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: [email protected] (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.
References
Andrews, R.K., Kroll, M.H., Ward, C.M., Rose, J.W., Scarborough,
R.M., Smith, A.I., Lopez, J.A., Berndt, M.C., 1996. Binding of a
novel 50-kilodalton alboaggregin from Trimeresurus albolabris
and related viper venom proteins to the platelet membrane
glycoprotein Ib-IX-V complex. Effect on platelet aggregation
and glycoprotein Ib-mediated platelet activation. Biochemistry
35, 12629–12639.
Andrews, R.K., Gardiner, E.E., Shen, Y., Berndt, M.C., 2003.
Structure-activity relationships of snake toxins targeting platelet
receptors, glycoprotein Ib-IX-V and glycoprotein VI. Curr.
Med. Chem. Cardiovasc. Hematol. Agents 1, 143–149.
B. Jennings et al. / Toxicon 46 (2005) 687–698 697
Andrews, R.K., Gardiner, E.E., Shen, Y., Whisstock, J.C., Berndt,
M.C., 2003. Glycoprotein Ib-IX-V. Int. J. Biochem. Cell Biol.
35, 1170–1174.
Brandt, W.F., Alk, H., Chauhan, M., von Holt, C., 1984. A simple
modification converts the spinning cup protein sequencer into a
vapour-phase sequencer. FEBS Lett. 174, 228–232.
Burgess, J.K., Lopez, J.A., Berndt, M.C., Dawes, I., Chesterman,
C.N., Chong, B.H., 1998. Quinine-dependent antibodies bind a
restricted set of epitopes on the glycoprotein Ib-IX complex:
characterization of the eptopes. Blood 92, 2366–2373.
Chen, Y.L., Tsai, I.H., 1995. Functional and sequence character-
ization of agkicetin, a new glycoprotein Ib antagonist isolated
from Agkistrodon acutus venom. Biochem. Biophys. Res.
Commun. 210, 472–477.
Clemetson, K.J., Polgar, J., Clemetson, J.M., 1998. Snake venom
C-type lectins as tools in platelet research. Platelets 9,
165–169.
Dambisya, Y.M., Lee, T.-L., Gopalakrishnakone, P., 1994. Action
of Calloselasma rhodostoma (Malayan pit viper) venon on
human blood coagulation and fibrinolysis using computerized
thromboelastography (CTEG). Toxicon 32, 1619–1626.
Dambisya, Y.M., Lee, T.-L., Gopalakrishnakone, P., 1995. Antic-
oagulant effects of Pseudechis australis (Australian brown
snake) venom on human blood: a computerized thromboelastic
study. Toxicon 33, 1378–1382.
Drickamer, K., 1993. Two distinct classes of carbohydrate-
recognition domains in animal lectins. J. Biol. Chem. 263,
9557–9560.
Escolar, G., Rao, G.H., Nieuwenhuis, H.K., White, J.G., 1996.
Ultrastructural expression of P-selectin on surface activated
platelets. Platelets 7, 297–301.
Fitzgerald, L.A., Leung, B., Phillips, D.R., 1985. A method for
purifying the platelet membrane glycoprotein IIb-IIIa complex.
Anal. Biochem. 151, 169–177.
Fukuda, K., Mizuno, H., Atoda, H., Morita, T., 2000. Crystal
structure of Flavocetin-A, a platelet glycoprotein Ib-binding
protein, reveals a novel cyclic tetramer of C-type lectin-like
heterodimers. Biochemistry 39, 1915–1923.
Gan, Z.-R., Gould, R.J., Jacobs, J.W., Friedman, P.A., Polokoff,
M.A., 1988. Echistatin. A potent platelet aggregation inhibitor
from the venom of the viper Echis carinatus. J. Biol. Chem. 263,
19827–19832.
Gould, R.J., Polokoff, M.A., Friedman, P.A., Huang, T.-F., Holt,
J.C., Cook, J.J., Niewiarowski, S., 1990. Disintegrins: a family
of integrin inhibitory proteins from viper venoms. Proc. Soc.
Exp. Biol. Med. 195, 168–171.
Hamako, J., Matsui, T., Suzuki, M., Ito, M., Makita, K., Fujimura,
Y., Ozeki, Y., Titani, K., 1996. Purification and characterization
of bitiscetin, a novel von Willebrand factor modulator protein
from Bitis arietans snake venom. Biochem. Biophys. Res.
Commun. 205, 273–279.
Harrison, P., 2000. Progress in the assessment of platelet function.
Br. J. Haematol. 111, 733–744.
Hirotsu, S., Mizuno, H., Fukuda, K., Chun, Q.M., Matsui, T.,
Hamako, J., Morita, T., Titani, K., 2001. Crystal structure of
bitiscetin, a von Willebrand factor-dependent platelet aggrega-
tion inducer. Biochemistry 40, 13592–13597.
Hutton, R.A., Warrell, D.A., 1993. Action of snake venom
components on the haemostatic system. Blood Rev. 7,
176–189.
Jagroop, I.A., Clatworthy, K., Lewin, J., Mikhailidis, D.P., 2000.
Shape change in human platelets: measurement with a
channelyzer and visualisation by electron microscopy. Platelets
11, 28–32.
Jennings, B.R., Spearman, C.W.N., Kirsch, R.E., Shephard, E.G.,
1999. A novel high molecular weight fibrinogenase from the
venom of Bitis arietans. Biochim. Biophys. Acta 1427,
82–91.
Jia, L.-G., Shimokawa, K.-I., Bjarnason, J.B., Fox, J.W., 1996.
Snake venom metalloproteinases: structure, function and
relationship to the ADAMs family of proteins. Toxicon 34,
1269–1276.
Kalvaria, I., Rabinowitz, S., Frith, L.O.C., Kirsch, R.E., 1986.
Fibrinogen synthesis in the rat; role of the C terminal end of the
gamma chain of fragment D1. Thromb. Res. 43, 287–291.
Kang, I.-C., Chung, K.-H., Lee, S.-J., Yun, Y., Moon, H.-M., Kim,
D.-S., 1998. Purification and molecular cloning of a platelet
aggregation inhibitor from the snake (Agkistrodon halys
brevicaudus) venom. Thromb. Res. 91, 65–73.
Kawasaki, T., Taniuchi, Y., Hisamichi, N., Fujimura, Y., Suzuki,
M., Titani, K., Sakai, Y., Kaku, S., Satoh, N., Takenaka, T.,
Handa, M., Sawai, Y., 1995. Tokaracetin, a new platelet
antagonist that binds to platelet glycoprotein Ib and inhibits von
Willebrand factor-dependent shear-induced platelet aggrega-
tion. Biochem. J. 308, 947–953.
Kawasaki, T., Fujimura, Y., Usami, Y., Suzuki, M., Miura, S.,
Sakurai, Y., Makita, K., Taniuchi, Y., Hirano, K., Titani, K.,
1996. 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, 10635–10639.
Kowalska, M.A., Tan, L., Holt, J.C., Peng, M., Karczewski, J.,
Calvete, J.J., Niewiarowski, S., 1998. Alboaggregins A and B.
Structure and interaction with human platelets. Thromb.
Haemost. 79, 609–613.
Litjens, P.E.H.M., Akkerman, J.-W.N., van Willigen, G., 2000.
Platelet integrin aIIbb3: target and generator of signalling.
Platelets 11, 310–319.
Markland Jr.., F.S., 1997. Snake venoms. Drugs 54 (Suppl. 3), 1–10.
Matsui, T., Hamako, J., Matsushita, T., Nakayama, T., Fujimura, Y.,
Titani, K., 2000. Binding site on human von Willebrand factor
of bitiscetin, a snake venom-derived platelet aggregation
inducer. Biochemistry 41, 7939–7946.
Mizuno, H., Fujimoto, Z., Koizumi, M., Kano, H., Atoda, H.,
Morita, T., 1997. Structure of coagulation factors IX/X-binding
protein, a heterodimer of C-type lectin domains. Nat. Struct.
Biol. 4, 438–441.
O’Brien, J.R., Heywood, J.B., 1996. Effects of aggregating agents
and their inhibitors on the mean platelet shape. J. Clin. Pathol.
19, 148–153.
Peng, M., Holt, J.C., Niewiarowski, S., 1994. Isolation, character-
ization and amino acid sequence of echicitin b subunit, a
specific inhibitor of von Willebrand factor and thrombin
interaction with glycoprotein Ib. Biochem. Biophys. Res.
Commun. 205, 68–72.
Phillips, D.R., Charo, I.F., Parise, L.V., Fitzgerald, L.A., 1988. The
platelet membrane glycoprotein IIb-IIIa complex. Blood 71,
813–843.
Polgar, J., Magnenat, E.M., Peitsch, M.C., Wells, T.N.C., Saqi,
M.S.A., Clemetson, K.J., 1997. Amino acid sequences of the a
B. Jennings et al. / Toxicon 46 (2005) 687–698698
subunit and computer modelling of the a and b subunits of
echicetin from the venom of Echis carinatus (saw-scaled viper).
Biochem. J. 323, 533–537.
Ruan, C., Du, X., Xi, X., Castaldi, P.A., Berndt, M.C., 1987. A
murine antiglycoprotein Ib complex monoclonal antibody, SZ2,
inhibits platelet aggregation induced by both ristocetin and
collagen. Blood 69, 570–577.
Sakurai, Y., Fujimura, Y., Kokubo, T., Imamura, K., Kawasaki, T.,
Handa, M., Suzuki, M., Matsu, T., Titani, K., 1998. The cDNA
cloning and molecular characterization of a snake venom
glycoprotein Ib-binding protein, mamushigin, from Agkistro-
don halys blomhoffii venom. Thromb. Haemost. 79,
1199–1207.
Sen, U., Vasudevan, S., Subbarao, G., McClintock, R.A., Celikel,
R., Ruggeri, Z.M., Varughese, K.I., 2001. Crystal structure of
the von Willebrand factor modulator botrocetin. Biochemistry
40, 345–352.
Shebuski, R.J., Ramjit, D.R., Bencen, G.H., Polokoff, M.A., 1989.
Characterization and platelet inhibitory activity of bitistatin, a
potent arginine-glycine-aspartic acid-containing peptide from
the venom of the viper Bitis arietans. J. Biol. Chem. 264,
21550–21556.
Tait, J.F., Smith, C., Wood, B.L., 1999. Measurement of phosphati-
dylserine exposure in leukocytes and platelets by whole-blood flow
cytometry with annexin V. Blood Cells Mol. Dis. 25, 271–278.
Taniuchi, Y., Kawasaki, R., Fujimura, Y., Suzuki, M., Titani, K.,
Sakai, Y., Kaku,S., Hisamichi, N., Satoh, N., Takenaka, T., Handa,
M., Sawai, Y., 1995. Flavocetin-A and -B, two high molecular
mass glycoprotein Ib binding proteins with high affinity purified
from Trimeresurus flavoviridis venom, inhibit platelet aggregation
at high shear stress. Biochim. Biophys. Acta 1244, 331–338.
Taub, R., Gould, R.J., Garsky, V.M., Ciccarone, T.M., Hoxie, J.,
Friedman, P.A., Shattil, S.J., 1989. A monoclonal antibody
against the platelet fibrinogen receptor contains a sequence that
mimics a receptor recognition domain in fibrinogen. J. Biol.
Chem. 264, 259–265.
Xia, Z., Wong, T., Liu, A., Kasirer-Friede, A., Brown, E., Frojmovic,
M.M., 1996. Optimally functional fluorescein isothiocyanate-
labelled fibrinogen for quantitative studies of binding to activated
platelets and platelet aggregation. Br. J. Haematol. 93, 204–214.
Yamada, D., Sekiya, F., Morita, T., 1996. Isolation and
characterization of carinactivase, a novel prothrombin activator
in Echis carinatus venom with a unique catalytic mechanism.
J. Biol. Chem. 271, 5200–5207.