574

Biochimica et Biophysica Acta, 5 8 6 ( 1 9 7 9 ) 5 7 4 - - 5 8 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28985

INTERACTION OF LECTINS WITH HUMAN PLATELETS EFFECTS ON P L A T E L E T STIMULATION BY THROMBIN AND RISTOCETIN

P A N K A J G A N G U L Y , N A N C Y L. G O U L D a n d P A R A M J E E T S I D H U

Laboratory of Hematology, St. Jude Children's Research Hospital, 332 N. Lauderdale Memphis, TN 38101 and Department of Biochemistry, University of Tennessee, Memphis, TN 38101 (U.S.A.) (Received January 10th, 1979)

Key words: Lectin; Platelet stimulation; Thrombin; Ristocetin; Agglutinin; (Wheat germ)

Summary Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but n o t secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin El. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 • l 0 s binding sites for the lectins with an apparent dissociation constant of 3.0 • 10 -7 M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was w i t h o u t effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.

Abbreviations: SDS, sodium dodecyl sulfate; GP-I, surface glycoprotein of h u m a n platelets with an apparent molecular weight of 150 000; EDTA, ethylenediaminetetraaeetate.

575 Introduction Lectins are plant proteins which bind to specific receptors on erythrocytes, l y m p h o c y t e s and a variety of other cells [1]. Binding of some of these proteins often cause agglutination of cells or leads to differentiation of l y m p h o c y t e s [1,2]. The interactions of lectins and cells may often be blocked with specific and simple sugars. This observation has led to the suggestion that lectins bind to specific saccharides on the surface of cells and has provided a tool for exploring molecular aspects of cell membranes [ 1,3]. In this paper, we report the interactions of wheat germ agglutinin and lentil lectin with human platelets. In other studies, we have investigated the effects of these lectins on the interaction of thrombin and ristocetin with platelets. Data are presented which suggest that the binding sites of wheat germ agglutinin and thrombin are independent while ristocetin and wheat germ agglutinin interact at overlapping sites. Materials and Methods Blood was collected from human volunteers or rats in plastic syringes utilizing 0.1 vol. of 3.8% sodium citrate as the anticoagulant. The red cells were removed by differential centrifugation and the platelet-rich plasma was collected. If necessary, platelets were washed as described [4] and resuspended in phosphate-buffered saline. Platelets were fixed with formaldehyde b y the method of Allain et al. [ 5]. Platelet aggregation was measured in a dual-channel aggregometer (Payton Associates, Buffalo, NY) [6]. Usually, the experimental sample was analyzed in one channel while an appropriate control was run in the other. The release reaction was measured with platelets preloaded with [14C]serotonin (60 Ci/ mol, Amersham, Arlington Heights, IL) [6]. Aliquots of these platelets were incubated with different amounts of the stimulant for 5 min and then 0.1 ml of 10% formaldehyde was added. The plastic tubes were 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 as follows: (Test supernatant count -- Control supernatant count) × 100 Control b u t t o n count The lectins or purified thrombin were labeled with 12sI by the chloramine T method [7]. A b o u t 90% of the initial enzymatic activity of thrombin as determined b y the fibrinogen-clotting assay could be recovered after labeling. The erythrocyte agglutinating activity of the lectins were identical before and after labeling. Further, the electrophoretic patterns in the presence of sodium dodecyl sulfate (SDS) of the labeled materials were similar to those of the unlabeled compounds. The specific radioactivity of the lectins varied with different preparations b u t was in the range of 3.5--8.8 • 10 s cpm/t~g. The Millipore filtration m e t h o d for the determination o f binding of thrombin to platelets has been described in previous papers [7,8]. All filters and plastic tubes were soaked overnight in 0.5% bovine serum albumin before use. The binding

576 of lectins to platelets was determined following the same method. The nonspecific binding of the lectins to platelets were determined by including 0.1 M N-acetylglucosamine for wheat germ agglutinin or a-methyl-D-mannoside for lentil lectin in the incubation mixtures. Additional details are provided in the figure legends. Gel electrophoresis in 7.5% polyacrylamide slabs was carried o u t by the m e t h o d of Laemmli [9]. Platelets or proteins were solubilized in 1% SDS and reduced with 1%/3-mercaptoethanol. The sample was heated in a boiling water bath for 5 min. Electrophoresis was carried o u t in a Bio-Rad apparatus at 25 mA until the d y e marker reached the b o t t o m of the gel. After electrophoresis, the gel was stained with Coomassie blue for proteins and periodic acid-Schiff reagent for glycoproteins. To determine the binding of wheat germ agglutinin, the gel strip containing the separated platelet proteins was washed with phosphate-buffered saline and 0.5 ml of 125I-labeled wheat germ agglutinin containing 8.4 • 106 cpm was layered on it. The gel was held overnight in a humid chamber at room temperature and then the free wheat germ agglutinin was washed o f f with phosphate-buffered saline. The gel was cut into 2-mm slices and the distribution of radioactivity determined. The results shown are representative of three independent determinations. Wheat germ agglutinin, purified by affinity chromatography, was obtained from U.S. Biochemicals, Cleveland, OH. It showed a single band in SDS gel electrophoresis. Lentil lectin was purchased from Sigma Chemical Co., St. Louis, MO. It contained both lentil A and lentil B which are known to be identical both in terms of erythrocyte binding and mitogenicity for l y m p h o c y t e s [10]. Prostaglandin E1 was a generous gift from Dr. John Pike, Upjohn Co., Kalamazoo, MI. Ristocetin was obtained from Pacific Hemostasis, Los Angeles, CA. Bovine thrombin (Parke Davis, Detroit, MI) was purified by ion-exchange chromatography to a minimum specific activity of 2500 NIH U/mg [11]. All other chemicals were of reagent grade and obtained commercially. Results

Platelet stimulation by wheat germ agglutinin Wheat germ agglutinin caused aggregation of fresh human platelets which was inhibited b y 50 mM N-acetylglucosamine (Fig. 1). The minimal concentration of the lectin required for aggregation varied with platelets from person to person b u t was in the range of 1 0 - 5 0 pg/ml. Platelets fixed with formaldeh y d e - b o u n d wheat germ agglutinin (see later) b u t did not aggregate with even 100 pg/ml of the agglutinin. However, fixed erythrocytes, similar to fresh erythrocytes, were readily agglutinated b y the lectin. These results suggested that wheat germ agglutinin may stimulate platelets b y a mechanism other than agglutination. Wheat germ agglutinin caused secretion of [~4C]serotonin from fresh platelets (Fig. 2). Aggregation and the release reaction of platelets are independent b u t interrelated processes. To determine whether the release reaction was dependent on aggregation of platelets by the lectin, the effect of ethylenediaminetetraacetate (EDTA) was tested. Like thrombin [12], the aggregation of platelets b y wheat germ agglutinin was inhibited b y EDTA {Fig. 3). While the

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Fig. 1. I n h i b i t i o n o f l e c t i n - i n d u c e d a g g r e g a t i o n o f p l a t e l e t s b y N - a c e t y l g l u c o s a m i n e . 10 # g o f w h e a t g e r m a g g l u t i n i n was a d d e d at zero t i m e t o a final v o l u m e o f 5 0 0 #1 o f p l a t c l e t s a n d t h e a g g r e g a t i o n w a s foll o w e d w i t h t i m e . In t h e l o w e r c u r v e , t h e p l a t e l e t s a m p l e c o n t a i n e d 50 m M N - a c e t y l g l u c o s a m i n e . Platelet c o u n t 2.5 • 1 0 8 / m l . Fig. 2. L a c k o f e f f e c t of E D T A o n t h e release of s e r o t o n i n f r o m p l a t e l e t s (2.5 • 1 0 8 / m l ) b y w h e a t g e r m a g g l u t i n i n . S e r o t o n i n - l o a d e d p l a t e l e t s in p l a s m a w e r e i n c u b a t e d w i t h d i f f e r e n t a m o u n t s of lectin in a t o t a l v o l u m e o f 0.5 m l for 5 rain at r o o m t e m p e r a t u r e , o, c o n t r o l ; a, 5 m M E D T A .

control curve showed about 70% aggregation, in the presence of 4.5 mM EDTA, the change in light transmission was less than 10%. However, the release of serotonin from platelets by wheat germ agglutinin remained unaffected in the presence of 5 mM EDTA (Fig. 2). These results clearly show that the agglutinin can stimulate platelets by a mechanism independent of agglutination.

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578 Further, prostaglandin El, a known inhibitor of thrombin-induced platelet stimulation [13], also inhibited platelet aggregation by the lectin (Fig. 4). These results show that the action of wheat germ agglutinin on platelets closely mimics thrombin.

Binding o f wheat germ agglutinin to platelets Wheat germ agglutinin labeled with '2sI was layered on gels containing the platelet components separated by electrophoresis. The gel was processed as described under Materials and Methods and cut into equal slices. The distribution of radioactivity showed a major peak (Fig. 5), the position of which corresponded to a prominent glycoprotein in stained gels with an apparent molecular weight of 150 000 (GP-I). The binding of wheat germ agglutinin to this glycoprotein was inhibited by 40% in the presence of 0.1 M N-acetylglucosamine. These data confirm earlier reports that wheat germ agglutinin binds mainly to GP-I o f h u m a n platelets [ 14--16 ]. The binding of '2SI-labeled wheat germ agglutinin to formaldehyde fixed platelets is shown in Fig. 6. The curve shows saturation kinetics data similar to those found in studies of binding of phytohemagglutinins to platelets or erythrocytes [2,10]. These data were plotted by the m e t h o d of Steck and Wallach [ 17 ] to determine the number of binding sites for wheat germ agglutinin on the platelet surface and the affinity. From a number of experiments, it was observed there are about 6 • l 0 S sites/platetet for wheat germ agglutinin with an apparent dissociation constant of 3 • 10 -7 M (Fig. 6, inset). Since wheat germ agglutinin is at least bivalent, these values are only relative approximations. In these calculations, the molecular weight of wheat germ agglutinin was considered to be 35 000 and its extinction coefficient ~/A1% Thus, c~280nm ~ ! 14.3 [18] platelets fixed in formaldehyde are capable of binding the lectin although they did not agglutinate in the aggregometer.

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SLICE NUMBER F i g . 5. B i n d i n g o f w h e a t g e r m a g g l u t i n i n t o p l a t e l e t c o m p o n e n t s s e p a r a t e d b y gel e l e c t r o p h o r e s i s . A f t e r w a s h i n g w i t h b u f f e r , t h e gel l a n e w a s i n c u b a t e d w i t h a l a y e r o f 12 S I - l a b e l e d w h e a t g e r m a g g l u t i n i n a n d t h e n f r e e l e e t i n w a s r e m o v e d b y w a s h i n g . T h e gel w a s c u t i n t o 2 - r a m s l i c e s a n d t h e r a d i o a c t i v i t y d e t e r mined. The position of the major peak corresponds to an apparent molecular weight of 150 000.

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WGAADDEDpg/ml Fig. 6. Binding of w h e a t g e r m agglutinin ( W G A ) to fixed p l a t e l e t s . Platelets ( 2 . 5 - 1 0 8 ) w e r e i n c u b a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f 125 T-labeled w h e a t g e r m a g g l u t i n l n for 15 rain at r o o m t e m p e r a t u r e in a t o t a l v o l u m e 1 m l . T h e a m o u n t of lectin b o u n d to p l a t e l e t s was d e t e r m i n e d b y Millipore filtration. T h e inset s h o w s a d o u b l e - r e c i p r o c a l p l o t of the s a m e d a t a for t h e d e t e r m i n a t i o n o f t h e n u m b e r o f b i n d i n g sites a n d t h e a p p ~ e n t d i s s o c i a t i o n c o n s t a n t . F o r clarity o f p r e s e n t a t i o n , only r e p r e s e n t a t i v e p o i n t s h a v e b e e n shown. -7

Lentil lectin did not stimulate platelets [10]. Platelets fixed in formaldehyde bound lentil lectin rapidly and tightly. Each platelet contained about 8 • l 0 s sites for this lectin with an apparent dissociation constant of 3 . 2 . 1 0 - T M (Fig. 7). Thus, the number of binding sites as well as the affinity of binding of lentil lectin to fixed platelets are quite similar to those of wheat germ agglutinin.

Effect o f lectins on thrombin binding to fixed platelets Results reported in this paper as well as by others indicate that wheat germ agglutinin binds primarily to GP-I on the platelet surface [14--16]. Recent results suggest that this glycoprotein also acts as the receptor for thrombin [6,16,19,20]. We considered it worthwhile to explore the effect of wheat germ

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