Toxicon, Vol. 16, pp. 503-508.
0041-0101/78]0901-0503502.00/0
© Pergamon Press Ltd. 1978. Printed in Great Britain
RED BLOOD CELL A G G R E G A T I O N BY TRIMERESURUS MUCROSQUAMATUS SNAKE VENOM CHAOHO OUYANG a n d CHE-MING TENG Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
(Accepted for publication 23 January 1978) C. OUYANG and C.-M. TENG. Red blood cell aggregation by Trimeresurus mucrosquamatus snake venom. Toxicon 16, 503-508, 1978.--T. mucrosquamatus venom induced aggregation of rabbit and human RBC's at a concentration higher than 25 tag/ml. This aggregation was inhibited by E D T A and could be reversed by Ca 2+. a-Methylgalactoside, but not a-methylglucoside, competitively inhibited the venom-induced RBC aggregation. Trypsin and neuraminidase pretreatment of RBC did not potentiate this aggregating effect. It was concluded that venom-induced aggregation was due to a lectin-like binding to saccharide (galactosyl)receptors of the RBC membrane. At a sublethal i.v. dose (1 mg/kg), the venom did not cause significant changes of rabbit RBC counts. INTRODUCTION IT IS k n o w n t h a t c a r d i o t o x i n a n d p h o s p h o l i p a s e A c a n a f f e c t r e d b l o o d cell ( R B C ) , c a u s i n g h e m o l y s i s d i r e c t l y , o r i n d i r e c t l y t h r o u g h t h e f o r m a t i o n o f l y s o l e c i t h i n (LEE et al., 1970; CONDREA a n d DE VRIES, 1965). W e r e p o r t h e r e a n o t h e r k i n d o f a c t i o n o n R B C b y s n a k e v e n o m - - R B C a g g r e g a t i o n in a l e c t i n - l i k e effect b y t h e v e n o m o f T. mucrosquamatus. MATERIALS A N D METHODS
Materials The venom of Trimeresurus mucrosquamatus was collected at our laboratory, centrifuged, lyophilized and stored in a desiccator below - 2 0 ° C . ~-Methylglucoside, ~-methylgalactoside, concanavalin A, trypsin, neuraminidase and disodinm ethylenediamine tetraacetic acid (EDTA) were obtained from Sigma Chem. Co. Concentrations indicated in data were final concentrations in RBC suspension.
Preparation of RBC suspension Blood was collected from the rabbit ear marginal vein through a 19 gauge needle and was mixed with 3"8~o sodium citrate (9 : 1, v/v). After centrifugation at 350 g and 4°C for 10 min, plasma was removed. RBC were washed with imidazole-buffered saline (pH 7'4), centrifuged (repeated this washing procedure two times) and then resuspended in imidazole-saline (pH 7.4) to give a 1 ~ suspension.
Preparation of platelet-rich plasma Blood was collected from rabbit marginal ear vein through a 19-gauge needle and was mixed with 3'8 ~ sodium citrate (14:1 v/v). Citrated blood was immediately centrifuged for 10 min at 90 g at room temperature. After removal of the platelet-rich plasma the remaining blood was recentrifuged an additional 20 rain at 1500 g and the platelet-poor plasma was obtained and mixed with the platelet-rich plasma to give a platelet count of about 300,000/mm 3. RBC or platelet aggregation was measured by the turbidimetric method (O'BgXEN, 1962; Bogr~ and CROSS, 1963) using an EEL 169 Aggregometer (600 nm) connected to a Coming Recorder (Model 840). RBC suspension or platelet-rich plasma was pre-warmed at 37°C for 5 min in a siliconized glass cuvette and stirred for 1 min. To initiate the reaction, 100 lal of the test solution was added with an Eppendorff micropipette. The percent aggregation was calculated as following: initial O.D.--final O.D. after aggregation Aggregation ( ~ ) = × 100. initial O.D.--O.D. of saline buffer 503
504
CHAOHO OUYANG and CHE-MING TENG
In experiments with human RBC, the aggregation of RBC was observed with a phase-contrast microscope because there was no large aggregation formed by the venom.
Changes of RBC counts in vivo After intravenous injection of the venom into the rabbit marginal ear vein, 1 ml samples of blood (citrated) were withdrawn at various intervals. RBC's were counted using a Linson Celloscope (Model 401) according to the procedure suggested by the manufacturer. RESULTS
Patterns of R B C aggregation V a r i o u s c o n c e n t r a t i o n s o f T. mucrosquamatus v e n o m were tested u p o n R B C suspensions; a series o f typical a g g r e g a t i o n p a t t e r n s is shown in Fig. 1. T h e m i n i m a l effective conc e n t r a t i o n for inducing R B C a g g r e g a t i o n was a b o u t 25 pg/ml. The higher the c o n c e n t r a t i o n s ,
,L
RBC 25
50
o
I00
E
2oo oi 4z_J 500
I000
FIG. l . PATTERNS OF RBC AGGREGATION WITH VARIOUS CONCENTRATIONS OF VENOM.
T. mucrosquamatus
The arrow indicates where the venom was added. the greater was the extent a n d rate o f aggregation. Figure 2 shows the c o n c e n t r a t i o n aggregation relationships o f R B C a n d platelet aggregations. In c o n t r a s t to platelet aggregation, no decrease o f R B C a g g r e g a t i o n could be detected with high c o n c e n t r a t i o n s o f the venom, even u p to 2000 pg/ml.
Effects o f C a 2+ and EDTA on RBC aggregation C a l c i u m ion (2.5 m M to 25 m M ) c o u l d n o t accelerate o r p o t e n t i a t e the R B C - a g g r e g a t i o n i n d u c e d by v e n o m in the c o n c e n t r a t i o n s o f 20-2000 pg/ml. However, E D T A at a concen-
RBC Aggregation by T. mucrosquamatus Venom
505
IOO
RBC
~0
TU-
80
70
60
50 c~ 40
0041-0101/78]0901-0503502.00/0
© Pergamon Press Ltd. 1978. Printed in Great Britain
RED BLOOD CELL A G G R E G A T I O N BY TRIMERESURUS MUCROSQUAMATUS SNAKE VENOM CHAOHO OUYANG a n d CHE-MING TENG Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
(Accepted for publication 23 January 1978) C. OUYANG and C.-M. TENG. Red blood cell aggregation by Trimeresurus mucrosquamatus snake venom. Toxicon 16, 503-508, 1978.--T. mucrosquamatus venom induced aggregation of rabbit and human RBC's at a concentration higher than 25 tag/ml. This aggregation was inhibited by E D T A and could be reversed by Ca 2+. a-Methylgalactoside, but not a-methylglucoside, competitively inhibited the venom-induced RBC aggregation. Trypsin and neuraminidase pretreatment of RBC did not potentiate this aggregating effect. It was concluded that venom-induced aggregation was due to a lectin-like binding to saccharide (galactosyl)receptors of the RBC membrane. At a sublethal i.v. dose (1 mg/kg), the venom did not cause significant changes of rabbit RBC counts. INTRODUCTION IT IS k n o w n t h a t c a r d i o t o x i n a n d p h o s p h o l i p a s e A c a n a f f e c t r e d b l o o d cell ( R B C ) , c a u s i n g h e m o l y s i s d i r e c t l y , o r i n d i r e c t l y t h r o u g h t h e f o r m a t i o n o f l y s o l e c i t h i n (LEE et al., 1970; CONDREA a n d DE VRIES, 1965). W e r e p o r t h e r e a n o t h e r k i n d o f a c t i o n o n R B C b y s n a k e v e n o m - - R B C a g g r e g a t i o n in a l e c t i n - l i k e effect b y t h e v e n o m o f T. mucrosquamatus. MATERIALS A N D METHODS
Materials The venom of Trimeresurus mucrosquamatus was collected at our laboratory, centrifuged, lyophilized and stored in a desiccator below - 2 0 ° C . ~-Methylglucoside, ~-methylgalactoside, concanavalin A, trypsin, neuraminidase and disodinm ethylenediamine tetraacetic acid (EDTA) were obtained from Sigma Chem. Co. Concentrations indicated in data were final concentrations in RBC suspension.
Preparation of RBC suspension Blood was collected from the rabbit ear marginal vein through a 19 gauge needle and was mixed with 3"8~o sodium citrate (9 : 1, v/v). After centrifugation at 350 g and 4°C for 10 min, plasma was removed. RBC were washed with imidazole-buffered saline (pH 7'4), centrifuged (repeated this washing procedure two times) and then resuspended in imidazole-saline (pH 7.4) to give a 1 ~ suspension.
Preparation of platelet-rich plasma Blood was collected from rabbit marginal ear vein through a 19-gauge needle and was mixed with 3'8 ~ sodium citrate (14:1 v/v). Citrated blood was immediately centrifuged for 10 min at 90 g at room temperature. After removal of the platelet-rich plasma the remaining blood was recentrifuged an additional 20 rain at 1500 g and the platelet-poor plasma was obtained and mixed with the platelet-rich plasma to give a platelet count of about 300,000/mm 3. RBC or platelet aggregation was measured by the turbidimetric method (O'BgXEN, 1962; Bogr~ and CROSS, 1963) using an EEL 169 Aggregometer (600 nm) connected to a Coming Recorder (Model 840). RBC suspension or platelet-rich plasma was pre-warmed at 37°C for 5 min in a siliconized glass cuvette and stirred for 1 min. To initiate the reaction, 100 lal of the test solution was added with an Eppendorff micropipette. The percent aggregation was calculated as following: initial O.D.--final O.D. after aggregation Aggregation ( ~ ) = × 100. initial O.D.--O.D. of saline buffer 503
504
CHAOHO OUYANG and CHE-MING TENG
In experiments with human RBC, the aggregation of RBC was observed with a phase-contrast microscope because there was no large aggregation formed by the venom.
Changes of RBC counts in vivo After intravenous injection of the venom into the rabbit marginal ear vein, 1 ml samples of blood (citrated) were withdrawn at various intervals. RBC's were counted using a Linson Celloscope (Model 401) according to the procedure suggested by the manufacturer. RESULTS
Patterns of R B C aggregation V a r i o u s c o n c e n t r a t i o n s o f T. mucrosquamatus v e n o m were tested u p o n R B C suspensions; a series o f typical a g g r e g a t i o n p a t t e r n s is shown in Fig. 1. T h e m i n i m a l effective conc e n t r a t i o n for inducing R B C a g g r e g a t i o n was a b o u t 25 pg/ml. The higher the c o n c e n t r a t i o n s ,
,L
RBC 25
50
o
I00
E
2oo oi 4z_J 500
I000
FIG. l . PATTERNS OF RBC AGGREGATION WITH VARIOUS CONCENTRATIONS OF VENOM.
T. mucrosquamatus
The arrow indicates where the venom was added. the greater was the extent a n d rate o f aggregation. Figure 2 shows the c o n c e n t r a t i o n aggregation relationships o f R B C a n d platelet aggregations. In c o n t r a s t to platelet aggregation, no decrease o f R B C a g g r e g a t i o n could be detected with high c o n c e n t r a t i o n s o f the venom, even u p to 2000 pg/ml.
Effects o f C a 2+ and EDTA on RBC aggregation C a l c i u m ion (2.5 m M to 25 m M ) c o u l d n o t accelerate o r p o t e n t i a t e the R B C - a g g r e g a t i o n i n d u c e d by v e n o m in the c o n c e n t r a t i o n s o f 20-2000 pg/ml. However, E D T A at a concen-
RBC Aggregation by T. mucrosquamatus Venom
505
IOO
RBC
~0
TU-
80
70
60
50 c~ 40
