 530 min incubation at 37°C.
poly-L-Glu effected a slow process of clot lysis stimulation with its peak appearing about 1 hr after the administration, with WBCLT 2 3 2 _ 26 min before the administration, tending to diminish to 225 + 42, 190+ 15 and 211 __+23 min 20, 60 and 90min after the administration, respectively, and fibrin resolving power in plasma euglobulin, which had been
2 8 _+ 1 1 m m 2 b e f o r e t h e a d m i n i s t r a t i o n , c h a n g i n g t o 53_+16, 78_13 and 65_ ll mm 2(n=10;P
