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Evidence for multiple mechanisms of interaction between wheat germ agglutinin and human platelets
256
Biochimica et Biophysica Acta, 6 2 7 ( 1 9 8 0 ) 2 5 6 - - 2 6 1 © Elsevier/North-Holland Biomedical Press
BBA 29154
EVIDENCE FOR MULTIPLE MECHANISMS OF INTERACTION BETWEEN WHEAT GERMAGGLUTININ AND HUMAN PLATELETS
PANKAJ GANGULY
and N A N C Y
G. F O S S E T T
Division of Hematology, St. Jude Children's Research Hospital, 332 No. Lauderdale, Memphis, TN 38101 and Department of Biochemistry, University o f Tennessee Center for the Health Sciences, Memphis, TN 38163 (U.S.A.) (Received May 29th, 1979)
Key words: Agglutinin; Lectin binding; Platelet interaction; (Wheat germ)
Summary Wheat germ agglutinin induced aggregation * and secretion of serotonin from human platelets in plasma. This aggregation of platelets was blocked by ethylenediaminetetraacetate, azide or prostaglandin E,. The secretion of serotonin was n o t affected by ethylenediaminetetraacetate but was inhibited by prostaglandin E,. Thus, wheat germ agglutinin acts on platelets in plasma as a true aggregating agent. Washed platelets showed increased light transmission when treated with the lectin which was n o t blocked by ethylenediaminetetraacetate or prostaglandin E,. The capacity to inhibit platelet clumping by the above agents was restored if plasma was added back to the cell suspension. Washed platelets did not release serotonin under the conditions of cell clumping. Thus, in contrast to platelets in plasma, washed platelets are agglutinated ** by the lectin. Platelets fixed in formaldehyde were not agglutinated by the lectin in the aggregometer but agglutination was observed in the microtiter plate. This agglutination may be mediated by interplatelet bridging. These results show that the same agent may act on platelets by different mechanisms depending on the state of the cell and its environment.
* Aggregation is c o n s i d e r e d t o be clumping of platelets which is d e p e n d e n t on extracellula~ calcium and is a c c o m p a n i e d b y the release o f s e r o t o n i n w h i c h is i n d e p e n d e n t of calcium. This aggregation has usually b e e n c o n s i d e r e d as ' s e c o n d a r y aggregation'. ** Agglutination is defined as clumping of l~latelets which is i n d e p e n d e n t of calcium and is n o t accompanied with true release. A g g r e g a t i o n and a g g l u t i n a t i o n m a y n o t be distinguished from the aggregom eter tracings.
257 Introduction Lectins are plant proteins which bind to specific saccharide groups of surface components of cells [1]. The binding of a lectin is often associated with marked cellular and functional changes. For example, erythrocytes agglutinate, lymphocytes are converted from resting cells to dividing cells and t u m o r cells exhibit the property of contact inhibition of growth [2,3]. The interaction of different lectins with platelets has been investigated [ 4,5]. In general, it has been observed that lectins with galactose or N-acetylglucosamine specificities (Ricinus communis lectin, wheat germ agglutinin) are strong stimulators, whereas those with glucose-mannose specificity (Lens culinaris lectin) are p o o r activators of platelets. Some of the lectins have been reported to interact with platelets as true aggregating agents and their mechanism of action is strikingly similar to that of thrombin [6,7]. In this paper we report that wheat germ agglutinin can interact with platelets by different mechanisms depending on the state of the cell and its environment. Materials and Methods Blood was collected from laboratory volunteers in 0.38% sodium citrate as the anticoagulant. The red cells were removed by slow centrifugation and the platelet-rich plasma was collected. When necessary, platelets were washed as described [8] and resuspended in phosphate-buffered saline (pH 7.4). Platelets were fixed with formaldehyde by the m e t h o d of MacFarland et al. [9]. Platelet aggregation was measured in a dual-channel aggregometer (Payton Associates, Buffalo, NY) [10]. The experimental sample was analyzed in one channel while an appropriate control was run in the other. Usually, 50 pl stimulant was added to 450 pl platelet suspension. The release reaction was measured with platelets preloaded with [~4C]serotonin (60 Ci/mol, Amersham, Arlington Heights, IL). Aliquots of these platelets were incubated with different amounts of wheat germ agglutinin for 5 min at room temperature and then 0.1 ml 10% formaldehyde was added. The tubes were then spun at 12 000 rev./min for 2 min in an Eppendorf centrifuge. The amount of radioactivity in the supernatant was expressed as percent release with respect to controls. Platelet agglutination was routinely assayed at room temperature in microtiter plates using standard methods [12]. Human platelets fixed in formaldehyde were washed and finally suspended in Ca 2÷- and Mg2÷-free phosphatebuffered saline (pH 7.4). The lectin was serially 2-fold diluted and then 100-pl platelets (6 • 108/ml) was added to each well. The final volume in all wells was 200 pl. The plate was agitated for a few seconds and then sealed. All assays were done at least in duplicate and read after overnight incubation. Most of the experiments described in this study were carried out with wheat germ agglutinin purified by affinity chromatography and obtained from U.S. Biochemicals (Cleveland, OH). It showed a single band in SDS gel electrophoresis. A limited number of experiments were carried o u t with wheat germ agglutinin obtained from Sigma Chemical Co. (St. Louis, MO). This lectin had a lower platelet-agglutinating activity although each experiment was internally consistent. Prostaglandin E~ was a generous gift from Dr. John Pike (Upjohn
258 Co., Kalamazoo, MI). All other chemicals were of reagent grade and obtained commercially. Results and Discussion Wheat germ agglutinin induced aggregation of fresh platelets in plasma (Fig. 1). The final concentration of lectin needed to cause 70--80% aggregation was 20--50 pg/ml. This aggregation was inhibited by 5 mM EDTA, 1.5% NaN3 or 5/~g/ml prostaglandin El. The inhibition of aggregation by EDTA could not be overcome when the lectin concentration was increased to 200 pg/ml. Wheat germ agglutinin also caused the release of serotonin from platelets in plasma {Fig. 2). This secretion was not affected by 5 mM EDTA but was inhibited by 5 pg/ml prostaglandin El. Thus, aggregation of platelets in plasma by wheat germ agglutinin is dependent on extracellular calcium while secretion of serotonin is independent of calcium. Platelet aggregation by wheat germ agglutinin was not significantly affected by 200 #g/ml apyrase which inhibited aggregation by ADP. Thus, the action of wheat germ agglutinin on platelets is in many ways similar to that of thrombin. Washed platelets suspended in buffer showed a marked increase in light transmission in response to wheat germ agglutinin (Fig. 3). Compared to platelets in plasma, washed platelets required less lectin for a similar change in light transmission. Binding of the lectin to plasma glycoproteins may explain these results. There was no significant initial decrease in light transmission upon the addition of wheat germ agglutinin to washed platelets. It appears that platelets in buffer do not undergo the morphological alteration when stimulated by the lectin. The aggregation of washed platelets was not blocked by EDTA or prostaglandin El. Furthermore, washed platelets did not release serotonin even at a lectin concentration five times higher than that necessary for platelet clumping (Fig. 2). The same platelets released serotonin in response to thrombin. In contrast, Greenberg and Jamieson [4]. reported secretion of serotonin
L
o~
1 MINUTE Fig. 1. W h e a t g e r m agglutLnin-induced a g g r e g a t i o n profiles o f 2.5 • 1 0 8 / m l h u m a n p l a t e l e t s in Plasma. (a) C o n t r o l . (b) R e s p r e s e n t a t i v e c u r v e in t h e p r e s e n c e o f 5 m M final c o n c e n t r a t i o n E D T A or 5 ~ g / m l p r o s t a g l a n d i n E 1 o r 1.5% azide. 75 ~g l e c t i n ( S i g m a ) w a s a d d e d at t h e a r r o w a n d the c h a n g e in light t r a n s m i s s i o n was f o l l o w e d w i t h t i m e .
259 I
[
I
i
]
20
40
60
80
IO0
40
uJ
~ 2o
w o2
o
?
LECTIN CONCENTRATION ~g/ml F i g . 2. T h e s e c r e t i o n o f s e r o t o n i n f r o m p l a t e l e t s b y w h e a t g e r m a g g l u t i n i n u n d e r d i f f e r e n t c o n d i t i o n s , o , c o n t r o l p l a t e l e t s in p l a s m a . T h e c u r v e r e m a i n e d u n c h a n g e d w h e n 5 m M E D T A w a s i n c l u d e d in t h e p l a t e l e t s a m p l e . D a t a s h o w n are a v e r a g e s o f f o u r i n d i v i d u a l d e t e r m i n a t i o n s , o, p l a t e l e t s in p l a s m a w i t h 1 0 #g/ m l p r o s t a g l a n d i n E 1 . W a s h e d p l a t e l e t s in b u f f e r ( 0 ) d i d n o t r e l e a s e s e r o t o n i n a l t h o u g h 8 0 % c h a n g e in l i g h t transmission was observed.
and ADP from washed platelets when exposed to wheat germ agglutinin although platelet clumping was not affected by the destruction of the released ADP. It is evident that wheat germ agglutinin acts on washed platelets by a mechanism which is different from that for platelets in plasma. It appears that platelets in plasma are aggregated by this lectin while the change in light transmission observed with washed platelets is due to agglutination of cells. Addition of plasma to a suspension of washed platelets restored the capacity of EDTA to inhibit aggregation by the lectin (Fig. 3). These results show that the capacity to inhibit platelet aggregation by EDTA is dependent on the presence of plasma and is not due to some u n k n o w n alteration produced in platelets during the process of washing. Jones and Evans [13] observed that gel-filtered platelets, but not platelets in plasma, were aggregated by concanavalin A and suggested the presence of concanavalin A-reacting components in plasma as a possible explanation. It is interesting to note that the aggregation of Ehrlich
b
d 1 MINUTE Fig. 3. Agglutination of 2.5 • 108/mi w a s h e d platelets by 50 #g wheat germ agglutinin (Sigma) and restoration of the inhibitory activity of 5 m M E D T A b y plasma. (a) Control curve with or without E D T A , no plasma; (b) 50 #l plasma; (c) 100 #l plasma, and (d) 2 5 0 #I plasma. T h e final volume in all cases was 0.5 ml.
260
ascites cells by concanavalin A was reduced b u t not abolished by plasma. The presence of lectin-binding glycoproteins cannot completely explain the differential effect of wheat germ agglutinin on platelets in plasma and in buffer. Wheat germ agglutinin does not require bivalent cations for its action [14]. In the aggregometer, platelets fixed in formaldehyde did n o t show a change in light transmission on the addition of wheat germ agglutinin even to a concentration of 100 pg/ml. The results were similar in the presence or absence of plasma. However, fixed platelets could be agglutinated by the lectin in a microtiter plate (Fig. 4). Cell agglutination is markedly inhibited by chemical fixation. We have shown that these cells are capable of binding the lectin [7]. The fixed cells are n o t viable and the mobility of the lectin receptors on the membrane will be restricted so that they cannot redistribute into clusters favoring cell agglutination. Thus, it appears reasonable to suggest that fixed platelets are agglutinated in the microtiter plate by wheat germ agglutinin involving the classical mechanism of interplatelet bridge formation. Washed platelets showed a strong change in light transmission upon the addition of the lectin. However, this alteration in light transmission was neither inhibited by EDTA nor did these platelets secrete serotonin in response to the agglutinin. As an agglutinating agent, wheat germ agglutinin was much more effective on washed platelets than fixed platelets perhaps due to the unrestricted mobility of the lectin receptors. Platelets in plasma were activated by wheat germ agglutinin in the absence of aggregation. In this system, the lectin mimicked the action of thrombin on platelets. The most c o m m o n l y used measurement of platelet stimulation by any agent is aggregation as determined in the aggregometer. However, any alteration in the turbidity of the solution will produce a change in light transmission. This study clearly shows that only limited information as to the mechanism leading to the change in light transmission may be obtained from simple aggregation experiments. Furthermore, data presented in this paper clearly establish the role of plasma on the mechanism of platelet stimulation. In the presence of plasma, platelets were aggregated by wheat germ agglutinin; but in buffer platelets agglutinated. This observation is true not only for human platelets b u t for rat platelets as well [15]. Thus, the same stimulating agent may act on platelets by different mechanisms depending on the state of the cell and its environment.
Fig. 4. A g g l u t i n a t i o n o f f i x e d p l a t e l e t s in the m i c r o t i t e r plate b y w h e a t germ agglutinin in duplicate, The l e c t i n was serially 2-fold diluted and t h e n 100-~tl p l a t e l c t s (6 • 1 0 8 / m l ) was a d d e d t o a final v o l u m e o f 2 0 0 /~I. T h e final l e c t i n ( S i g m a ) c o n c e n t r a t i o n in the s e c o n d well is 1 0 0 p g / m l and t h e first and last w e l l s are c o n t r o l s w i t h o u t lectin.
261
Acknowledgments This study was supported by grants HL 16720 and CA 21765 from the National Institutes of Health and by ALSAC. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sharon, N. and Lis, H. (1972) Science 177,949--959 Kornfeld, S., Rogers, J. and Gregory, W. (1971) J. Biol. Chem. 246, 6581--6586 Burger, M.M. and Noonan, K.D. (1970) Nature 228, 512--515 Greenberg, J.H. and Jamieson, G.A. (1974) Biochim. Biophys. Acta 345, 231--242 Clemetson, K.J,, Pfueller, S.L., Luscher, E.F. and Jenkins, C.S.P. (1977) Biochim. Biophys. Acta 464, 493- - 50 8 Majerus, P.W. and Brodie, G.N. (1972) J. Biol. Chem. 247, 4253--4257 Ganguly, P., Gould, N. and Sidhu, P. (1979) Biochim. Biophys. Acta 586, 574--583 Ganguly, P. (1972) J. Biol. Chem. 247, 1809--1816 MacFarland, E.D., Stlbbe, J., Kirby, E.F., Zucker, M.B., Grant, R.A. and McPherson, J. (1975) Thromb. Diath. Haemorrh. 34, 306--313 Ganguly, P. (1975) Br. J. Haematol. 2 9 , 6 1 7 - - 6 2 6 Ganguly, P. (1977) Biochim. Biophys. Acta 498, 21--27 Yamada, K.M., Yamada, S.S. and Pastan, I. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3158--3161 Jones, B.M. and Evans, P.M. (1976) Biochim. Biophys. Acta 448, 368--378 Nagata, Y. and Burger, M.M. (1974) J. Biol. Chem. 249, 3116 --3122 Ganguly, P. and Fossett, N.G. (1980) Thromb. Res., in the press
Biochimica et Biophysica Acta, 6 2 7 ( 1 9 8 0 ) 2 5 6 - - 2 6 1 © Elsevier/North-Holland Biomedical Press
BBA 29154
EVIDENCE FOR MULTIPLE MECHANISMS OF INTERACTION BETWEEN WHEAT GERMAGGLUTININ AND HUMAN PLATELETS
PANKAJ GANGULY
and N A N C Y
G. F O S S E T T
Division of Hematology, St. Jude Children's Research Hospital, 332 No. Lauderdale, Memphis, TN 38101 and Department of Biochemistry, University o f Tennessee Center for the Health Sciences, Memphis, TN 38163 (U.S.A.) (Received May 29th, 1979)
Key words: Agglutinin; Lectin binding; Platelet interaction; (Wheat germ)
Summary Wheat germ agglutinin induced aggregation * and secretion of serotonin from human platelets in plasma. This aggregation of platelets was blocked by ethylenediaminetetraacetate, azide or prostaglandin E,. The secretion of serotonin was n o t affected by ethylenediaminetetraacetate but was inhibited by prostaglandin E,. Thus, wheat germ agglutinin acts on platelets in plasma as a true aggregating agent. Washed platelets showed increased light transmission when treated with the lectin which was n o t blocked by ethylenediaminetetraacetate or prostaglandin E,. The capacity to inhibit platelet clumping by the above agents was restored if plasma was added back to the cell suspension. Washed platelets did not release serotonin under the conditions of cell clumping. Thus, in contrast to platelets in plasma, washed platelets are agglutinated ** by the lectin. Platelets fixed in formaldehyde were not agglutinated by the lectin in the aggregometer but agglutination was observed in the microtiter plate. This agglutination may be mediated by interplatelet bridging. These results show that the same agent may act on platelets by different mechanisms depending on the state of the cell and its environment.
* Aggregation is c o n s i d e r e d t o be clumping of platelets which is d e p e n d e n t on extracellula~ calcium and is a c c o m p a n i e d b y the release o f s e r o t o n i n w h i c h is i n d e p e n d e n t of calcium. This aggregation has usually b e e n c o n s i d e r e d as ' s e c o n d a r y aggregation'. ** Agglutination is defined as clumping of l~latelets which is i n d e p e n d e n t of calcium and is n o t accompanied with true release. A g g r e g a t i o n and a g g l u t i n a t i o n m a y n o t be distinguished from the aggregom eter tracings.
257 Introduction Lectins are plant proteins which bind to specific saccharide groups of surface components of cells [1]. The binding of a lectin is often associated with marked cellular and functional changes. For example, erythrocytes agglutinate, lymphocytes are converted from resting cells to dividing cells and t u m o r cells exhibit the property of contact inhibition of growth [2,3]. The interaction of different lectins with platelets has been investigated [ 4,5]. In general, it has been observed that lectins with galactose or N-acetylglucosamine specificities (Ricinus communis lectin, wheat germ agglutinin) are strong stimulators, whereas those with glucose-mannose specificity (Lens culinaris lectin) are p o o r activators of platelets. Some of the lectins have been reported to interact with platelets as true aggregating agents and their mechanism of action is strikingly similar to that of thrombin [6,7]. In this paper we report that wheat germ agglutinin can interact with platelets by different mechanisms depending on the state of the cell and its environment. Materials and Methods Blood was collected from laboratory volunteers in 0.38% sodium citrate as the anticoagulant. The red cells were removed by slow centrifugation and the platelet-rich plasma was collected. When necessary, platelets were washed as described [8] and resuspended in phosphate-buffered saline (pH 7.4). Platelets were fixed with formaldehyde by the m e t h o d of MacFarland et al. [9]. Platelet aggregation was measured in a dual-channel aggregometer (Payton Associates, Buffalo, NY) [10]. The experimental sample was analyzed in one channel while an appropriate control was run in the other. Usually, 50 pl stimulant was added to 450 pl platelet suspension. The release reaction was measured with platelets preloaded with [~4C]serotonin (60 Ci/mol, Amersham, Arlington Heights, IL). Aliquots of these platelets were incubated with different amounts of wheat germ agglutinin for 5 min at room temperature and then 0.1 ml 10% formaldehyde was added. The tubes were then spun at 12 000 rev./min for 2 min in an Eppendorf centrifuge. The amount of radioactivity in the supernatant was expressed as percent release with respect to controls. Platelet agglutination was routinely assayed at room temperature in microtiter plates using standard methods [12]. Human platelets fixed in formaldehyde were washed and finally suspended in Ca 2÷- and Mg2÷-free phosphatebuffered saline (pH 7.4). The lectin was serially 2-fold diluted and then 100-pl platelets (6 • 108/ml) was added to each well. The final volume in all wells was 200 pl. The plate was agitated for a few seconds and then sealed. All assays were done at least in duplicate and read after overnight incubation. Most of the experiments described in this study were carried out with wheat germ agglutinin purified by affinity chromatography and obtained from U.S. Biochemicals (Cleveland, OH). It showed a single band in SDS gel electrophoresis. A limited number of experiments were carried o u t with wheat germ agglutinin obtained from Sigma Chemical Co. (St. Louis, MO). This lectin had a lower platelet-agglutinating activity although each experiment was internally consistent. Prostaglandin E~ was a generous gift from Dr. John Pike (Upjohn
258 Co., Kalamazoo, MI). All other chemicals were of reagent grade and obtained commercially. Results and Discussion Wheat germ agglutinin induced aggregation of fresh platelets in plasma (Fig. 1). The final concentration of lectin needed to cause 70--80% aggregation was 20--50 pg/ml. This aggregation was inhibited by 5 mM EDTA, 1.5% NaN3 or 5/~g/ml prostaglandin El. The inhibition of aggregation by EDTA could not be overcome when the lectin concentration was increased to 200 pg/ml. Wheat germ agglutinin also caused the release of serotonin from platelets in plasma {Fig. 2). This secretion was not affected by 5 mM EDTA but was inhibited by 5 pg/ml prostaglandin El. Thus, aggregation of platelets in plasma by wheat germ agglutinin is dependent on extracellular calcium while secretion of serotonin is independent of calcium. Platelet aggregation by wheat germ agglutinin was not significantly affected by 200 #g/ml apyrase which inhibited aggregation by ADP. Thus, the action of wheat germ agglutinin on platelets is in many ways similar to that of thrombin. Washed platelets suspended in buffer showed a marked increase in light transmission in response to wheat germ agglutinin (Fig. 3). Compared to platelets in plasma, washed platelets required less lectin for a similar change in light transmission. Binding of the lectin to plasma glycoproteins may explain these results. There was no significant initial decrease in light transmission upon the addition of wheat germ agglutinin to washed platelets. It appears that platelets in buffer do not undergo the morphological alteration when stimulated by the lectin. The aggregation of washed platelets was not blocked by EDTA or prostaglandin El. Furthermore, washed platelets did not release serotonin even at a lectin concentration five times higher than that necessary for platelet clumping (Fig. 2). The same platelets released serotonin in response to thrombin. In contrast, Greenberg and Jamieson [4]. reported secretion of serotonin
L
o~
1 MINUTE Fig. 1. W h e a t g e r m agglutLnin-induced a g g r e g a t i o n profiles o f 2.5 • 1 0 8 / m l h u m a n p l a t e l e t s in Plasma. (a) C o n t r o l . (b) R e s p r e s e n t a t i v e c u r v e in t h e p r e s e n c e o f 5 m M final c o n c e n t r a t i o n E D T A or 5 ~ g / m l p r o s t a g l a n d i n E 1 o r 1.5% azide. 75 ~g l e c t i n ( S i g m a ) w a s a d d e d at t h e a r r o w a n d the c h a n g e in light t r a n s m i s s i o n was f o l l o w e d w i t h t i m e .
259 I
[
I
i
]
20
40
60
80
IO0
40
uJ
~ 2o
w o2
o
?
LECTIN CONCENTRATION ~g/ml F i g . 2. T h e s e c r e t i o n o f s e r o t o n i n f r o m p l a t e l e t s b y w h e a t g e r m a g g l u t i n i n u n d e r d i f f e r e n t c o n d i t i o n s , o , c o n t r o l p l a t e l e t s in p l a s m a . T h e c u r v e r e m a i n e d u n c h a n g e d w h e n 5 m M E D T A w a s i n c l u d e d in t h e p l a t e l e t s a m p l e . D a t a s h o w n are a v e r a g e s o f f o u r i n d i v i d u a l d e t e r m i n a t i o n s , o, p l a t e l e t s in p l a s m a w i t h 1 0 #g/ m l p r o s t a g l a n d i n E 1 . W a s h e d p l a t e l e t s in b u f f e r ( 0 ) d i d n o t r e l e a s e s e r o t o n i n a l t h o u g h 8 0 % c h a n g e in l i g h t transmission was observed.
and ADP from washed platelets when exposed to wheat germ agglutinin although platelet clumping was not affected by the destruction of the released ADP. It is evident that wheat germ agglutinin acts on washed platelets by a mechanism which is different from that for platelets in plasma. It appears that platelets in plasma are aggregated by this lectin while the change in light transmission observed with washed platelets is due to agglutination of cells. Addition of plasma to a suspension of washed platelets restored the capacity of EDTA to inhibit aggregation by the lectin (Fig. 3). These results show that the capacity to inhibit platelet aggregation by EDTA is dependent on the presence of plasma and is not due to some u n k n o w n alteration produced in platelets during the process of washing. Jones and Evans [13] observed that gel-filtered platelets, but not platelets in plasma, were aggregated by concanavalin A and suggested the presence of concanavalin A-reacting components in plasma as a possible explanation. It is interesting to note that the aggregation of Ehrlich
b
d 1 MINUTE Fig. 3. Agglutination of 2.5 • 108/mi w a s h e d platelets by 50 #g wheat germ agglutinin (Sigma) and restoration of the inhibitory activity of 5 m M E D T A b y plasma. (a) Control curve with or without E D T A , no plasma; (b) 50 #l plasma; (c) 100 #l plasma, and (d) 2 5 0 #I plasma. T h e final volume in all cases was 0.5 ml.
260
ascites cells by concanavalin A was reduced b u t not abolished by plasma. The presence of lectin-binding glycoproteins cannot completely explain the differential effect of wheat germ agglutinin on platelets in plasma and in buffer. Wheat germ agglutinin does not require bivalent cations for its action [14]. In the aggregometer, platelets fixed in formaldehyde did n o t show a change in light transmission on the addition of wheat germ agglutinin even to a concentration of 100 pg/ml. The results were similar in the presence or absence of plasma. However, fixed platelets could be agglutinated by the lectin in a microtiter plate (Fig. 4). Cell agglutination is markedly inhibited by chemical fixation. We have shown that these cells are capable of binding the lectin [7]. The fixed cells are n o t viable and the mobility of the lectin receptors on the membrane will be restricted so that they cannot redistribute into clusters favoring cell agglutination. Thus, it appears reasonable to suggest that fixed platelets are agglutinated in the microtiter plate by wheat germ agglutinin involving the classical mechanism of interplatelet bridge formation. Washed platelets showed a strong change in light transmission upon the addition of the lectin. However, this alteration in light transmission was neither inhibited by EDTA nor did these platelets secrete serotonin in response to the agglutinin. As an agglutinating agent, wheat germ agglutinin was much more effective on washed platelets than fixed platelets perhaps due to the unrestricted mobility of the lectin receptors. Platelets in plasma were activated by wheat germ agglutinin in the absence of aggregation. In this system, the lectin mimicked the action of thrombin on platelets. The most c o m m o n l y used measurement of platelet stimulation by any agent is aggregation as determined in the aggregometer. However, any alteration in the turbidity of the solution will produce a change in light transmission. This study clearly shows that only limited information as to the mechanism leading to the change in light transmission may be obtained from simple aggregation experiments. Furthermore, data presented in this paper clearly establish the role of plasma on the mechanism of platelet stimulation. In the presence of plasma, platelets were aggregated by wheat germ agglutinin; but in buffer platelets agglutinated. This observation is true not only for human platelets b u t for rat platelets as well [15]. Thus, the same stimulating agent may act on platelets by different mechanisms depending on the state of the cell and its environment.
Fig. 4. A g g l u t i n a t i o n o f f i x e d p l a t e l e t s in the m i c r o t i t e r plate b y w h e a t germ agglutinin in duplicate, The l e c t i n was serially 2-fold diluted and t h e n 100-~tl p l a t e l c t s (6 • 1 0 8 / m l ) was a d d e d t o a final v o l u m e o f 2 0 0 /~I. T h e final l e c t i n ( S i g m a ) c o n c e n t r a t i o n in the s e c o n d well is 1 0 0 p g / m l and t h e first and last w e l l s are c o n t r o l s w i t h o u t lectin.
261
Acknowledgments This study was supported by grants HL 16720 and CA 21765 from the National Institutes of Health and by ALSAC. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sharon, N. and Lis, H. (1972) Science 177,949--959 Kornfeld, S., Rogers, J. and Gregory, W. (1971) J. Biol. Chem. 246, 6581--6586 Burger, M.M. and Noonan, K.D. (1970) Nature 228, 512--515 Greenberg, J.H. and Jamieson, G.A. (1974) Biochim. Biophys. Acta 345, 231--242 Clemetson, K.J,, Pfueller, S.L., Luscher, E.F. and Jenkins, C.S.P. (1977) Biochim. Biophys. Acta 464, 493- - 50 8 Majerus, P.W. and Brodie, G.N. (1972) J. Biol. Chem. 247, 4253--4257 Ganguly, P., Gould, N. and Sidhu, P. (1979) Biochim. Biophys. Acta 586, 574--583 Ganguly, P. (1972) J. Biol. Chem. 247, 1809--1816 MacFarland, E.D., Stlbbe, J., Kirby, E.F., Zucker, M.B., Grant, R.A. and McPherson, J. (1975) Thromb. Diath. Haemorrh. 34, 306--313 Ganguly, P. (1975) Br. J. Haematol. 2 9 , 6 1 7 - - 6 2 6 Ganguly, P. (1977) Biochim. Biophys. Acta 498, 21--27 Yamada, K.M., Yamada, S.S. and Pastan, I. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3158--3161 Jones, B.M. and Evans, P.M. (1976) Biochim. Biophys. Acta 448, 368--378 Nagata, Y. and Burger, M.M. (1974) J. Biol. Chem. 249, 3116 --3122 Ganguly, P. and Fossett, N.G. (1980) Thromb. Res., in the press