Purification and characterization of platelet aggregation inhibitors from snake venoms.
ABSTRACT Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species. A small platelet aggregation inhibitor (p1.AI), multisquamatin (Mr = 5,700), was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC. A larger p1.AI, contortrostatin (Mr = 15,000), was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix. Both p1.AIs inhibit ADP-induced human, canine and rabbit platelet aggregation using platelet rich plasma (PRP). Multisquamatin has an IC50 of 97 nM, 281 nM and 333 nM for human, canine and rabbit PRP, respectively. Contortrostatin has an IC50 of 49 nM, 120 nM and 1,150 nM for human, canine and rabbit PRP, respectively. In a competitive binding assay using 125I-7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits platelet aggregation) both contortrostatin and multisquamatin demonstrated GPIIb/IIIa specific binding to human and canine platelets. The IC50 for contortrostatin displacement of 7E3 binding to human and canine GPIIb-/IIIa is 27 nM and 16 nM, respectively and for multisquamatin it is 3 nM and 63 nM, respectively. Our results indicate that both p1.AIs inhibit platelet aggregation by binding with high affinity to GPIIb/IIIa.
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PURIFICATION AND CHARACTERIZATION OF PLATELET AGGREGATION
INHIBITORS FROM SNAKE VENOMS
Mohit Trikha', Willia 9 E. Rote2, Peter J. Manley', Benedict R.
Lucchesi and Francis S. Markland'
1Department of Biochemi/try and Molecular Biology, University of
Southern California, School of Medicine, Los Angeles, CA and
2Department of Pharmacology, University of Michigan Medical
School, Ann Arbor, MI
(Received 7 9 July 7 993 by Editor H. Pirkle; accepted 7 November 7993)
Abstract Proteins that inhibitglycoprotein
platelet aggregation have been purified from the venom of
two snake species. A small platelet aggregation inhibi-
tor (pl.AI), multisquamatin (Mr=5,700), was purified from
venom by hydrophobic interaction
HPLC and two steps on Cl8 reverse phase HPLC. A larger
pl.AI, contortrostatin (Mr=15,000), was purified by a
similar HPLC procedure from the venom of Agkistrodon
Both pl.AIs inhibit ADP-induced hu-
man, canine and rabbit platelet aggregation using plate-
let rich plasma (PRP). Multisquamatin has an IC5U of 97
nM, 281 nM and 333 nM for human, canine and rabbrt PRP,
respectively. Contortrostatin has an IC
nM and 1,150 nM for human, canine an
respectively. In a competitive binding
7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits
platelet aggregation) bothcontortrostatin andmultisqua-
matin demonstrated GPIIb/IIIa specific binding to human
and canine platelets. The IC
displacement of 7E3 binding to uman and canine GPIIb-
/IIIa is 27 nM and 16 nM, respectively and for multisqua-
matin it is 3 nM and 63 nM, respectively.
indicate that both pl.AIs inhibit platelet aggregation by
binding with high affinity to GPIIb/IIIa.
of 49 nM, 120
561 rabbit lSf;L
Key words: platelet aggregation inhibitors, protein purification,
snake venoms, GPIIb/IIIa, fibrinogen.
Corresponding author: Dr. Francis Markland, USC School of Medicine,
CRL 106, Los Angeles, CA 90033. Phone (213) 224-7981.
Vol. 73, No. 1
Platelets serve an important role in mediating coronary artery
thrombosis and rethrombosis in the genesis of acute myocardial
infarction (1). Thus inhibition of platelet aggregation may
provide an effective adjunctive approach for prevention of coronary
artery reocclusion after successful thrombolytic
aggregation involves the interaction of the platelet membrane
glycoprotein (GP) IIb/IIIa receptor with plasma
GPIIb/IIIa belongs to the superfamily of integrin cell surface
Integrins are heterodimers composed of a and Q
subunits that are noncovalently associated.
to be involved in cell-cell and cell-substratum interactions (3).
It has been shown that both the a and the fl subunits are required
for fibrinogen binding (3). Integrins serve as receptors for
extracellular matrix proteins such as fibronectin, fibrinogen,
vitronectin, collagen and laminin (3). Some of these interactions
have been shown to be mediated via an Arg-Gly-Asp (RGD) sequence
present in the matrix proteins (3). For platelet aggregation an
RGD sequence present in fibrinogen is essential for the interaction
with GPIIb/IIIa (3).
They have been shown
A number of disintegrins
(pl.AIs) have been isolated from snake venoms (4-9).
small proteins, rich in disulfide bonds and contain the RGD
sequence. Recently pl.AIs have been characterized with respect to
the amino acid sequence around the RGD site (10). The RGD sequence
is present in all known pl.AIs except barbourin, the pl.AI purified
from Sisturus m. barbouri, which has a Lys-Arg-Asp (KGD) sequence
instead of RGD and seems to be highly GPIIb/IIIa specific (8).
or platelet aggregation inhibitors
In this communication, we describe two pl.AIs, contortrostatin and
multisqamatin, that have been purified from different snake venoms.
Contortrostatin was purified
from the venom of Agkistrodon
contortrix contortrix (Southern copperhead) and multisquamatin was
purified from the venom of Echis multisquamatus.
the inhibitory activity of both proteins on platelets obtained from
humans and two animal species. We have also studied the ability of
these pl.AIs to compete for binding sites with a monoclonal
antibody (7E3) directed to GPIIb/IIIa (11).
present evidence indicating that contortrostatin and multisquamatin
inhibit platelet aggregation by binding to the integrin receptor,
We have studied
In this report we
MATERIALS AND METHODS
obtained from Biotoxins, Inc., St. Cloud, FL; venom from Echis
multisquamatus was obtained from Latoxan, Rosans, France.
chemicals were of the highest grade available.
assay kit using bicinchoninic acid was employed to determine
protein concentrations (12).
venom from Agkistrodon contortrix contortrix was
Hioh Performance Liauid Chromatosraphv IHPLC):
For hydrophobic interaction (HIC)-HPLC a Perkin Elmer 410 LC pump
was employed with a LC-95 UV/VIS detector and for reverse phase
HPLC a Spectra Physics LC 8810 pump was employed with an SP 8450
Vol. 73, No. 1
and for RP-HPLC at 215 nm. A polypropyl aspartamide (250 x 21 mm)
column (Poly LC, Columbia, MD) was used for hydrophobic interaction
HPLC. Cl8 (218TP54 and 218TP510) columns were used for reverse
phase (RP) HPLC (Vydac, Hesperia, CA).
Absorbance for HIC-HPLC was monitored at 280 nm
Purification of contortrostatin. the platelet assresation inhibitor
from Ackistrodon contortrix contortrix venom:
Contortrostatin was purified fromAgkistrodon contortrix contortrix
(Southern copperhead) venom using a three step HPLC procedure. For
the first step of purification crude venom (1 g) was dissolved in
0.1 M phosphate buffer containing 1 M ammonium sulphate, pH 6.8
(buffer A) and applied to the polypropyl aspartamide HIC-HPLC
column. Elution was achieved as follows: 50 minutes isocratically
with 100% buffer A; a linear gradient for 90 minutes to 0.1 M
phosphate, pH 6.8 (buffer B); 40 minutes isocratic at 100% buffer
Fractions of 10 ml were collected in a Pharmacia Frac 100
fraction collector at 4'C using a flow rate of 5 ml/min. Fractions
containing pl.AI activity were pooled and concentrated by ultra-
filtration using an Amicon stir cell with a YM3 membrane.
purification was achieved by Cl8 RP-HPLC. The Cl8 column (218TP510)
was equilibrated with 95% of 0.1% TFA in water (solvent A) and 5%
of 80% acetonitrile in 0.1% TFA in water (solvent B). Elution was
achieved as follows: isocratic at 95% solvent A and 5% solvent B
for 10 minutes; a linear gradient to 40% solvent B in 65 minutes;
linear gradient to 100% solvent B in 20 minutes; isocratic at 100%
solvent B for 25 minutes. Fractions were collected manually every
minute at a flow rate of 7 ml/minute.
activity were pooled and rerun on the same Cl8 RP-HPLC column using
a shallower gradient. Elution was achieved as follows: isocratic
at 80% solvent A and 20% solvent B for 20 minutes; a linear
gradient to 30% solvent B over 90 minutes; and a 25 minute linear
gradient to 100% solvent B.
Fractions containing pl.AI
Purification of multisauamatin, the platelet assresation inhibitor
from Echis multisquamatus venom:
Multisquamatin was purified from 1 gram of Echis multisquamatus
venom by a three step HPLC procedure.
venom (1 g) was treated as Agkistrodon contortrix contortrix venom
and applied to the HIC-HPLC column.
identical to the first step employed for the purification of
Pl.AI activity coeluted with platelet aggregatory
activity in the HIC flow through.
separate platelet inhibitory activity from platelet aggregatory
activity. Fractions from the HIC flow through were pooled and
concentrated using an Amicon stir cell with a YM3 membrane and
subsequently applied to a Cl8 RP-HPLC column (218TP510).
was achieved as follows: 10 minute isocratic at 95% solvent A and
5% solvent B; a linear gradient to 30% solvent B over 110 minutes;
and a linear gradient from 30% to 100% solvent B over 30 minutes;
isocratic at 100% solvent B for 30 minutes.
were collected at a flow rate of 7 ml/minute at 4OC.
fractions were pooled, concentrated using a Savant Rotovac, and
rerun on the same Cl8 RP-HPLC column.
follows: 10 minute isocratic at 95% solvent A and 5% solvent B; a
linear gradient to 30% solvent B over 80 minutes; a linear gradient
In the first step crude
Elution conditions were
Cl8 RP-HPLC was employed to
Fractions of 10.5 ml
Elution was achieved as
from 30% to 100% solvent B over 10 minutes;
solvent B for 10 minutes.
The peak containing
Vol. 73, No. 1
isocratic at 100%
pl.AI activity was
Assav of nlatelet assresation inhibitorv activitv:
Column fractions obtained during purification were assayed for
pl.AI activity using fresh human platelet rich plasma (PRP)
prepared from blood obtained from human volunteers who had no
medication for at least two weeks. Blood (36 ml) was drawn into 4
ml of 0.1 M citrate and centrifuged at 150 x g for 20 minutes. The
PRP, was removed and the remaining
centrifuged at 10,000 RPM to obtain platelet poor plasma (PPP).
Platelet counts were adjusted to 250,000 platelets/+
Coulter counter. A Bio-Data one channel aggregometer and a Helena
Inhibition of ADP-induced platelet aggregation was
monitored at 37OC by adding venom fractions one minute prior to the
addition of ADP (lo-20 PM final concentration).
ting pl.AI activity were pooled and further purified.
canine PRP was prepared by the same procedure and used in the
studies described below.
SDS-nolvacrvlamide se1 electroDhoresis (SDS-PAGE):
A Tris-Tricine 16.5% gel was used according to the protocol of
Schagger and von Jagow under reducing and nonreducing conditions
(13). The gel was run using a Biorad minigel system and stained
with silver (14) or Coomassie blue R250.
Measurement of GPIIb/IIIa snecific bindinq:
Measurement of contortrostatin
platelet GPIIb/IIIa receptor was carried out using PRP prepared
from blood obtained from human volunteers or male mongrel dogs. PRP
was prepared as described above and the platelet count was
determined with a
Laboratories, Inc., Houston, TX). PRP (180 ~1) was incubated with
20 ~1 of varying concentrations
multisquamatin at room temperature. Radiolabelled antibody (
7E3 IgG, 20 ~1, 18 mg/ml, 80,000 cpm), specific for GPIIb/IIIa, was
then added and the mixture incubated for 30 minutes. To establish
equilibrium binding, 50 7~1 aliquots of the binding assay mixture
were layered over 200 7.~1 of 30% sucrose in 0.4 ml microcentrifuge
tubes and spun at 10,000 RPM for 4 minutes in a swinging bucket
rotor to separate platelet-bound antibody from free antibody. The
pellet and the supernatant were separated and counted in a Pa
Minaxi 5000 series gamma counter. The number of molecules of F&;d
7E3 bound per platelet in the presence and absence of pl.AI was
calculated by using the following formula:
and multisquamatin binding to
counter (Texas International
of either contortrostat$. or
f4) x 0.9 nc 7E3 x 3.76 x 1012 molecules 7E3/nq
1 = Pellet
(1) + (2)
Vol. 73, No. 1 VENOM DISINTEGRINS
pl x 45 pl
Purification of contortrostatin:
Contortrostatin was purified from Southern copperhead venom using
a three step chromatographic approach: HIC-HPLC, and two steps on
For the first step of purification crude venom was
disolved in a minimal volume of HIC buffer A and applied to the
HIC-HPLC column. Proteins were eluted by a decreasing gradient of
ammonium sulfate and were detected at 280 nm (Fig. 1 A).
activity was observed in the flow through.
pl.AI activity were pooled and concentrated for the second step of
purification. In this step pooled fractions were applied to a Cl8
RP-HPLC column equilibrated with 0.1% TFA and 4% acetonitrile.
Elution was achieved by an increasing gradient of acetonitrile
(Fig. 2 A). Contortrostatin eluted at 28% acetonitrile (66 min).
For the final step of purification, fractions containing pl.AI
activity were applied to the Cl8 RP-HPLC column this time
equilibrated with 0.1% TFA and 16% acetonitrile. Using a shallower
gradient to 24% acetonitrile (Fig. 3 A), contortrostatin eluted as
a sharp peak at 22% acetonitrile (82 min). The minor peak eluting
just before contortrostatin also contained pl.AI activity and had
a similar molecular weight to that of contortrostatin.
low yield this peak was not further characterized.
mg of three step purified contortrostatin were obtained per gram of
Due to the
Yields of l-2
Purification of multissuamatin:
Multisquamatin was purified from Echis multisquamatus
similar chromatographic approach to that used for the purification
Crude venom was directly applied to the HIC-
HPLC column under the same conditions used for Southern copperhead
venom. Elution was achieved by a decreasing gradient of ammonium
sulfate (Fig. 1 B). The major protein peak eluting in the flow
through possessed platelet aggregating activity.
contains pl.AI activity but it was difficult to detect due to the
coeluting platelet aggregating activity.
flow through peak possessed pl.AI activity we applied aliquots to
an analytical Cl8 RP-HPLC column (218TP54). One of the major peaks
eluting from the Cl8 RP-HPLC column possessed pl.AI activity
thereby indicating that the flow through peak from the HIC-HPLC
column contains pl.AI activity.
column were pooled, concentrated, and applied to the preparative
Cl8 RP-HPLC column equilibrated with 0.1% TFA and 4% acetonitrile.
Elution was obtained by an increasing gradient to 80% acetonitrile
(Fig.2 B). Pl.AI activity was obtained in the major peak eluting
at 14% acetonitrile (61 mins).
minants multisquamatin was rerun on the Cl8 RP-HPLC column (Fig. 3
B). Multisquamatin eluted at 68 min (16% acetonitrile). Yields of
lo-11 mg of three step purified multisquamatin were obtained per
gram of crude venom.
venom by a
This peak also
To confirm that the HIC
Active fractions from the HIC
In order to remove minor conta-
Vol. 73, No. 1
0 20 40 60 80 100 120 140 160
0 20 ,o
80 100 120 I.0 160 I80
the HIC-HPLC column under the same conditions as used for
Agkistrodon contortrix contortrix venom. Refer to Mate-
rials and Methods for elution conditions. Arrows identify
peaks containing pl.AI activity.
of crude venoms by HIC-HPLC.
contortrix was injected onto a HIC-HPLC
(B) E&is multisquamatus venom was applied to
20 40 50
0 20 40
a0 100 120 140 160 ((10
Cl8 RP-HPLC separation.
HPLC of Agkistrodon contortrix contortrix venom were
applied to a Cl8 RP-HPLC column.
from HIC-HPLC of Echis multisquamatus venom were applied
to the Cl8 RP-HPLC column.
thods for elution conditions.
activity are indicated by the arrows.
(A) Pooled fractions from HIC-
(B) Pooled fractions
Refer to Materials and Me-
Peaks containing pl.AI
Vol. 73, No. 1
Final step of purification of contortrostatin and multi-
(A) Contortrostatin was further purified by
rerunning on the Cl8 RP-HPLC column but with a shallower
gradient. It eluted at 82 min (22% acetonitrile) and was
collected manually. Multisquamatin was further purified
by rerunning on the Cl8 column using the same gradient as
in the second step. It eluted at 68 min (16% acetonit-
rile) and was collected manually. Refer to Materials and
Methods for elution conditions. Arrows indentify peaks
containing pl.AI activity.
Reducing and nonreducing SDS-PAGE were used to assess homogeneity
and to calculate molecular mass of purified pl.AIs (Fig. 4). SDS-
PAGE analysis of contortrostatin revealed that it has a molecular
mass of approximately 15,000 Da under nonreducing conditions and
5,000-7,000 Da under reducing conditions, thereby suggesting that
it is composed of two or three subunits.
although unlikely, is that the large difference in migration may be
attrib ted to differential uptake of SDS under nonreducing and
reducin conditions (5). Multisquamatin, on the other hand, has a
molecular mass of 6,400 Da under nonreducing conditions and 5,700
Da under reducing conditions.
Echistatin, the pl.AI purified from
the venom of Echis carinatus (Mr=5.4 kDa) (S), exhibits a similar
differential migration pattern to that of multisquamatin under
reducing and nonreducing SDS-PAGE.
migration could be attributed to differential detergent binding
based on structural differences under reducing and nonreducing
This pattern in SDS-PAGE
Vol. 73, No. 1
SDS-PAGE of contortrostatin
and 6 are low
doublet; 16.9kDa, horse heart myoglobin;
8.1 myoglobin CNBr fragment
(F2); and 2.5kDa, myoglobin
of reduced and nonreduced contortrostatin,
Lanes 4 and 5 are 1 pg of nonreduced
massie blue R250.
1 (Fl); 6.2kDa, myoglobin
(F3). Lanes 2 and 3 are 1 pg
and reduced multi-
The gel was stained with Coo-
for 20 PM ADP-induced
aggrega ion is considerably
rabbit platelet aggregation
only 1.9 pg/ml for multisquamatin.
of platelet assresation:
in human, canine, and rabbit PRPs (Fig. 5).
(0.73 pg/ml) and multisquamatin
human platelet aggregation
canine platelet aggregation
and 1.6 pg/ml for multisquamatin.
for contortrostatin mediated inhibition of rabbit platelet
is 17.3 pg/ml ?& contortrostatin,
inhibit ADP-induced platelet
(0.55 pg/ml) inhibited 10
by 50% (IC5
). The IC50
.8 pg/ml for
for 20 PM ADP-induced
Vol. 73. No. 1
, t *o
012J I Sl 78 a 10
for contortrostatin inhibition
platelet aggregation. (*) Determkkk?a~~ of human,
circles represent human PRP, solid circles represent
canine PRP, and empty triangles represent rabbit PRP.
Varying concentrations of controtrostatin were preincu-
bated with PRP prior to the addition of ADP. Contortros-
tatin has an IC50 of 0.73 pg/ml,
for inhibition of human, canine and rabbit platelet ag-
gregation, respectively. (B) Determination of IC50 for
multisquamatin inhibition of human, canine and rabbit
The symbols are the same as for
contortrostatin. Varying concentrations of multisqua-
matin were preincubated with PRP as for contortrostatin.
Multisquamatin has an IC50 of 0.55 pg/ml, 1.6 pg/ml and
1.9 pg/ml for human, canine and rabbit platelet aggrega-
tion, respectively. Each point represents the average of
and 17.3 pg/ml
Determination of GPIIb/IIIa specific bindins of pl.AIs usins the
The murine monoclonal antibody, 7E3, is a potent inhibitor of human
and canine platelet aggregation (11).
inhibit platelet aggregation
binding to GPIIb/IIIa, we employed competitive binding studies
using 7E3. These studies demonstrated specific platelet GPIIb/IIIa
receptor binding for the two pl.AIs with both human and canine
platelets (Fig. 6).
The concentration of contortrostatin
inhibit 50% of 7E3 binding to human GPIIb/IIIa (IC50) is 0.4 pg/ml.
Multisquamatin has an IC5
for human GPIIb/IIIa of 0.018 pg/ml.
for contortros atin
a is 0.24 yg/ml and 0.36 pg/ml, respectively.
studies indicate that both contortrostatin
inhibit platelet aggregation by binding to GPIIb/IIIa.
To determine if contor-
and multisquamatin for canine
Vol. 73, No. 1
? 3 00000 -
wooo - - \
W DOO: ~
' t ,
! I 40000
‘x. ‘.._ -
(A) Binding of contortrostatin and multisquamatin to
human GPIIb/IIIa in the presence of a fixed saturating
concentration of 7E3.
Solid line with solid triangle
represents contortrostatin and dotted line with solid
circle represents multisquamatin.
trostatin displacement of 7E3 is 0.4
pg/ml. (B) Binding of contor-
trostatin and multisquamatin to canine platelet GPIIb-
/IIIa in the presence of a fixed saturating concentration
of 7E3. The symbols are as in A. The IC
trostatin displacement of 7E3 is 0.25 pg/m and for mul-
tisquamatin it is 0.36 pg/ml. Refer to Materials and Me-
thods for a detailed description. Each point represents
the average of at least three determinations.
The IC50 for contor-
Recently platelet aggregation inhibitors have been purified from
venoms of snakes of the Crotalidae and Viperidea families (4-10).
The first pl.AI to be reported was trigramin from Trimeresurus
gramineus venom (4). This pl.AI was shown to bind to the GPIIb/IIIa
complex on the platelet surface (4). All pl.AIs purified thus far,
with the exception of barbourin (8), possess the RGD sequence.
This sequence motif has been implicated as being involved in the
inhibition of integrin mediated interactions (1).
number of investigators have examined the efficacy of linear and
cyclic RGD peptides (15, 16) or cyclic KGD peptides (17) as
However, there appears to be increasing
evidence that pl.AIs may have unique surface geometry which
facilitates interactions with integrins by mechanisms other than
those solely based on the RGD site. Recently it has been reported
Vol. 73, No. 1
that some snake venom metalloproteinases, which have no structural
resemblance to mammalian matrix-degrading metalloproteinases, con-
tain a domain which bears a striking resemblance to the pl.AI
sequence (18-21). Although the pl.AI domain is devoid of the RGD
sequence in at least one of the metalloproteinases
proteinase is a potent inhibitor of ADP- and collagen-induced
Furthermore, the finding that a mutated,
chemically synthesized pl.AI derivative of echistatin (alanine
substituted for arginine in the RGD sequence) still possessed some
biological activity (22), suggests that other regions in the
protein may be involved in binding and that there may be some
flexibilty in the RGD binding site.
due to their small size, do not possess the molecular topography of
the pl.AIs and, therefore, cannot interact with the multiplicity of
mechanisms used by pl.AIs.
The synthetic RGD peptides,
In view of the potential clinical use of pl.AIs as antithrombotic
agents we have purified two pl.AIs, contortrostatin and multi-
from Agkistrodon contortrix
multisquamatin are potent inhibitors of human, rabbit, and canine
platelet aggregation. Interestingly, studies in collaboration with
Dr. Joan Brugge and Edwin A. Clark, at Ariad Pharmaceuticals Inc.,
Cambridge, MA, have shown that contortrostatin does not inhibit
platelet release reactions (unpublished results).
amino acid sequence of contortrostatin shows significant identity
with applaggin (6).
However, there appears to be an interesting
difference in that the sequence for contortrostatin begins nine
amino acid residues downstream from the applaggin start site
(unpublished results). Presumably the amino terminal deletion has
effect pl.AI activity
z&tortrostat% and applaggin are v%yzimilar
Multisquamatin appears to be similar to echistatin (5) based on the
multisquamatin appears to start three amino acids upstream from the
start site (unpublished results).
multisquamatin (lo-11 mg per gram of crude venom) is to our
knowledge the highest yield of any pl.AI reported thus far.
appears that multisquamatin
hydrophobic because they do not bind to the HIC-HPLC column.
Furthermore, they elute early in the Cl8 RP-HPLC gradient which
separates proteins based on their hydrophobicity (23).
supported by their amino acid composition
hydrophobic amino acids (unpublished results).
the amino terminus of
The yield for
are not very
is low in which
multisquamatin inhibit platelet aggregation by binding specifically
to the GPIIb/IIIa integrin receptor.
GPIIb/IIIa ELISA (24),
in which the extent of purified GPIIb/IIIa
multisquamatin and contortrostatin effectively block GPIIb/IIIa
binding (data not shown).
Addititonally, the partial amino acid
sequences of both pl.AIs indicate considerable identity with other
snake venom pl.AIs which are known to bind to GPIIb/IIIa. Finally,
contortrostatin and multisquamatin block 7E3 binding to GPIIb/IIIa
lines of evidence
indicate that contortrostatin and
Thus, in a fibrinogen-
VENOM DISINTEGRINS Vol. 73, No. 1
binds to GPIIb/IIIa, thereby inhibiting platelet aggregation (11).
In the presence of low concentrations of either contortrostatin or
multisquamatin 7E3 binding to platelets was significantly inhibited
7E3 is a murine monoclonal antibody that specifically
An interesting observation is the relatively high IC58 for inhibi-
tion of rabbit platelet aggregation by contortrostatin.
of 17.3 pg/ml is considerably higher than its IC
and 1.8 pg/ml for inhibition of human and canine p 5_O
However the IC
inhibition of human, canine and rab it platelet aggregation are
very similar. This apparent difference raises two possible
questions. First, does contortrostatin have a different mechanism
of inhibition of platelet aggregation than multisquamatin? Second,
is the rabbit GPIIb/IIIa different than
human and canine platelets? Further study into the mechanisms
involved in contortrostatin and multisquamatin mediated inhibition
could provide more insight into the mechanistic differences between
rabbit GPIIb/IIIa and its counterpart
of 0.73 pg/m
values for multisquamatin
GPIIb/IIIa present on
in human and canine
Two snake venom pl.AIs, kistrin (9) and bitistatin (25), have
demonstrated a potential role as antithrombotic agents for use in
thrombolytic therapy by
thrombolysis in conjunction with recombinant tissue plasminogen
activator. Based on the low IC50 values of contortrostatin we have
tested its in vivo efficacy as an antithrombotic agent.
canine carotid reoccluding arterial thrombosis model, preliminary
results with contortrostatin have been promising.
with APSAC contortrostatin efficiently sustains opening of the
carotid artery, whereas APSAC alone is unable to prevent the rapid
reocclusion of the carotid artery. Further in vivo characteriza-
tion of both contortrostatin and multisquamatin is in progress.
and sustaining arterial
Based on the experiments described above both contortrostatin and
multisquamatin have been shown to be potent
GPIIb/IIIa mediated platelet aggregation.
We would like to thank Gee Kim and Simy Loayza for technical
assistance, and Ms. Ginny Kortes, R.N., USC Comprehensive Cancer
Center, for providing phlebotomy services required for these
studies. We would also like to thank the American Heart Associa-
tion-Greater Los Angeles Affiliate (AHA-GLAA Grant number 938 GI)
(FM), the National Cancer Institute (Grant No. R03CA54861) (FM) and
the National Heart, Lung and Blood Institute (Grant No. HL19782-15)
(BL) for partial support of this project.
1. ZUCKER, M.B. The functioning
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Vol. 73, No. 1
VENOM DISINTEGRINS 51
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