A novel 25 kDa protein from the venom of Bitis arietans

with similarity to C-type lectins causes fibrinogen-dependent

platelet agglutination

Brent Jennings, Wendy Spearman, Enid Shephard*

Department of Medicine, UCT/MRC Liver Research Centre, University of Cape Town, Observatory 7925, South Africa

Received 6 April 2005; revised 13 July 2005; accepted 14 July 2005

Available online 15 September 2005

Abstract

Snake venoms affect blood coagulation and platelet functions in various ways. Venom from the Viperidae and Crotalidae

family of snakes contains biologically active proteins that possess coagulant and anticoagulant activities, as well as platelet

aggregating and inhibitory activities. Many of these proteins belong to the C-type lectin family. C-type lectins from viper

venoms can act by prohibiting the interaction between platelet receptors, such as GPIIbIIIa and the GPIb/V/IX complex, and

their ligands. We report on the purification of a novel 25 kDa protein, Ba25, from Bitis arietans with a primary structure that

possesses similarity to other C-type lectins from viper venom. This protein has a profound effect on the clotting of whole blood,

as well as being able to cause agglutination of platelets in platelet rich plasma without degranulation of the cells, but not of

washed platelets in the absence of fibrinogen. Ba25 interacts with the platelet via the GPIb/V/IX, as well as the GPIIbIIIa

receptor, and causes an increase in binding of fibrinogen to platelets. These results suggest that Ba25 may be a potent mediator

of platelet–platelet interactions, and other coagulatory mechanisms.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Agglutination; C-type lectin; GPIIbIIIa; Platelet; Snake venom; vWF

1. Introduction

The primary role of the numerous and varied proteins

within snake venom is to act in unison to immobilise prey.

Snakes belonging to the Viperidae and Crotalidae families

produce venom with hemorrhagic activity through the

action of proteins classified as proteases, fibrinogenases,

haemorrhagins, disintegrins, metalloproteases or C-type

lectins. These various proteins modulate the function of

platelets, endothelial cells, fibrinogen, coagulation factors

and other processes within the clotting pathway. Disruption

of clotting is a consequence of both the proteolytic

activation of coagulation factors and cleavage of fibrinogen

0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2005.07.011

* Corresponding author. Fax: C27 21 4486815.

E-mail address: enid@uctgsh1.uct.ac.za (E. Shephard).

by metalloproteases and the effects on the subsequent

function of platelets (Hutton and Warrell, 1993; Markland,

1997), while cleavage of basal membrane proteins in vessel

walls is due to haemorrhagins, metalloproteases with a zinc-

binding domain. The disintegrins, which are also a major

component of many snake venoms, contain an RGD (Arg-

Gly-Asp) or similar active sequence, which inhibits the

binding capacity of integrin adhesion receptors on platelets

and other cells (Gould et al., 1990). RGD-containing venom

proteins such as echistatin from Echis carinatus (Gan et al.,

1988), and salmosin from Agkistrodon halys brevicaudus

(Kang et al., 1998) bind to the GPIIbIIIa, blocking the

binding of ligands to this receptor, thus inhibiting platelet

aggregation. Proteins with both a metalloprotease and

disintegrin domain exist in snake venom and have been

classified as the ADAMS class of proteins (Jia et al., 1996).

Such proteins have dual purpose through the ability to bind

Toxicon 46 (2005) 687–698

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B. Jennings et al. / Toxicon 46 (2005) 687–698688

receptors and subsequently cleave the receptor and

extracellular matrix to which the receptor is bound. These

proteins predominantly affect platelet–collagen interactions.

C-type lectins, a family of proteins with heterodimeric

structures and calcium-dependent carbohydrate-binding prop-

erties are abundant in venom with haemorrhagic activity and

bind either specific platelet receptors or coagulation factors

(Drickamer, 1993). The C-type lectins are so called because of

their requirement for calcium ions for expression of their

activity. Most of the snake C-type lectins have a heterodimeric

structure and a high degree of homology, but display diverse

mechanisms of action (Clemetson and Polgar, 1998).

Botrocetin (Sen et al., 2001) and bitiscetin (Hamako et al.,

1996; Matsui et al., 2000) act by binding to the vWF, forming

an active complex that binds to GPIb on the platelet

membrane, serving as a bridging agent and resulting in platelet

aggregation. Echicetin (Peng et al., 1994; Polgar et al., 1997),

agkicetin (Chen and Tsai, 1995), flavocetin-A and -B

(Taniuchi et al., 1995; Fukuda et al., 2000), tokaracetin

(Kawasaki et al., 1995), Crotalus horridus horridus GPIb-

binding protein (Andrews et al., 1996) and mamushigin

(Sakurai et al., 1998) are all C-type lectins that bind to platelet

GPIb, preventing vWF binding, consequently inhibiting

platelet aggregation. Alboaggregins A and B from Trimer-

esurus albolabris, are also able to stimulate release of platelet

granule content in addition to aggregating the cells by binding

to the GPIb (Kowalska et al., 1998). Other C-type lectins in

snake venom such as the coagulation factor IX/factor

X-binding protein from T. flavoviridis (Mizuno et al., 1997)

and carinactivase from E. carinatus (Yamada et al., 1996) bind

to clotting factors, which prohibits aggregation of platelets.

The venom of the puff adder, Bitis arietans, has been

shown to contain disintegrins such as bitistatin, which binds

to the platelet GPIIb/IIIa (Shebuski et al., 1989). It also

contains the C-type lectin biticetin and Ba100, a C-type

lectin with fibrinogenase activity (Jennings et al., 1999).

This study investigates the existence of platelet-binding

proteins within the venom of the South African puff adder.

A GPIIb/IIIa affinity column isolated a single novel protein

from the venom that agglutinated platelets only in the

presence of fibrinogen, and inhibited blood clotting in whole

blood. This protein has a heterodimeric structure and an

apparent molecular mass of 25 kDa, a pI of 7.4 and an

N-terminal amino acid sequence similar to that of many

other C-type lectins from crotalids and vipers (Andrews

et al., 1996; Mizuno et al., 1997; Yamada et al., 1996;

Hirotsu et al., 2001; Kawasaki et al., 1996).

2. Materials and methods

2.1. Materials

Lyophilised snake venom was obtained from puff adders

found in the Western Cape region of South Africa.

Radioactive serotonin was purchased from Amersham

Pharmacia Biotech. PAC-1 (an IgM that binds to GPIIb/IIIa

at or near the fibrinogen-binding site) and FACS lysing

solution were from Becton Dickinson (BD Biosciences).

Anti-CD41 (clone P2, which reacts with GPIIb in the intact

GPIIb/IIIa complex), CD61 (clone SZ21, which reacts with

GPIIIa), CD42b (clone SZ2, which reacts with GPIb),

CD62P (clone CLB-Thromb/6, reacting with P-selectin) and

annexin-V (a protein reacting with phosphatidylserine) were

purchased from Coulter Immunotech. Human fibrinogen

was purified in our laboratory (Kalvaria et al., 1986;

Jennings et al., 1999). Equine tendon collagen was

purchased from Helena Biosciences (UK). Labelled chicken

anti-fibrinogen antibody was purchased from Diapensia

(Sweden). Other chemicals were purchased from Sigma

Chemical Co.

2.2. Purification and N-terminal amino acid sequencing

of Ba25

Lyophilised crude venom (100 mg) was dissolved in

3 mL 0.1 M acetic acid (glacial, Riedel-deHaen), clarified

by centrifugation at 400!g for 10 min and subjected to gel

filtration as described previously (Jennings et al., 1999).

Fractions were eluted from the column in 0.1 M acetic acid

and protein was detected by absorbance at 280 nm (Hitachi

U-2000 spectrophotometer). Three pools (A–C, Fig. 1a) of

the individual fractions were made, then dialysed into Tris

saline buffer (TS150 C/M, 10 mM Tris pH 7.4, 150 mM

NaCl, 1 mM CaCl2, 1 mM MgCl2) before being loaded onto

a GPIIb/IIIa-affinity column, prepared in our laboratory. For

this, the GPIIbIIIa receptor was isolated from outdated

platelets (Fitzgerald et al., 1985) and concentrated by

ultrafiltration on a Diaflo PM10 membrane, before being

loaded onto a fibrinogen affinity column made by coupling

purified fibrinogen to Sepharose CL-4B according to

manufacturers specifications. The fibrinogen-depleted GPII-

bIIIa was then coupled to Sepharose CL-4B, according to

manufacturer’s instructions.

Pools A, B and C were passed through the GPIIbIIIa

column under gravity after which the column was washed

with 10 column volumes of TS150 C/M. Bound protein was

eluted with 0.1 M sodium acetate buffer, pH 4, containing

1 M NaCl. Protein in each fraction (1 mL) was estimated by

measuring the absorbance at 280 nm. Peak fractions were

pooled and immediately dialysed into 1/10—strength

phosphate-buffered saline (PBS), then lyophilised and

stored at K70 8C. Prior to use the lyophilised protein was

dissolved using a volume of Milli-Q (Millipore) purified

water equal to 1/10th the volume prior to lyophilization.

Protein concentration was determined using the Biorad

assay, and adjusted to 0.5 mg/mL with PBS.

SDS-PAGE was performed as described in (Jennings

et al., 1999) using a 10% gel under reducing and non-

reducing conditions. Non-reduced Ba25 was dialysed into

purified water and lyophilised before amino acid sequence

3

B. Jennings et al. / Toxicon 46 (2005) 687–698 689

analysis was performed as described previously (Brandt

et al., 1984).

2.3. Isoelectric point determination of Ba25

The isoelectric point of Ba25 was determined on a 5%

polyacrylamide gel plate with 3% gel linkage and 2.2%

(w/v) ampholine, with a broad pH range between 3.5 and

9.3, as described previously (Jennings et al., 1999), and

stained with 0.12% Coomassie blue.

2.4. FITC-labelling of Ba25

Ba25 was labelled with FITC by incubating 2 mg of

Ba25 in 1 mL PBS with 10 mg FITC in 1 mL DMSO. The

mixture was vortexed for 5 min at room temperature then

dialysed into PBS to remove unreacted FITC. Ba25–FITC

concentration was adjusted to 0.5 mg/mL PBS.

2.5. Thromboelastography

Thromboelastography (TEG) was performed using a

thromboelastograph coagulation analyser (Haemoscope

Corp.) pre-warmed to 37 8C according to manufacturers

instructions and as previously described (Jennings et al.,

1999). Various concentrations of Ba25 were added to

350 mL of freshly drawn blood in the absence of antic-

oagulants in a cuvette in the apparatus and carefully mixed.

A TEG profile obtained in the absence of Ba25 was included

in parallel as a control for each run to ensure that the

parameters were within the normal range. All reactions were

tested in triplicate.

2.6. Isolation of platelets

Whole blood was collected from healthy volunteers, on

no medication affecting platelet aggregation, into antic-

oagulant (CCD, 93 mM citrate, 7 mM citric acid, pH 6.5,

140 mM dextrose with 0.35% BSA). Platelet rich plasma

Fig. 1. Isolation and N-terminal sequence of Ba25. (a). Gel filtration

of crude venom. Pooled fractions of protein were combined. (b)

SDS-PAGE analysis (10% polyacrylamide gel, reducing con-

ditions) of GPIIbIIIa concentrate prior to (lane a) and after (lane

c) removal of fibrinogen on the anti-fibrinogen affinity column. (c).

SDS-PAGE analysis (5–13% polyacrylamide gradient gel) on a

GPIIbIIIa affinity column. Lane 1, molecular weight markers

(weights in kDa on the left); Lane 2, protein from pool C prior to

loading on the GPIIbIIIa column; Lane 3, Protein eluted from the

column called Ba25; Lane 4, Ba25 in the presence of reducing

agent. (d). Ba25 shows N-terminal sequence homology with other

C-type lectins from snake venom. Amino acids that are identical to

those in the 18 kDa sequence from Ba25 are boxed. The percentage

similarity is indicated on the right of the figure. (1) mamushigin, (2)

coagulation factor IX/X binding protein, (3) alboaggregin A, (4)

CHH-B, (5) echiscetin, (6) botrocetin, (7) bitiscetin, (8) the short

subunit of Ba25, (9) Ba100.

B. Jennings et al. / Toxicon 46 (2005) 687–698690

(PRP) was prepared by centrifugation of anticoagulated

blood at room temperature for 20 min at 300!g. Washed

platelets were obtained by pelleting the platelets from PRP

by centrifugation at 1800!g for 20 min at room tempera-

ture. The pellet was carefully rinsed three times with 2 mL

of modified Tyrode Hepes buffer (THB, 10 mM HEPES,

140 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.42 mM

NaH2PO4, 5.5 mM glucose, pH 7.4) containing 1 mM

CaCl2, 1 mM MgCl2 and 0.1% BSA (THB C/M/B). The

pellet was then resuspended in this buffer to the previous

volume of PRP. Cells were counted on a Coulter H-1

counter and made up to 2.25!108 cells/mL unless

otherwise indicated.

2.7. Light microscopy

To observe the reaction of platelets with Ba25, 0.4 or

0.1 mM Ba25 was added to PRP for 4 min at 37 8C with

gentle mixing. The platelets were then fixed with 0.5%

formalin. PRP aggregated with 10 mM ADP for 4 min then

fixed with 0.5% formalin served as a negative control.

Fixation was at room temperature for 10 min, after which

aliquots were pipetted onto glass microscope slides and

covered with cover slips prior to viewing using a 100!magnification oil-immersion objective lens in a Zeiss

Axioscop microscope.

2.8. Platelet aggregometry

Aggregometry was performed with either freshly

prepared washed platelets in THB C/M/B or PRP using a

Chrono-long whole blood aggregometer according to

manufacturers instructions. Platelet poor plasma (PPP), or

THB C/M/B was used as a blank. The agglutination of

washed cells was measured relative to that induced by

0.1 U/mL thrombin, while agglutination of platelets in PRP

was measured relative to response to 10 mM ADP. Washed

platelets (in the presence and absence of fibrinogen) or PRP

was warmed to 37 8C. Four-hundred and fifty microliters

were then added to aggregometer tubes containing various

concentrations of Ba25 in 50 mL and agglutination was

measured for 4 min. For experiments, which contained anti-

CD42b, anti-CD61 or anti-CD41, 350 mL PRP (or washed

platelets) was pre-incubated with antibody at a final

concentration of 40 mg/mL for 5 min at 37 8C in the

aggregometer prior to the addition of 50 mL Ba25 at various

concentrations to initiate agglutination, which was recorded

for 4 min.

2.9. Flow cytometry

To investigate Ba25 binding to platelets, washed

platelets (50 mL) were incubated (15 min at room tempera-

ture) with various concentrations of Ba25–FITC in the

presence or absence of various concentrations of ADP. In

experiments using monoclonal antibodies to investigate

Ba25 binding to platelets, reactions contained PRP diluted

10-fold with THB C/M/B (1/10 PRP). In the case of anti-

CD61, anti-CD41 and anti-CD42b, 60 mL 1/10 PRP aliquots

were added to 6 mL (6 mg) of antibody for 10 min at room

temperature. In the case of the chicken anti-fibrinogen

antibody, 50 mL 1/10 PRP aliquots were added to 10 mL

antibody for 20 min at room temperature. For measurement

of PAC-1 binding, washed platelets, warmed to 37 8C, were

incubated in the presence and absence of 1 mM Ba25 and

various concentrations of ADP for 4 min. 10 mL aliquots

were then removed from each sample into tubes containing

20 mL PAC-1 and incubated at room temperature for

15 min.

At the end of the reactions all samples were processed by

the addition of 500 mL FACS Lysing solution (BD

Biosciences) prior to acquisition of 5000 platelets and

analysis of fluorescent platelets using an EPICS XL

(Beckman Coulter) flow cytometer within 30 min.

3. Platelet activation assays

Platelet a-granule release was measured as the

expression of P-selectin, CD62P, on the surface of the

platelet by flow cytometry. Washed platelets (50 mL) at 1!107 cells per mL were warmed to 37 8C, then added to tubes

containing various concentrations of Ba25 in the presence

and absence of 0.2 mg/mL fibrinogen in a final volume of

100 mL for 3 min at 37 8C, after which 10 mL of anti-CD62P

or isotype IgG1-PE antibody (negative control) were added.

Tubes were incubated for 5 min at 37 8C before being fixed

using the Q-Prep (Coulter) system. Positive controls for

a-granule release contained thrombin at a final concen-

tration of 0.25 U/mL.

Platelet dense granule secretion was measured as

described by Andrews et al. (1996). Briefly, platelets in

PRP were loaded with 3H-5HT, washed and then stimulated

with an agonist in the presence and absence of 0.5 mg/mL

fibrinogen. Radioactivity was measured in the supernatant

and expressed as percentage release relative to that released

by 0.25 U/mL thrombin control.

Expression of phosphatidylserine on the surface of

washed platelets in the presence and absence of

0.5 mg/mL fibrinogen, or platelets in PRP, was measured

by the binding of annexin-V-FITC by flow cytometry as

described by Tait et al. (1999) using the binding of annexin-

V-FITC as a marker, either with washed cells or with PRP.

4. Results

4.1. Isolation of Ba25 and N-terminal amino-acid

sequencing

Crude venom was fractionated using an HW-50 column

(Jennings et al., 1999) and pooled fractions A, B and C were

B. Jennings et al. / Toxicon 46 (2005) 687–698 691

subjected to GPIIbIIIa affinity chromatography (Fig. 1a).

The platelet receptor GPIIbIIIa was isolated from outdated

platelets and contaminating fibrinogen removed prior to the

preparation of the GPIIbIIIa affinity column (Fig. 1b). No

proteins in pool A or B bound to the affinity column. The

nature of the protein in pool C that eluted from the column

was assessed by SDS polyacrylamide gel electrophoresis

under reducing and non-reducing conditions (Fig. 1c).

Under non-reducing conditions, a single band migrating to

an apparent molecular mass of 25 kDa was observed which

resolved into two bands migrating to apparent molecular

masses of 18 and 14 kDa, respectively, in the presence of

reducing agent. This protein was called Ba25, and was

found to have an isoelectric point of 7.4. The N-terminal

sequences of the two subunits of Ba25 indicate the presence

of the sequence region typical in C-type lectins (Fig. 1d).

Fig. 2. Thromboelastography. The TEG parameters were read directly off

the TEG (b). The effect of Ba25 on the reaction time (r), the clot formation

was measured. Blood that was drawn from four different donors yeilded r

The sequence similarity to the N-termini of other C-type

lectins from snakes is also shown.

4.2. Thromboelastography

Ba25 had a profound effect on clot formation as

measured by changes in the TEG parameters (Fig. 2a).

Increasing concentrations of Ba25 increased the time taken

for the initiation of clot formation (as measured by the r

time), and caused an increase in the time taken for the clot to

reach a specified strength (K time, measured from r time to

the point where the tracing amplitude reaches 20 mm)

(Fig. 2b). The rate of clot formation decreased, as measured

by the a-angle, while the maximum strength of the clot,

indicated by the maximum amplitude of the trace (MA), was

decreased with increasing concentration of Ba25. At

the tracings (a). Ba25 (or PBS control) was added to whole blood in

time (K), the clot strength (MA) and the speed of clot formation (a)

esults that were within 10% of these results.

B. Jennings et al. / Toxicon 46 (2005) 687–698692

concentrations of Ba25 above 300 nM, a clot failed to form

and a ‘flat line’ tracing was recorded by the TEG apparatus

indicating the blood remained in a completely fluid state.

The addition of 0.1 U/mL thrombin at this stage was found

to induce clotting. Blood from four different donors yielded

the same results.

4.3. Light microscopy

When viewed under the light microscope, resting

platelets, seen along their narrow-edge axis (dark arrows)

or broad-edge axis (light arrows) (Fig. 3a), are highly

refractile, discoid particles present in high numbers with no

signs of aggregation, or activation, such as ‘rounding-off’ or

clumping of cells. Platelets that have been stimulated with

ADP (Fig. 3b) are clumped into aggregates and exhibit a

granular appearance which is a result of fusion of granule

Fig. 3. Light microscopy of platelet agglutination in PRP. Resting platelets

arrows). PRP was aggregated with either 10 mM ADP (B) or 0.4 mM Ba2

viewing. Arrows show particularly clear examples of single cells. Bars re

membranes with each other and with the membranes of the

open canalicular system, as well as the outer cell membrane

(O’Brien and Heywood, 1996). Aggregation is also

associated with membrane spreading and a high degree of

platelet–platelet interaction. Platelets that have been

incubated for 4 min with 0.4 mM of Ba25 are clumped

into small aggregates even though their discoid shape has

been maintained (Fig. 3c, good examples evident at arrows)

with little sign of the granularity as seen in Fig. 3b. Thus, the

platelets appear to be undergoing cell–cell interactions, and

a high degree of adhesion with each other, but without the

usual signs of activation, such as shape change. 1 mM Ba25

caused large aggregates of platelets with a granular

appearance to form within 10 min (Fig 3d). The platelets

have lost their discoid shape and are present as more

rounded particles, appearing to be in a more advanced stage

of agglutination than in Fig. 3c. However, the individual

(A) viewed along their narrow axis (dark arrows), or broad axis (light

5 (C) for 4 min or 1 mM Ba25 for 10 min (D) before fixation and

present 5 mm.

B. Jennings et al. / Toxicon 46 (2005) 687–698 693

platelets are still clearly discernable (clear examples at

arrows), and it appears that the degree of cell spreading and

shape loss is less than for ADP treated platelets.

4.4. Platelet agglutination by Ba25

Ba25 agglutinated PRP in a concentration-dependent

manner (Fig. 4a). One micromolar Ba25 caused 100%

agglutination (relative to that caused by 10 mM ADP) after

3 min at 37 8C with 50% agglutination attained at 0.25 mM

Ba25. Washed platelets were not agglutinated by Ba25 at

Fig. 4. The agglutination of platelets by Ba25. (a) Various

concentrations of Ba25 were added to PRP for 3 min (results

expressed as the average of seven different experiments). (b) Ba25,

at a final concentration of 1 mM, was added to washed platelets in

the presence of various amounts of fibrinogen for 4 min. (c) PRP

incubated with 40 mg/mL anti-CD42b or control PBS before the

addition of various concentrations of Ba25 for 4 min. Results are

expressed as a percentage of maximum agglutination in response to

5 mM ADP (for PRP), or 0.1 U/mL thrombin (for washed platelets).

any concentration tested (up to 2 mM Ba25, results not

shown). However, when washed platelets were treated with

1 mM Ba25 in the presence of various concentrations of

fibrinogen, a fibrinogen-dependent increase in platelet

agglutination was seen (Fig. 4b). After 4 min of aggluti-

nation, platelets in the presence of 100 mg/mL of fibrinogen

and 1 mM Ba25 had undergone 80% agglutination (relative

to that caused by 0.1 U/mL thrombin), as opposed to zero in

the absence of fibrinogen. Pre-incubating PRP with 40 mg/

mL anti-CD42b inhibited platelet agglutination induced by

1 mM Ba25 by 92% (Fig. 4c). When washed platelets were

pre-incubated with anti-CD42b, their agglutination in

response to 1 mM Ba25 and 100 mg/mL fibrinogen was

inhibited by 96%.

No inhibition of platelet agglutination in PRP by 1 mM

Ba25 could be detected if the PRP was pre-incubated with

either anti-CD61 or anti-CD41 at 40 mg/mL. Both these

antibodies were able to inhibit platelet agglutination in

response to ADP (result not shown).

4.5. Assessment of Ba25 interaction with platelets

by flow cytometry

Ba25–FITC bound concentration dependently to washed

platelets and saturation was observed at a concentration of

2 mM Ba25 (Fig. 5). There was an increase in both the

number of platelets binding Ba25–FITC and an increase in

the number of receptors for Ba25–FITC as the amount of

Ba25–FITC offered to the platelets increased. Fig. 5 also

shows that platelets in the presence of 20 mM ADP

displayed increased percentage fluorescence as well as

mean fluoresence intensity. Binding of Ba25–FITC was

inhibited by unlabelled Ba25 (results not shown).

The use of PRP diluted tenfold negates the potential

problems of platelet aggregate formation during stimulation

Fig. 5. Ba25–FITC binding to platelets. Fifty microliter-aliquots of

washed platelets were incubated with various concentrations of

Ba25–FITC, in the presence and absence of 20 mM ADP, to a final

volume of 100 mL for 15 min at room temperature. The cells were

fixed using the Q-Prep system before being subjected to flow

cytometry within 30 min.

Fig. 6. Effect of Ba25 on the binding of antibodies to GPIIbIIIa and GPIb. 1/10 PRP was incubated with various concentrations of Ba25 for

10 min at 37 8C. Various antibodies (final concentration 100 mg/mL) were added to aliquots for 10 min at room temperature before fixation, and

processing for flow cytometry. Inserts show representative flow histograms with Ba25 concentrations indicated above the relevant curve. The

percentage of platelets binding to the antibody was in excess of 98% in each sample. Error bars represent the average of four separate

experiments.

Fig. 7. Chicken anti-fibrinogen binding to platelets increases in

response to Ba25. Various concentrations of Ba25 were added to

1/10 PRP for 5 min at 37 8C. Fifty microliter-aliquots were removed

and added to 10 mL FITC-labelled monoclonal chicken anti-

fibrinogen IgG for 20 min at room temperature. Experiments were

done in triplicate and processed for flow cytometry within 30 min.

B. Jennings et al. / Toxicon 46 (2005) 687–698694

and has thus been found to be suitable for analysis of

the response of platelets to stimuli using flow cytometry

(Xia et al., 1996).

Incubating platelets with Ba25 did not affect the ability

of anti-CD61, anti-CD41 or anti-CD42b antibodies to bind

to 100% of platelets. However, an approximate 1.4-fold

increase in the mean fluorescence intensity of the binding of

anti-CD61 and anti-CD41 to the GPIIbIIIa receptor was

observed when platelets in PRP were incubated with 1 mM

Ba25 (Fig. 6). In contrast, the mean fluorescence intensity of

anti-CD42b binding to platelets decreased 2.5-fold when

1 mM Ba25 was incubated with platelets in 1/10 PRP.

54% of platelets in 1/10 PRP were labelled with an anti-

fibrinogen antibody (Fig. 7). This binding of anti-fibrinogen

increased 1.6 fold with the addition of 1 mM Ba25. In

addition, the mean fluorescence intensity of anti-fibrinogen

binding increased 1.3 fold with the addition of 1 mM Ba25.

PAC-1 binds only to the activated form of the GPIIbIIIa

(Taub et al., 1989). The binding of PAC-1 to washed

platelets increased in response to ADP and was dose

dependent (Fig. 8). A concentration of 20 mM ADP resulted

in 36% of the platelets binding PAC-1. In the presence of

Fig. 8. PAC-1 binding to washed platelets in the presence and

absence of Ba25. Various concentrations of ADP were added to

washed platelets at 37 8C in the presence and absence of 1 mM Ba25

for 4 min. Ten microliter-aliquots were then removed from each

sample and incubated with 20 mL PAC-1 for 15 min at room

temperature. Experiments were done in duplicate and processed for

flow cytometry within 30 min.

B. Jennings et al. / Toxicon 46 (2005) 687–698 695

Ba25 (1 mM) only 15% of platelets labelled with PAC-1 and

the mean fluorescence intensity of PAC-1 binding to

platelets decreased 1.5 fold (Fig. 8).

4.6. Platelet activation assays

There was no indication of platelet granule release;

either a-granules (measured by P-selectin exposure)

(Escolar et al., 1996) or dense granules (measured as

radioactive serotonin release), in response to Ba25 (results

not shown) with washed platelets, either in the presence or

absence of fibrinogen. In addition, Ba25 failed to cause any

significantly detectable binding of annexin-V to platelets,

indicating that it did not stimulate translocation of

phosphatidylserine to the outer cell membrane (results not

shown).

5. Discussion

A single, pH neutral protein with a disulphide linked

heterodimeric structure and an apparent molecular mass of

25 kDa which we have called Ba25, has been isolated from

the venom of B. arietans, using the platelet integrin

GPIIbIIIa receptor immobilised on Sepharose. The use of

a GPIIbIIIa affinity column was employed as the intention

was to search for proteins in venom that bound to this

receptor, and the high level of purity to which the GPIIbIIIa

could be prepared (Fig. 1). The N-terminal sequence of the

two chains of Ba25 indicates it to be a new protein. There is

a similarity between the sequence of Ba25 and the sequence

of other C-type lectins from B. arietans, namely bitiscetin

(Hamako et al., 1996; Matsui et al., 2000), and the

fibrinogenase Ba100, which prevents platelet aggregation

by proteolytic cleavage of fibrinogen, thus rendering this

molecule unable to form fibrin clots (Jennings et al., 1999).

In addition, Ba25 shows a high degree of homology with

C-type lectins from other vipers and crotalids (Peng et al.,

1994; Polgar et al., 1997; Andrews et al., 1996; Sakurai

et al., 1998; Kowalska et al., 1998).

Ba25 appears to bind to the platelet receptor, GPIIbIIIa,

immobilised on Sepharose, although it is unclear if it binds

to this receptor in whole cells. This apparent ability is a

characteristic not shared with the other snake venom C-type

lectins. FITC-labeled Ba25 bound to intact resting platelets,

with an increase in binding with ADP stimulation. As ADP

stimulation of platelets activates the GPIIbIIIa receptor, this

increase in binding in response to ADP stimulation suggests

that Ba25 does associate with this receptor on intact

platelets. This interaction may be at, or at least near one

of the sites within GPIIbIIIa involved in the binding of

fibrinogen, as Ba25 inhibited the binding of the antibody

PAC-1 to ADP-stimulated platelets.

Monoclonal antibodies to various receptors on the

platelet and flow cytometry techniques were used to further

elucidate the position of Ba25 interaction with the platelet

membrane. Ba25 blocked the binding of the antibody SZ2—

known to bind to a distinct epitope within the N-terminal

domain of GP1ba—to platelets in PRP (Ruan et al., 1987;

Burgess et al., 1998). GPIba is a glycoprotein component of

the vWF receptor, and C-type lectins from other snake

venoms (Andrews et al., 1996; Sakurai et al., 1998) have

been shown to bind to this subunit within the receptor. Ba25

interaction with the vWF receptor was associated with both

an increase in the number of GPIIbIIIa receptors per resting

platelet, observed as an increase in the number of CD61 and

CD41 binding sites, and an increase in fibrinogen binding to

platelets (observed as an increase in binding of an antibody

to fibrinogen). Thus, binding of Ba25 to platelets appears to

promote the binding of fibrinogen to these cells. This

binding appears not to be close to the binding sites of the

antibody SZ21 to CD61 and antibody P2 to CD41. These

antibodies are known to bind to fibrinogen binding sites

within GPIIbIIIa (Phillips et al., 1988).

The antibody SZ2 inhibited agglutination of PRP and

washed platelets (in the presence or absence of fibrinogen)

in response to Ba25. The use of washed platelets negates any

vWF effects, and underscores the importance of fibrinogen

for platelet cross-linking required for the agglutination

reaction. Ba25-mediated platelet agglutination appears to be

a consequence of Ba25 interacting with the GP1ba receptor

in the region of binding of the monoclonal antibody SZ2,

which then promotes fibrinogen binding to the platelet.

Washed platelets were not agglutinated by Ba25 until

fibrinogen was added. Fibrinogen serves as a necessary

bridging molecule for linking receptors on adjacent

B. Jennings et al. / Toxicon 46 (2005) 687–698696

platelets; in its absence, agglutination of platelets would be

precluded even though Ba25 still interacts with the platelet

receptors on the cell surface. The failure of monoclonal

antibodies SZ21 (anti-CD61) and P2 (anti-CD41) to inhibit

agglutination in response to Ba25 further supports the

concept that Ba25 promotes fibrinogen binding to GPIIbIIIa

but not at the binding sites of these two monoclonal

antibodies within this receptor. Both these antibodies are

known to inhibit ADP-induced platelet aggregation. The

role of binding of Ba25 to the GPIIbIIIa receptor close to the

PAC-1 site in the agglutination reaction is not clear.

Thus, Ba25 induced agglutination of platelets appears to

be a process that is independent of activation of the platelet.

No CD62P expression (a-granule release) or release of

serotonin (dense granule release) occurred during the

interaction of Ba25 with platelets. There is also no evidence

of disruption in the phospholipid bilayer asymmetry, with

respect to phosphatidylserine, when Ba25 is incubated with

platelets, as no binding of annexin-V could be demonstrated.

The lack of activation of platelets in PRP by Ba25 is

reflected both in the inability of Ba25 to promote PAC-1

binding in the absence of ADP and light microscopy

evaluation of platelets treated with Ba25. The cells appear to

be partially agglutinated when treated with Ba25, and

distinctly different from platelets treated with other

aggregating agents, such as ADP (O’Brien and Heywood,

1996; Jagroop et al., 2000). Ba25 appears to enhance cell-

cell interactions only in the presence of fibrinogen, leading

to the agglutination of platelets that is measured in the

aggregometer. However, this agglutination is without the

major and rapid changes that platelets undergo when

stimulated with agonists such as ADP, namely membrane

spreading, expansion and degranulation. Further proof that

agglutination by Ba25 is activation independent is evident in

that incubation of platelets with 10 mg/mL of the cyclo-

oxygenase inhibitor, indomethacin, failed to cause any

decrease in agglutination in response to Ba25 (results not

shown).

TEG is one of the most useful tools to assess the effects

of agents acting on haemostatic mechanisms in whole blood,

and permits the analysis of the physical properties of the clot

during its formation (Harrison, 2000) and has been used in

several studies to assess the anticoagulant effects of snake

venom (Dambisya, et al., 1994; Dambisya et al., 1995;

Jennings et al., 1999). The ability of Ba25 to prevent clot

formation in whole blood in the TEG further supports the

results indicting that the interaction of Ba25 with platelets is

an activation independent event without the usual increase

of surface appearing, annexin-V binding phosphatidylser-

ine. Clotting could be induced at the end of the TEG reaction

by the addition of thrombin (results not shown). Ba25 had a

major effect on all the parameters measured by TEG with a

concentration above 0.3 mM causing complete failure of clot

formation. Reduction in MA shows that Ba25 reduced the

strength of the clot at lower concentrations, reflecting the

importance of functional cellular interactions in

the stabilisation of the forming thrombus. The reduction of

reaction time r in particular, suggests that Ba25 may be an

important inhibitor of early-stage blood clotting events,

such as cell-cell, or cell-surface interactions. No degranula-

tion of platelets takes place during TEG, and fibrinogen

remains clottable in response to thrombin. Peripheral blood

smears of whole blood after incubation with Ba25 show

small aggregates of platelets, in an inactivated state, without

the involvement of other cells, which accurately reflects the

condition of the cells within the TEG cuvette (results not

shown), supporting the light microscopy results shown in

Fig. 3.

These results indicate that one of the binding sites for

Ba25 is on or near the receptor GPIIbIIIa. Although useful

for the isolation of Ba25, this binding site appears to be less

important to the agglutination of platelets than the additional

binding of Ba25 to the GPIb. Binding to GPIb may increase,

via intracellular signalling mechanisms, the affinity for

GPIIbIIIa for fibrinogen (Litjens et al., 2000). Indeed, this is

an important ‘inside-out’ activation mechanism under

scrutiny for many of the GPIb-binding snake venom

proteins (Andrews et al., 2003a, b), but the first one

described is strictly dependent on fibrinogen for aggluti-

nation of platelets. The binding of fibrinogen to its receptor,

thus enhanced, leads to the agglutination of the platelets, in

the case of Ba25 without the release of their granule

contents. It is unclear as to how the interaction of Ba25 with

the platelet causes the increase in affinity for fibrinogen

binding, but may involve a conformational change in the

GPIIbIIIa. Ba25 may thus be a potent modulator of

coagulation effects relating to thrombus formation and

stabilisation. It may also have implications for the role of

fibrinogen binding during haemostasis and thrombosis.

Acknowledgements

We thank Helen Botes and Vivienne Woodburne for

assistance with the purification of Ba25 used in this study.

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