Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom

www.bba-direct.com

Biochimica et Biophysica Acta 1651 (2003) 30–40

Lebecetin, a potent antiplatelet C-type lectin from

Macrovipera lebetina venom

Sameh Sarraya,*, Najet Srairia, Mohamed Hatmib, Jose Luisc, Hechmi Louzird, Imed Regayaa,Hmida Slemae, Jacques Marvaldic, Mohamed El Ayeba, Naziha Marrakchia,f

aLaboratoire des Venins et Toxines, Institut Pasteur de Tunis, 13, place Pasteur, BP 74, 1002 Belvedere, Tunis, TunisiabUnite de Pharmacologie Cellulaire/INSERM U485, Institut Pasteur de Paris, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France

cCNRS UPRESA 6032, Faculte de Pharmacie, Laboratoire de Biochimie Cellulaire, 27 Bd Jean Moulin, 13385 Marseille CEDEX 5, FrancedLaboratoire d’Immunologie, Institut Pasteur de Tunis, BP 74-1002, Tunisia

eCentre National de Transfusion Sanguine, 11 Bis, Rue Djebel Lakhdar, Bab Saadoun, 1006 TunisiafFaculte de Medecine de Tunis, 9 Rue Professeur Zouheir Essafi, Tunisia

Received 13 November 2002; received in revised form 9 July 2003; accepted 14 July 2003

Abstract

A novel C-type lectin protein (CLP), lebecetin, was purified to homogeneity from the venom of Macrovipera lebetina by gel filtration on

a Sephadex G75 column and ion exchange chromatography on Mono S column. Lebecetin is a basic protein with a pHi = 9.9 and migrates in

SDS-PAGE as a single band or two distinct bands under nonreducing and reducing conditions, respectively. These results are further

confirmed by MALDI-TOF mass spectrometry that indicates a molecular mass of 29779 Da for native lebecetin and molecular masses of

15015 and 16296 Da for a and h subunits, respectively. The N-terminal amino acid sequences of lebecetin subunits show a high degree of

similarity with those of C-type lectin-like proteins. In addition, functional studies showed that lebecetin has a potent inhibitory effect on

platelet aggregation induced by thrombin in a concentration-dependent manner. In contrast, no inhibitory effect is observed when platelets are

exposed to thromboxane A2 (TxA2) mimetic (U46619) or arachidonic acid. Moreover, there was no effect either on blood coagulation or A,

B and O washed human erythrocytes agglutination. Furthermore, flow cytometric analysis revealed that fluoro-isothiocyanate (FITC)-

labelled lebecetin bound to human formalin fixed platelets in a saturable and concentration manner and this binding was specifically

prevented by anti-glycoprotein Ib (GPIb) mAb. These observations suggest that lebecetin is a C-type lectin-like protein that selectively binds

to platelet GPIb.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Macrovipera lebetina; C-type lectin-like venom protein; anti-aggregating protein; glycoprotein Ib antagonist

1. Introduction belong to two classes: disintegrins and C-lectin proteins

Snake venoms contain a variety of proteins that affect

mammalian proteins such as GPIb-IX-V, von Willebrand

factor (vWF), a2h1 and aIIbh3. These proteins are of

opposite functions: some of them promote while other block

platelet aggregation. Interestingly, a number of proteins

1570-9639/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S1570-9639(03)00232-2

Abbreviations: CLP, C-lectin protein; vWF, von Willebrand factor;

ADP, adenosine diphosphate; TxA2, thromboxane A2; IC50, concentration

leading to the half inhibitory effect; FITC, fluoro-isothiocyanate; GPIb,

glycoprotein Ib; TFA, trifluoroacetic acid; PRP, platelet-rich plasma

* Corresponding author. Tel: +216-71-843-755; fax: +216-71-791-833.

E-mail address: [email protected] (S. Sarray).

(CLPs). Disintegrins represent a family of low molecular

mass, cysteine-rich, RGD-containing, or not, polypeptides.

They inhibit integrins aIIb h3, avh3, a5h1, a4h1/a4h7and a5h1 [1–5].

The CLPs represent a separate large family of snake

venom proteins that exhibit different biological activi-

ties. They have many similar features, in particular

their inhibitory effects on both vWF binding to glyco-

protein Ib (GPIb) and vWF-mediated platelet aggrega-

tion. However, their specific binding sites on GPIb and

their actions are not necessarily identical. For example,

Botrocetin from Bothrops jararaca venom modifies

vWF, increasing the binding affinity of vWF for GPIb

S. Sarray et al. / Biochimica et Biophysica Acta 1651 (2003) 30–40 31

complex and promotes platelet aggregation [6,7]. Echi-

cetin from Echis carinatus venom binds to GPIb and

inhibits platelet aggregation [8]. Alboaggregins A and

B from Trimeresurus albolabris venom are also ago-

nists of GPIb, causing platelet agglutination without

any cofactors [9–11]. Agkicetin from Agkistrodon acu-

tus venom specifically blocks human umbilical vein

endothelial GPIb and inhibits angiogenesis in vivo

[12]. Other platelet GPIb-binding proteins include fla-

vocetins A and B from Trimeresurus flavoviridis ven-

om [13], tokaracetin from Trimeresurus tokarensis venom

[14] and EMS16 isolated from Echis multisquamatus

venom which is a selective inhibitor of the a2h1 integrin

[15].

Despite their different functions, these proteins represent

a structurally homologous group of proteins. They exist as a

multimeric form of two subunits, a and h, linked by a singleinter-chain disulfide bond. Each subunit of these proteins

constitutes a structural domain known as a carbohydrate

recognition domain (CRD). This structure was identified as

the minimum functional motif of Ca+ +-dependent animal

lectins [16].

In this article, we report the purification of a novel anti-

platelet aggregation protein termed lebecetin from the ven-

om of Macrovipera lebetina. As a result, this venom

component is of a heterodimeric structure and acts probably

as GPIb ligand.

2. Materials and methods

2.1. Materials

Venom was collected from M. lebetina snake of both

sexes in the serpentarium (Institut Pasteur, Tunis) and

immediately kept at � 18 jC. Collagen and adenosine

diphosphate (ADP) were a product of Chrono-Par. Human

thrombin was from Hoffman la Roche Diagnostica

(R-3235)l; Ca+ +-ionophore was purchased from Sigma.

U46619, a thromboxane A2 (TxA2) mimetic, was obtained

from Caymon Chemical. Luciferin and luciferase solution

was from Lumac (The Netherlands). Monoclonal antibod-

ies LJIb1 and LJCP8 were a generous gift from Dr.

Zavario M. Ruggeri (Room Research Center for Arterio-

sclerosis and thrombosis, Division of Experimental Hae-

mostasis and Thrombosis, Departments of Molecular and

Experimental Medicine and Vascular Biology, The Scripps

Research Institutes La Jolla, California 92037). vWF was

from immuno AG, Vienna, Austria. Applied Biosystem

supplied the reagents used for protein sequencing. MonoS

(HR 5/5) column for Fast Protein Liquid Chroma-

tography and Sephadex G-75 were purchased from Phar-

macia (Sweden) and reverse phase C8 column (5 Am;

4.6� 250 mm) was obtained from Beckman (USA). All

buffers and organic solvents were from standard commer-

cial sources.

2.2. Purification of lebecetin

Crude venom (267 mg) of M. lebetina was dissolved in

0.2 M ammonium acetate pH 6.8, applied to Sephadex G-

75 column equilibrated with the same buffer. The column

was eluted at a rate flow of 20 ml/h and 3-ml aliquots

were collected. Fraction II, containing anti-platelet aggre-

gation, was collected and lyophilised for further purifica-

tion. It was loaded on a Mono S (HR5/5) column

previously equilibrated with 50 mM HEPES/HCl pH 7.5

and eluted with linear 0–1 M NaCl gradient at a flow rate

of 1 ml/min. The fraction, containing the highest anti-

aggregation activity, was found in the last peak and has

been collected manually and dialysed against 5 mM

HEPES/HCl pH 7.5. Finally, this fraction was purified

on C8 reversed phase HPLC column equilibrated in 0.1%

trifluoroacetic acid (TFA), 10% acetonitrile and eluted with

a 60-min linear gradient of 10–80% acetonitrile at a flow

rate of 1 ml/min.

2.3. Gel electrophoresis

The homogeneity and apparent mass of the purified

lebecetin and its subunits were determined by SDS-PAGE

according to the method of Laemmli [17] using 15%

acrylamide gel without or with reduction by 2% h-mercaptoethanol. Molecular weight standards consisted

of phosphorylase b (93.3 kDa), BSA (67.6 kDa), oval-

bumin (42.7 kDa), carbonic anhydrase (30.2 kDa), soy-

bean trypsin inhibitor (20 kDa) and a-lactalbumine (14.4

kDa). Proteins were stained with Coomassie blue. Iso-

electric focusing was performed with a Phast system

(Pharmacia-LKB).

2.4. Protein assay

Protein concentration was determined by the BCA protein

assay (Pierce Chemical Co.) using bovine albumin as a

standard.

2.5. Carbohydrate analysis

Neutral sugars were determined as described by Dubois

et al. [18] using a standard solution containing D-glucose.

2.6. Separation of lebecetin subunits

Five nanomoles of purified lebecetin was dissolved in 40

Al of alkylation buffer (6 M guanidine–HCl; 0.5 M Tris/

HCl, 2 mM EDTA, pH 7.5) containing 1.4 Amol dithio-

threitol (DTT). After 1 h of reduction at 37 jC, 9 Amol of 4-

vinylpyridine (4VP) was added for alkylation. The reaction

was stopped after 5 min by addition of 14 Amol of DTT. The

S-alkylated protein chains were then desalted and separated

by reverse phase HPLC on a C8 column as described above

for protein purification.

S. Sarray et al. / Biochimica et Biophysica Acta 1651 (2003) 30–4032

2.7. NH2-terminal amino acid sequence analysis and

sequence identity search of lebecetin

The N-terminal amino acid sequences of S-alkylated

subunits were determined by Edman degradation in an

Applied Biosystem 476 A protein sequencer and phenyl-

thiohydantoin derivative of amino acids were identified by

on-line reverse HPLC using an RP-18 column. Sequence

homology was evaluated by a computer search in the protein

sequence database (BLAST search).

2.8. Mass spectrometry analysis

Samples were analyzed on a Voyager DE RP MALDI-

TOF mass spectrometer (perspective Biosystem, Inc., Fra-

mingham, MA). Lebecetin and its subunits were dissolved in

CH3CN/H2O (30:70) with 0.3% TFA. The matrix was pre-

pared as follows: alpha-cyano 4-hydroxycinnamic acid was

dissolved in 50% CH3CN in 0.3% TFA/H2O to obtain a

saturated solution at 10 Ag/Al. Peptide solution, 0.5 Al, wasplaced on the sample plate before addition of a 0.5-Al matrix

solution, and the mixture was then allowed to dry. Mass

spectra recorded in linear mode were externally calibrated

with suitable standards and then analyzed by the GRAMS/386

software.

2.9. Hemagglutination assay

Hemagglutination activity was assayed using human

erythrocytes and micro-agglutination slides according to the

Song method [19]. Serial dilutions of lebecetin in physiolog-

ical saline (9x NaCl) were used. Slides were placed on a

mini-shaker and 25 Al of a four-time washed 0.5% suspension

of human erythrocytes was added to each dilution. The slides

shacked for 1 min after erythrocyte addition were subse-

quently incubated for 1 h at 4 jC and the agglutination was

then read macroscopically.

2.10. Plasma clot retraction

As described by Li et al. [20], human platelet-rich plasma

(PRP; 0.6 ml) was mixed with 0.3 ml of lebecetin at various

concentrations of phosphate-buffered saline in noncoated

glass tubes. After incubation at 37 jC for 30 min, 0.1 ml of

thrombin (5 IU/ml final concentration) was added to each

tube, gently mixed and left for 2 h at 37 jC. The serum

volume of each tube was then measured and compared to a

control.

2.11. Platelet preparation

Platelets were prepared from whole blood obtained from a

3–4-kg male rabbit (HY/CR), with addition of 5 mM EDTA

as anticoagulant. Platelets were then separated from blood

and washed as previously described [21]. PRP was collected

after blood centrifugation at 375� g for 20 min and centri-

fuged for 15 min at 1800� g at 20 jC. The pellet was

washed twice with calcium-free Tyrode’s buffer (137 mM

NaCl, 2.7 mM KCl, 33 mM NaHCO3, 0.42 mM NaH2PO4, 1

mMMgCl2, 0.2 mM EGTA and 1% glucose or 2.5% gelatin)

adjusted to pH 7.38. Platelets were resuspended in a mod-

ified Tyrode’s buffer (pH 7.38) where EGTAwas substituted

by 2 mM CaCl2 and 1 mM MgCl2.

2.12. Platelet aggregation and ATP release

Platelet aggregation was measured by the turbidimetric

method [22] using a chronolog aggregometer with contin-

uous stirring at 1100 rpm. Platelets (0.4 ml) were pre-

incubated at 37 jC for 2 min with various concentrations

of lebecetin or its separated subunits prior stimulation by

various agents. Percent inhibition was calculated by com-

paring the value of the aggregation curves 5 min after the

addition of platelet agonists, taking the value of the control

curve as 100% aggregation. ATP release was measured by

bioluminescence technique [23] using a lumi aggregometer

(Coulter-France).

2.13. Preparation of formalin-fixed platelet suspension and

platelet agglutination

Formalin-fixed platelets were prepared as described by

Kirby and Mills [24]. Human platelet suspensions were

incubated with an equal volume of 2% formalin in Tris-

saline buffer for 1 h at room temperature. Platelets were then

washed twice with phosphate-buffered saline (PBS). Finally,

platelets were resuspended to a final concentration of

3� 108 platelets/ml in PBS containing 2 mg/ml BSA.

Platelets were pre-incubated for 2 min with variable

amounts of lebecetin in the presence of vWF (1 U/ml) and

ristocetine (20 Ag/ml), before recording the agglutination

with a chronolog aggregometer under continuous stirring at

1100 rpm. The effect of anti GPIb mAb on lebecetin activity

was tested after 1-min pre-incubation of the antibody with

fixed platelets.

2.14. Fluoro-isothiocyanate (FITC) conjugation of lebecetin

Lebecetin was conjugated with FITC according to Liu et

al. [25]. Briefly, protein dissolved in 0.1 M sodium bicar-

bonate was mixed continuously with FITC solution for 4

h and the reaction was stopped by addition of 1.5 M

hydroxylamine. The conjugated protein was separated from

free FITC using a Sephadex G-25 column at room temper-

ature. The concentration of FITC-protein was determined by

BCA protein assay kit. FITC-lebecetin retained an anti-

aggregating activity equivalent to unlabelled lebecetin.

2.15. Binding assay of FITC-lebecetin by flow cytometry

Thirty microliters of fixed platelets (2� 105 platelets/

ml) premixed with 65 Al of Tyrode’s solution was


incubated with 5 Al of FITC-lebecetin for 30 min at

room temperature. After incubation, mixtures were

centrifuged at 1000� g for 6 min and pellets were

Fig. 1. Purification of lebecetin. (A) Freshly milked M. lebetina venom was dilu

Sephadex G-75 column. Five fractions were collected and anti-aggregation activity

II, possessing anti-platelet aggregation, was lyophilised and applied to mono S co

performed at a flow rate of 1 ml/min by increasing the NaCl concentration in

absorbance at 280 nm. Anti-aggregation activity was determined as indicated

aggregation activity, was applied to a reverse phase C8 column. Elution was achi

insert is the SDS-PAGE pattern of the purified lebecetin. SDS-PAGE (15%) was p

with Coomassie blue. The standard proteins used for calibration of the apparent m

(67.6 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (30.2 kDa), soybean tryp

subsequently resuspended in 600 Al of PBS before flow

cytometric analysis. The platelet’s mean fluorescence

intensity was determined by fluorescence activated cell

ted twice with ammonium acetate 200 mM, pH 6.8, and then applied on

was tested. The overall yield of the column is about 85%. (B) The fraction

lumn (HR 5/5) equilibrated with HEPES 50 mM pH 7.5. The elution was

the same buffer as indicated (--). The protein content was monitored by

in Materials and methods. (C) Fraction II4, containing the highest anti-

eved in 60 min, using a gradient of acetonitrile (--) from 10% to 80%. The

erformed under nonreducing (NR) and reducing (R) conditions and stained

olecular weight were phosphorylase b (93.3 kDa), bovine serum albumin

sin inhibitor (20 kDa) and a-lactoalbumin (14.4 kDa).


sorter using excitation and emission of 488 and 525

nm, respectively. To determine the relative inhibitory

effect, anti-GPIb (LJIb1) and anti-GPIIb/IIIa (LJCP8)

Fig. 2. Chromatographic profile of reduced and alkylated lebecetin on an RP-H

chromatographic conditions were used: isocratic (10% B) for 10 min, followed by

Components A and B were 0.1% TFA (v/v) and 0.1% TFA (v/v) in 100% acetonitr

subunits.

were respectively added to fixed platelets during 30

min prior to the interaction of FITC-protein with

platelets.

PLC C8 column and MALDI-TOF mass spectrometry. (A) The following

10–80% B for 60 min and 80–100% for 2 min at a flow rate of 1 ml/min.

ile, respectively. (B) Mass spectrometry of lebecetin (b1), a (b2) and h (b3)


4. Results

4.1. Purification and characterization of lebecetin

The crude venom of M. lebetina was separated into five

fractions by gel filtration in a Sephadex G-75 column

yielding about 85% (Fig. 1A). Among these, fraction II

exhibiting anti platelet aggregation activity was collected

and fractionated again by ion exchange chromatography in

mono S column (Fig. 1B). Four fractions (II1, II2, II3 and

II4) were purified and showed to posses a molecular mass of

about 30 kDa, as estimated by SDS-PAGE. These fractions

exhibited an anti-aggregating activity (data not shown).

Fraction II4, containing the main anti-aggregating activity,

was purified by reversed phase HPLC in a C8 column. The

component eluted at a retention time of 38.53 min was

designated as lebecetin (Fig. 1C). The yields of purified

lebecetin after RP-HPLC were about 1% of the starting

material. Lebecetin is a basic protein with an isoelectric

point of about 9.9 (data not shown). The neutral sugar

content is about 20% for the purified protein. The reduced

and alkylated lebecetin was separated by chromatography on

Fig. 3. N-terminal sequence of subunit and comparison with other C-lectin prote

using an automatic sequencer of EDMAN degradation. (B) Alignment of N-termin

snake venom. References are: agkisacutacin [35], alboaggregin A [33], aggretin [

tokaracetin [14] and agkicetin [12].

C8 reverse column into two subunits, which were present in

an equal molecular ratio (Fig. 2A). As estimated by SDS-

PAGE (insert Fig. 1C), the lebecetin’s apparent molecular

mass was 29 kDa under nonreducing conditions. After

reduction, lebecetin showed two, 15 and 16 kDa, subunits.

This suggests that lebecetin is an heterodimer made of two

different subunits linked by disulfide bridge(s). Moreover,

mass spectrometry data (Fig. 2B) give the precise mass of

lebecetin (29,778.5 Da) and its two subunits a (15,016.7 Da)

and h (16,295.4 Da), demonstrating the heterodimeric com-

position of lebecetin structure.

4.2. N-terminal amino acid sequence of lebecetin

The N-terminal amino acid sequences of a and hlebecetin subunits were determined by polypeptide se-

quencing. These sequences show 52% identity (Fig. 3A).

Blast search results showed that the N-terminal sequences

of lebecetin subunits are homologous to the ones from

other snake venom CLPs, including the conserved cysteine

residues. The a and h subunits show 80% to 31% and

58% to 37% identity, respectively, when compared to the

in: (A) N-terminal sequencing of each subunit of lebecetin was performed

al amino acid sequences of a and h lebecetin subunits with other CLPs from

38], convulxin [27], botrocetin [6], echicetin [31,32], ECLV IX/X-bp [26],


sequences of the corresponding subunits of the other

CLPs. The lebecetin’s h subunit shares a highest identity

with the h chain of ECLV IX/X-bp (80%) [26]. However,

the N-terminal sequence of a lebecetin’s subunit exhibits

only 48% identity to that of convulxin a chain [27] (Fig.

3B). Consequently, we may assume that lebecetin is one of

the CLP superfamily.

4.3. Effect of lebecetin on enzymatic, hemagglutination and

thrombin-induced clot retraction and coagulation activities

Lebecetin was devoid of enzymatic activities such as

phospholipase A2, fibrinogenolytic and amidolytic activities

(data not shown). Moreover, lebecetin did not inhibit the

procoagulant activity of thrombin in the presence of human

plasma or fibrinogen. Also, it is unable to inhibit thrombin-

Fig. 4. Inhibition of platelet aggregation by lebecetin. (A) Inhibitory effect of leb

incubated with lebecetin for 2 min at 37 jC before adding 1 mM ADP (-o-) or 10

with lebecetin for 2 min before the addition of 0.04 IU/ml thrombin (-x-) or

experiments. (C) Effect of lebecetin on U46619- (1 AM) and collagen- (1 mg/ml

cells/ml) pre-incubated with different concentrations of lebecetin were exposed to

were determined; shown traces are results of experiments realized with 1.6 nM

platelets were incubated with a (-o-) or h (-x-) lebecetin subunits for 2 min befo

separate experiments.

induced clot retraction in PRP and the agglutination of

human erythrocytes (data not shown).

4.4. Effect of lebecetin on platelet aggregation

Lebecetin showed a potent inhibitory effect on platelet

aggregation in PRP induced by ADP or Ca+ +-ionophore

(Fig. 4A). The concentrations of lebecetin leading to 50% of

the inhibition of platelet aggregation (IC50) were 3.6 and

4.4 nM, respectively.

Lebecetin also inhibited either the thrombin- or the

collagen-induced aggregation of washed platelets in a con-

centration-dependent manner. The IC50 were about of 0.25

and 0.35 nM, respectively (Fig. 4B). However, under same

conditions, lebecetin did not inhibit the aggregation induced

by U46619.

ecetin on rabbit whole blood platelet aggregation: Platelet-rich plasma was� 3 mM Ca+ +-ionophore (-x-). (B) Rabbit washed platelets were incubated

1 mg/ml of collagen (-o-). Results are representative of three separate

) induced platelet aggregation and ATP release: Platelet suspensions (3.108

collagen (left panel) or U46619 (right panel); aggregation and ATP released

of lebecetin. Values in brackets indicate ATP amounts. (D) Rabbit washed

re the addition of 0.04 IU/ml thrombin. Results are representative of three


Rabbit washed platelets which aggregated and released

their granular contents in the presence of collagen and

U46619 were used to study the effects of lebecetin. As

shown in Fig. 4C, lebecetin failed to block both aggregation

and ATP release in response to U46619. In contrast, when

platelets were exposed to collagen, lebecetin inhibited in a

concentration-dependent manner both aggregation and ATP

release.

Reduced and alkylated lebecetin subunits have exhibited

separately an anti-aggregating activity when tested on

washed platelets in the presence of low concentration of

thrombin (0.04 IU/ml). The h subunit appears to be more

efficient than the a subunit. In fact, the IC50 were 1.55 and

0.7 nM for a and h subunits, respectively (Fig. 4D).

4.5. Effect of lebecetin on platelet agglutination

Lebecetin inhibited the agglutination of human platelets

by ristocetin in PRP. This effect was concentration-depen-

dent with an IC50 of about 0.45 nM (Fig. 5A). Furthermore,

Fig. 5. Inhibition of platelet agglutination by lebecetin. (A) Inhibition of agglutin

incubated with different concentrations of lebecetin for 2 min at 37 jC before add

lebecetin was incubated with fixed platelets for 2 min at 37 jC. Ristocetin (20 Agdetermined. A representative experiment is shown (n= 3). (C) Binding studies o

human platelets. Fixed platelets (2.105/ml) were incubated with various concentr

lebecetin concentration (nonspecific binding) (-o-) and then analysed by flow cy

the agglutination of washed human formalin-fixed platelets

induced by vWF (1 IU/ml) in the presence of ristocetin was

inhibited by lebecetin (Fig. 5B), with an IC50 of 0.65 nM.

In order to evaluate the importance of Ca+ + in this

process, we have tested the effect of EDTA and EGTA on

lebecetin-induced fixed platelet agglutination. Indeed,

EDTA and EGTA significantly reduced lebecetin activity

to 40% on platelet agglutination (data not shown).

4.6. Effect of FITC-lebecetin binding to formalin-fixed

platelets

Using FITC-conjugated lebecetin as a probe, we have

started to study the molecular mechanism of lebecetin. The

nonspecific binding was evaluated using high concentration

of native lebecetin. FITC-lebecetin is a potent anti-platelet

aggregating protein as native lebecetin (data not shown).

FITC-lebecetin bound in a concentration-dependent manner

to human fixed platelets and almost reached a saturable

binding at a concentration of 90 Ag/ml (3.1 AM).

ation of human whole blood by lebecetin: human platelet-rich plasma was

ing ristocetin (20 Ag/ml). (B) Effect of lebecetin on formalin fixed platelets:

/ml) and vWF (1 U/ml) were then added for 5 min and inhibition was then

f lebecetin to fixed platelets: binding isotherm of FITC-lebecetin to fixed

ation of FITC-lebecetin (-x-) in the presence or absence of higher native

tometry. Each point is the average of triplicate determinations.


4.7. Effect of various inhibitors on FITC-lebecetin binding

The concentration of FITC-lebecetin (3.1 AM) used in a

competition binding studies induces the maximal antipla-

telet aggregation. At 50 Ag/ml, anti GPIb mAb LJIb1 and

anti-GPIIb/IIIa (LJCP8) are used in separate experiments

using FITC lebecetin at 3.1 AM. Anti-GPIIb/IIIa (LJCP8)

did not compete with FITC-labelled lebecetin. In the

contrary, anti-GPIb mAb LJIb1, used at the same concen-

tration, was able to inhibit 50% of FITC-labelled lebecetin

to fixed platelets (data not shown). This result indicates

that lebecetin binds specifically with high affinity on GPIb

receptor.

5. Discussion

Ca+ +-dependent CLPs isolated from snake venom are

defined as nonenzymatic proteins. They are heterodimers

comprising of a and h subunits with a molecular mass value

of about 14–16000 Da. The subunits share a high degree of

sequence similarity with each other and with the other snake

venom CLPs. These proteins are recognized to contain a

CRD characterized by several conserved amino acid residues

in their structure. Despite their striking structural similarity,

the proteins of this group show a variety of biological effects

that depend on the structure of the carbohydrate recognition

site’s nature [28]. We report here the purification and the

characterization of a CLP, called lebecetin, isolated from the

venom of M. lebetina.

The crude venom of M. lebetina prevents platelet aggre-

gation induced by various agonists including thrombin. This

venom is known to contain small lebetin peptides with an

anti-platelet activity [29,30]. Besides them, we have identi-

fied at least three proteins with the same molecular mass of

about 29 kDa. One of them, lebecetin, has been purified. It

is a potent anti-platelet and anti-agglutination protein as

revealed by IC50 in the nanomolar range. Lebecetin was

homogeneous as examined by SDS-PAGE and mass spec-

trometry. It behaved as a molecule of 29,779 Da under

nonreducing conditions and separated into two bands, a and

h, of 15,016 and 16,296 Da, respectively, under reducing

conditions. Lebecetin exists as an heterodimeric glycopro-

tein composed of a and h subunits linked probably with a

disulfide bridge, as observed for other CLPs [6,9,31–35].

The putative role of the glycosylation in the biological

properties of lebecetin is not so far elucidated. Thus, it will

be interesting to elucidate the contribution of glucidic motif

to its biological activities. Lebecetin subunits were separated

by HPLC after reduction and alkylation and then sequenced.

Comparing the alignment of amino acid sequences of

lebecetin’s subunits, we found that the N-terminal sequences

of a and h subunits of lebecetin are highly homologous to

one another, and with those of CLPs, including GPIb-

binding proteins [6–8,12]. Thus, lebecetin may belong to

the CLP family.

Lebecetin inhibits platelet aggregation and ATP release

triggered by ADP, Ca+ +-ionophore, collagen or thrombin

but not by arachidonic acid or the TxA2 mimetic, U46619.

This selective inhibition rules out the possibility that,

whatever agonists used, lebecetin may inhibit GPIIb/IIIa–

fibrinogen interaction required for platelet aggregation [36].

One of the hurdles in understanding the structure–function

relationships of snake venom CLPs is to recognize whether

one or both subunits are required for biological activity.

Kawasaki et al. [37] suggested that the platelet GPIb-

binding site of these GPIb-BPs resides on the h subunit

but not the a subunit. On the other hand, Polgar et al. [32]

proposed that the a subunit may play a major role on the

cross-linking GPIb molecules in the same way as the natural

ligand, vWF, based on the observation that the amino acid

sequencing of h subunits is highly homologous to each

other and only GPIb ligand, such as AL-B (25-kDa alboag-

gregin), can agglutinate platelets. However, the reduced and

alkylated a and h subunits of lebecetin retained their

biological activities, suggesting the importance of the pri-

mary structure of lebecetin’s subunits. Therefore, the exact

participation of each subunit in binding is not clearly

elucidated.

The specific binding of lebecetin to GPIb is supported

by several lines of evidence. Lebecetin prevents selective-

ly a further aggregation induced by ristocetin in the

presence of human vWF. Flow cytometric analysis reveals

that lebecetin binds to fixed platelets in a dose-dependent

and saturable manner and that this binding is blocked

specifically by an anti-GPIb mAb (LJIb1). However, this

binding is unaffected by an anti-GPIIb-IIIa mAb (LJCP8),

which acts as a fibrinogen receptor (GPIIb/IIIa complex)

antagonist.

Further studies, which include the GPIb-binding site of

lebecetin, by using a panel of monoclonal antibodies

presenting different specificities with GPIb, will allow

us to elucidate the molecular mechanism of platelet

aggregation in terms of GPIb-vWF and provide a molec-

ular design suitable for the development of future anti-

thrombotic agents targeting at blocking vWF binding to

GPIb.

Acknowledgements

We thank Prof. Koussay Dellagi (Institut Pasteur de

Tunis) for his continuous interest in this study and for his

support. Prof. Zavario M. Ruggeri (Department of Molecular

and Experimental Medicine and Vascular Biology, The

Scripps Research Institutes La Jolla, California) is greatfully

acknowledged for his generous gift of monoclonal anti-

bodies. We would also like to thank Dr. Elalamy Ismaıl

(Institut Pasteur de Paris) for helpful advices. Dr. Benlasfar

Zakaria and Ben Zakour Lotfi (laboratoire veterinaire,

Institut Pasteur de Tunis) are acknowledged for providing

viper venoms.


References

[1] S. Niewiarowski, M.A. McLane, M. Kloczewiak, G.J. Stewart, Dis-

integrins and other naturally occurring antagonists of platelet fibrino-

gen receptors, Semin. Hematol. 31 (1994) 289–300.

[2] M.A. McLane, C. Marcinkiewicz, S. Vijay-Kumar, I. Wierzbicka-Pa-

tynowski, S. Niewiarowski, Viper venom disintegrin and related mol-

ecules, Proc. Soc. Exp. Biol. Med. 219 (1998) 109–119.

[3] C. Marcinkiewicz, L.A. Rosenthal, D.M. Mosser, T.J. Kunicki, S.

Niewiarowski, Immunological characterization of eristostatin and

echistatin binding sites on alpha IIb beta 3 and alpha v beta 3 integ-

rins, Biochem. J. 317 (1996) 817–825.

[4] C. Marcinkiewicz, J.J. Calvete, M.M. Marcinkiewicz, M. Raida, S.

Vijay-Kumar, Z. Huang, R.R. Lobb, S. Niewiarowski, EC3, a novel

heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4

and alpha5 in an RGD-independent manner, J. Biol. Chem. 274

(1999) 12468–12473.

[5] C. Marcinkiewicz, J.J. Calvete, S. Vijay-Kumar, M.M. Marcinkie-

wicz, M. Raida, P. Shick, R.R. Lobb, S. Niewiarowski, Structural

and functional characterization of EMF10, a heterodimeric disintegrin

from Eristocophis macmahoni venom that selectively inhibits alpha 5

beta 1 integrins, Biochemistry 38 (1999) 13302–13309.

[6] Y. Fujimura, K. Titani, Y. Usami, M. Suzuki, R. Oyama, T. Matsui, H.

Fukui, M. Sugimoto, Z.M. Ruggeri, Isolation and characterization of

two structurally and functionally distinct from botrocetin, the platelet

co agglutinin from the venom of Bothrops jararaca, Biochemistry 30

(1991) 1957–1964.

[7] M.S. Read, S.V. Smith, M.A. Lamb, K.M. Brinkhous, Role of

botrocetin in platelet agglutination: formation of an activated com-

plex of botrocetin and von Willebrand factor, Blood 74 (1989)

1031–1035.

[8] M. Peng, F.A. Emig, A. Mao, W. Lu, E.P. Kirby, S. Niewiarowski,

M.A. Kowalska, Interaction of echicetin with a high affinity throm-

bin-binding site on platelet glycoprotein GPIb, Thromb. Haemost. 74

(1995) 954–957.

[9] M. Peng, W. Lu, L. Beviglia, S. Niewiarowski, E.P. Kirby, A

snake venom protein that inhibits binding of von Willebrand Fac-

tor and alboaggregins to platelet glycoprotein Ib, Blood 81 (1993)

2321–2328.

[10] M. Peng, W. Lu, E.P. Kirby, Alboaggregins-B: a new platelet agonist

that binds to platelet membrane glycoprotein Ib, Biochemistry 30

(1991) 11529–11536.

[11] M. Peng, W. Lu, E.P. Kirby, Characterization of three alboaggregins

purified from Trimeresurus albolabris venom, Thromb. Haemost. 67

(1992) 702–707.

[12] C.H. Yeh, W.C. Wang, T.T. Hsieh, T.F. Huang, Agkicetin, a snake

venom-derived glycoprotein Ib antagonist, disrupts von Willebrand

factor–endothelial cell interaction and inhibits angiogenesis, J. Biol.

Chem. 275 (2000) 18615–18618.

[13] Y. Taniuchi, T. Kawasaki, Y. Fujimura, M. Suzuki, K. Titani, K.

Sakai, S. Kaku, N. Hisamichi, V. Satoh, T. Takenaka, M. Handa, Y.

Swai, Flavocetin-A and -B, two high molecular mass glycoprotein Ib

binding proteins with high affinity purified from Trimeresurus flavor-

idis venom, inhibit platelet aggregation at high shear stress, Biochim.

Biophys. Acta 1244 (1995) 331–338.

[14] T. Kawasaki, Y. Taniuchi, N. Hisamichi, Y. Fujimura, M. Suzuki, K.

Titani, S. Kaku, N. Satoh, T. Takenaka, M. Handa, Y. Swai, Tokar-

acetin, a new platelet antagonist that binds to platelet glycoprotein Ib

and inhibits von Willebrand factor-dependent shear-induced platelet

aggregation, Biochem. J. 308 (1995) 947–953.

[15] C. Marcinkiewicz, R.R. Lobb, M.M. Marcinkiewicz, J.L. Daniel, J.

Bryan Smith, C. Dangelmair, P.H. Weinreb, D.A. Beacham, S.

Niewiarowski, Isolation and characterization of EM16, a C-lectin

type protein from Echis multisquamatus venom, a potent and se-

lective inhibitor of the a2h1 integrin, Biochemistry 39 (2000)

9859–9867.

[16] K. Drickamer, M.E. Taylor, Biology of animal lectins, Annu. Rev.

Cell Biol. 9 (1993) 237–264.

[17] U.K. Laemmli, Cleavage of structural proteins during the assembly of

the head of bacteriophage T4, Nature 277 (1970) 680–685.

[18] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Col-

orimetric method for determination of sugars and related substances,

Anal. Chem. (1956) 350–356.

[19] P.L. Song, P. Xin, A lectin-like peptide isolated from the venom

of the Chinese spider Selenocosmia huwena, Toxicon 33 (1995)

875–882.

[20] Y.S. Li, K.F. Liu, Q.C. Wang, Mechanism of action of the platelet

function inhibitor from Vipera russelli siamensis snake venom, Tox-

icon 24 (1986) 875–883.

[21] N.G. Ardlie, D.W. Perry, M.A. Packham, J.F. Mustard, Adenosine

diphosphate induced platelet aggregation in suspensions of washed

rabbit platelet, Proc. Soc. Exp. Biol. Med. 13 (1970) 1021–1023.

[22] G.V.R. Born, M.J. Cross, The aggregation of blood platelets, J. Phys-

iol. 168 (1963) 178–195.

[23] H. Holmsen, E. Storm, H.J. Day, Determination of ATP and ADP in

blood platelets: a modification of the firefly luciferase assay for plas-

ma, Anal. Biochem. 46 (1972) 489–501.

[24] E.P. Kirby, D.C.B. Mills, The interaction of bovine factor VIII with

human platelets, J. Clin. Invest. 56 (1975) 491–502.

[25] C.Z. Liu, Y.W. Wang, M.C. Shen, T.F. Huang, Analysis of human

platelet glycoprotein IIb– IIIa by fluorescein isothiocyanate-conju-

gated disintegrins with flow cytometry, Thromb. Haemost. 39

(1994) 1915–1923.

[26] Y.L. Chen, I.H. Tsai, Functional and sequence characterization of

coagulation factor X-binding protein from the venom of Echis car-

inatus leucogaster, Biochemistry 35 (1996) 5264–5271.

[27] M. Leduc, C. Bon, Cloning of subunits of convulxin, a collagen-like

platelet aggregating protein from Crotalus durissus terrificus venom,

Biochem. J. 333 (1998) 389–393.

[28] Y. Komori, T. Nikai, T. Tohkai, H. Sugihara, Primary structure

and biological activity of snake venom lectin (APL) from Agkis-

trodon p. piscivorus (Eastern cottonmouth), Toxicon 37 (1999)

1053–1064.

[29] R. Barbouche, N. Marrakchi, P. Mansuelle, M. Krifi, E. Fenouillet, H.

Rochat, M. El Ayeb, Novel anti-platelet aggregation polypeptides

from Vipera lebetina venom: isolation and characterization, FEBS

Lett. 392 (1996) 6–10.

[30] R. Barbouche, N.Marrakchi, K.Mabrouk,M. Krifi, J. VanRietschoten,

E. Fenouillet, M. El Ayeb, H. Rochat, Anti-platelet activity of the

peptides composing the lebetin 1 family, a new cl of inhibitors of

platelet aggregation, Toxicon 36 (1998) 1939–1947.

[31] M. Peng, J.C. Holt, S. Niewiarowski, Isolation, characterization and

amino acid sequence of echicetin beta subunit, a specific inhibitor of

von Willebrand factor and thrombin interaction with glycoprotein Ib,

Biochem. Biophys. Res. Commun. 205 (1994) 68–72.

[32] J. Polgar, E.M. Magnenat, M.C. Peitsch, T.N.C. Wells, M.S.A. Saqi,

K.J. Clemetson, Amino acid sequence of the alpha subunit and com-

puter modelling of the alpha and beta subunits of echicetin from the

venom of Echis carinatus (saw-scaled viper), Biochem. J. 323 (1997)

533–537.

[33] M.A. Kowalska, L. Tan, J.C. Holt, M. Peng, J. Karczewski, J.J.

Calvete, S. Niewiarowski, Alboaggregins A and B. Structure and

interaction with human platelets, Thromb. Haemost. 79 (1998)

609–613.

[34] Y. Usami, Y. Fujimura, M. Suzuki, Y. Ozeki, K. Nishio, H. Fukui, K.

Titani, Primary structure of two chain botrocetin, a von Willebrand

factor modulator purified from the venom of Bothrops jararaca, Proc.

Natl. Acad. Sci. U. S. A. 90 (1993) 928–932.

[35] X. Cheng, Y. Qian, Q. Liu, B.X. Li, M. Zhang, J. Liu, Purification,

characterization, and cDNA cloning of a new fibrinogenolytic venom

protein, Agkisacutacin, from Agkistrodon acutus venom, Biochem.

Biophys. Res. Commun. 265 (1999) 530–535.

[36] J.J. Calvete, Platelet integrin GPIIb/IIIa structure– function correla-


tions. An update and lessons from other integrins, Proc. Soc. Exp.

Biol. Med. 222 (1999) 29–38.

[37] T. Kawasaki, Y. Fujimura, Y. Usami, M. Suzuki, S. Miura, Y. Sakurai,

K. Makita, Y. Taniuchi, K. Hirano, K. Titani, Complete amino acid

sequence and identification of the platelet glycoprotein Ib-binding site

of jararaca GPIb-BP, a snake venom protein isolated from Bothrops

jararaca, J. Biol. Chem. 271 (1996) 10635–10639.

[38] C.H. Chung, L.C. Au, T.F. Huang, Molecular cloning and sequence

analysis of aggretin, a collagen-like platelet aggregation inducer, Bio-

chem. Biophys. Res. Commun. 263 (1999) 723–727.

Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom

Documents

Transcript of Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom