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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1582-1589
Antithrombotic Effect of Crotalin, a Platelet Membrane Glycoprotein
Ib Antagonist From Venom of Crotalus atrox
By
Mei-Chi Chang,
Hui-Kuan Lin,
Hui-Chin Peng, and
Tur-Fu Huang
From the Team of Biomedical Science, Chang-Gung Institute of Nursing,
Taoyuan, Taiwan; and the Pharmacological Institute, College of
Medicine, National Taiwan University, Taipei, Taiwan.
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ABSTRACT |
A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with
a molecular weight of 30 kD was purified from the snake venom of
Crotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 µg/mL (83 nmol/L). It was also active in
inhibiting ristocetin-induced platelet aggregation of platelet-rich
plasma (IC50, 6.3 µg/mL). 125I-crotalin bound
to human platelets in a saturable and dose-dependent manner with a kd
value of 3.2 ± 0.1 × 10 7 mol/L, and its
binding site was estimated to be 58,632 ± 3,152 per platelet. Its
binding was specifically inhibited by a monoclonal antibody, AP1 raised
against platelet GPIb. Crotalin significantly prolonged the latent
period in triggering platelet aggregation caused by low concentration
of thrombin (0.03 U/mL), and inhibited thromboxane B2
formation of platelets stimulated either by ristocetin plus von
Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was
intravenously (IV) administered to mice at 100 to 300 µg/kg, a
dose-dependent prolongation on tail bleeding time was observed. The
duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 µg/kg. In addition, its in vivo
antithrombotic activity was evidenced by prolonging the latent period
in inducing platelet-rich thrombus formation by irradiating the
mesenteric venules of the fluorescein sodium-treated mice. When
administered IV at 100 to 300 µg/kg, crotalin dose dependently
prolonged the time lapse in inducing platelet-rich thrombus formation.
In conclusion, crotalin specifically inhibited vWF-induced platelet
agglutination in the presence of ristocetin because crotalin
selectively bound to platelet surface receptor-glycoprotein Ib,
resulting in the blockade of the interaction of vWF with platelet
membrane GPIb. In addition, crotalin is a potent antithrombotic agent
because it pronouncedly blocked platelet plug formation in vivo.
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INTRODUCTION |
PLATELET ADHESION and aggregation play
important roles in hemostasis and thrombosis. Physiologically,
platelets arrest bleeding from damaged blood vessels by sealing wound
and initiating the repairing processes. Thrombin-mediated platelet
activation and aggregation is partially related to glycoprotein Ib
(GPIb)1,2 and this membrane glycoprotein also plays a role
in regulating the interactions of platelets with their environment via
linking with membrane-associated cytoskeleton.2
Additionally, the interaction of platelet GPIb with von Willebrand
factor (vWF) is a critical event, allowing platelet adhesion,
aggregation, and subsequent thrombus formation in the vessels either
with high shear rates or with damaged endothelium.3-5 The
role of GPIb in the adhesion of platelets to endothelial cell matrix
has been studied with monoclonal antibodies (MoAbs). An MoAb against
GPIb inhibited the platelet adhesion to endothelial cell matrix,
whereas an MoAb against vWF which inhibits the interaction of vWF with
platelets has a less pronounced effect, indicating that GPIb has
another role in platelet adhesion, apart from serving as the binding
site for vWF.6
Many of the currently investigated antithrombotic drugs interfere with
the process of platelet aggregation.7-9 However, the inhibition of platelet adhesion may be an important and efficient process for preventing the platelet-rich thrombus formation in the
damaged vessels with high shear rate. In addition, thrombin is an
important stimulator for platelet aggregation under normal physiological and pathological conditions.10-12 Thrombin is
an important mediator of platelet aggregation in stenosed canine coronary arteries.13 Therefore, an inhibitor of thrombin
and vWF platelet interaction may be used in the prevention of
thrombosis. Several snake venom constituents affecting blood
coagulation and platelet aggregation, especially Arg-Gly-Asp
(RGD)-containing peptides, have been extensively
studied.14,15 In addition, GPIb agonists as well as GPIb
antagonists have been isolated and characterized.16-20 We
report here the purification of a protein, crotalin, from Crotalus
atrox venom that specifically inhibits ristocetin-induced
agglutination or aggregation without affecting the aggregation induced
by adenosine diphosphate (ADP) or collagen. Of particular interest is
that crotalin, when administered intravenously (IV), markedly delays
platelet-rich thrombus formation of the irradiated mesenteric venules
in a fluorescein sodium-treated mice model. Among the antithrombotic
agents tested, crotalin appears not only to be the most efficacious
agent in prolonging the time lapse for inducing platelet-rich thrombus
formation in this model, but also exhibits this antiplatelet activity
with a longer duration.
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MATERIALS AND METHODS |
Materials.
C atrox venom was obtained from LATOXAN (Rosans,
France). All of DEAE-Sephadex A-50, Sephadex G-75, Mono-S,
and Superose columns were purchased from Pharmacia (Uppsala,
Sweden). Halysin was purified from
Agkistrodon halys venom as previously described.21
ADP, U46619, collagen (type I, bovine achilles tendon), and fluorescein sodium were purchased from Sigma Chemical Co (St Louis, MO).
MoAb AP1 raised against platelet GPIb was generously provided by Dr
Robert Montgomery (Blood Center of Southeastern Wisconsin Milwaukee)
and 7E3, an MoAb raised against platelet GPIIb/IIIa complex, was
supplied from Dr Barry Coller (The Mount Sinai Medical Center, New
York, NY).
Purification of vWF.
According to the modified method of a previous report,22
vWF was purified from human plasma through cryoprecipitation,
polyethylene glycol (PEG) sedimentation, and gel
filtration with a Sepharose CL-4B column, and the purity of vWF was
assessed by 5% sodium dodecyl sulfate (SDS)-gel electrophoresis.
Human platelet aggregation.
Blood was collected from healthy human volunteers, who did not take any
medication within the 2 weeks before the study, and anticoagulated with
3.8% sodium citrate (9:1, vol/vol). Citrated blood was immediately
centrifuged for 10 minutes at 120g and 25°C, and the
supernatant (platelet-rich plasma) was obtained. Human washed platelet
suspension was prepared as previously described.22,41 Washed platelets were suspended in modified Tyrodes' solution (pH 7.3)
(in mmol/L: NaCl, 136.9; CaCl2, 2; KCl, 2.7;
MgCl2, 1.0; NaH2PO4, 0.4;
NaHCO3, 11.9; glucose, 11.1) containing bovine serum albumin (3.5 mg/mL) and adjusted to about 3 × 108
platelets/mL.
The turbidimetric method,23 using a Lumi-Aggregometer
(Chrono-Log, Havertown, PA), was used to measure platelet
aggregation. The extent of aggregation was expressed in light
transmission unit.
Assay of thromboxane B2 formation.
EDTA (2 mmol/L) and indomethacin (50 µmol/L) were added to platelet
suspension at 6 minutes after the addition of thrombin (0.03 U/mL).
After centrifugation with an Eppendorf Centrifuge (Model
5414; Hamburg, Germany) at 14,000 cpm for 2 minutes, thromboxane B2 level of the supernatant was determined by thromboxane
B2 EIA Kit (Amersham, Buckinghamshire, UK).
Radiolabeling of crotalin using enzymobead.
The vial of enzymobead reagent (Bio-Rad, San Francisco,
CA) was hydrated with 50 µL distilled water at 4°C
for 1 hour, followed by adding 50 µL phosphate buffer, 50 µg
crotalin, 0.5 mCi Na125I, and 25 µL  -D-glucose (1%).
The mixture was incubated at room temperature for 25 minutes.
125I-conjugated crotalin was eluted through a Sephadex G-10
column (0.7 × 23 cm; bed volume, 10 mL) with phosphate buffer. The
radioactivity of 125I-labeled crotalin was detected with
radiocounter (Capintec, CRC-7) continuously.
Binding of 125I-crotalin to platelets.
It was performed according to previously described
methods24,25 with slight modification. Aliquot of 0.4 mL of
platelet suspension (4.5 × 108/mL) were incubated in
the presence of Tyrode solution (control), AP1, or excess unlabeled
crotalin at room temperature for 2 minutes. Then,
125I-crotalin in various concentrations was added, and the
mixture was gently shaken and incubated for 6 minutes. Platelet
suspension mixture, 0.4 mL, was overlayed on sucrose solution (20%,
wt/vol) and centrifuged for 5 minutes at 14,000 rpm (Eppendorf
centrifuge, 5415). The radioactivities of supernatant (200 µL) and
the cut-off tips containing platelet pellet were separately measured by
using a  -counter (Beckman, Fullerton, CA). The
specific 125I-crotalin binding to the platelet pellet was
calculated by subtracting the nonspecific radioactivity bound from the
total radioactivity bound. The number of crotalin binding sites per
platelet and dissociation constant (kd) were calculated by the method
of Scatchard.26
Measurement of bleeding time in mice.
Bleeding time of mice was measured by a modification of the method
described by Dejana et al.27 Saline or various
concentrations of crotalin was injected intravenously through a lateral
vein of the mouse (ICR, with an average body weight of 20 g). A sharp cut of 2 to 3 mm from tail tip of mouse was made 10 minutes
(except as noted) after injection. The tail was then immediately placed into a tube filled with saline, and kept at 37°C for measuring bleeding time.
Platelet-rich thrombosis model in mice.
Normal male mice (ICR) were used. The mice weighing
between 18 and 20 g were anesthetized with sodium pentobarbital (50 mg/kg) by intraperitoneal injection. The method of this thrombogenic animal model has been described in detail.21,28,29 In
brief, an external jugular vein was cannulated for the administration of dye and crotalin, and a mesenteric preparation was then mounted on a
transilluminator and continuously rinsed with warm saline. The room
temperature around microscope was kept at 37°C. Mesenteric venules
with a diameter around 20 µm were selected for thrombogenic assay.
Fluorescein sodium (100, 150, or 200 µg/mL) was first injected
through a cannula, and microvessels were irradiated by filtered light
10 minutes later. Simultaneously, a video timer was started. Platelet
adhesion and aggregation were observed through a television monitor and
time lapse for inducing the formation of platelet-rich thrombus, as
reflected by cessation of blood flow, was taken. In the
epi-illumination system, the light from a 100-W mercury lamp was
excited by a filter (B-2A; Nikon) with a dichronic mirror (DM510;
Nikon, Tokyo, Japan). The filtered light then irradiated a microvessel through an objective lens (20×). In this experimental model, platelet thrombus formation was induced and found to consist exclusively of platelets with pseudopod formation and degranulated appearance, accompanied with moderately damaged endothelial
cells.21
Statistics.
All data are presented as mean ± SEM (n). Student's t-test
was used to assess the statistical differences.
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RESULTS |
Purification and physicochemical characterization of crotalin.
Crotalin was purified from C atrox venom through columns of
DEAE-Sephadex A-50 and Sephadex G-75 and refractionated by fast protein
liquid chromatography using Mono-S (Fig 1A)and Superose columns (Fig 1B). The purified fraction migrated as a
single band and the apparent molecular weight was estimated to be 30 kD
under nonreducing or reducing conditions by SDS-polyacrylamide (15%) gel electrophoresis (Fig 1B, inlet) and named crotalin.
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| Fig 1.
(A) Rechromatography of crotalin on Mono-S column. The
active fraction was dissolved in 0.02 N ammonium acetate, pH 5.0, and loaded on Mono-S column. Elution was carried out with a gradient from 0 to 0.75 mol/L NaCl as indicated (---) at a flow rate of 0.5 mL/min. The
active fraction (*) was collected. (B) Gel filtration chromatography on
Superose HR 10/30 column. The active fraction above was applied (5 mg)
to this column equilibrated with 0.05 mol/L phosphate buffer (pH 7.2).
The column was eluted at a flow rate of 0.25 mL/min with the same
buffer. The absorbance profile monitored at 280 nm was shown. The
active fraction (*) which was eluted at 70 minutes was named
crotalin.
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Amino acid analysis showed that crotalin is a polypeptide, consisting
of about 260 amino acid residues (Asp/Asn 36, Glu/Gln 25, Ser 14, Gly
21, His 9, Thr 8, Ala 14, Arg 22, Pro 10, Tyr 6, Val 16, Ile 18, Leu
28, Cys 7, Phe 10, and Lys 16). However, the N-terminal amino acid
sequence of crotalin was found to be blocked.
Inhibition of ristocetin-induced platelet aggregation.
Crotalin showed a marked inhibitory effect on ristocetin (1.0 mg/mL)-induced human washed platelet aggregation with IC50
of 2.4 µg/mL (Fig 2). This inhibitory
effect was independent of the incubation time of crotalin with
platelets. Furthermore, crotalin was specific for ristocetin-induced
platelet aggregation because it had little effect on the platelet
aggregations caused by ADP (20 µmol/L) plus 200 µg/mL of
fibrinogen, U46619 (1 µmol/L), or collagen (10 µg/mL). Crotalin
also inhibited ristocetin (1 mg/mL)-induced platelet aggregation of
platelet-rich plasma with IC50 value of 6.3 µg/mL (Fig
2), indicating that it might have an antithrombotic effect in vivo.
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| Fig 2.
Dose-response relationship of crotalin on platelet
aggregation induced by 1 mg/mL of ristocetin in human washed platelets in the presence of 10 µg/mL of vWF ( ) or in platelet-rich plasma ( ). Values are presented as mean ± SEM (n = 4).
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Similarly, crotalin (10 µg/mL) apparently did not affect platelet
aggregation caused by high concentration of thrombin (>0.05 U/mL).
However, crotalin (20 to 100 µg/mL)
prolonged the latent period in triggering platelet
aggregation in a dose-dependent manner, with a slight inhibition on the
maximal aggregation (<30%) inhibition). The MoAb AP1 (40 to 80 µg/mL) showed a similar effect to that of crotalin (data not shown).
Effect of crotalin on thromboxane B2 formation of
platelets caused by thrombin and other agonists.
As shown in Table 1, crotalin at a lower
concentration (5 to 10 µg/mL) inhibited thromboxane B2
formation of platelets stimulated by ristocetin and vWF. At higher
concentration (20 µg/mL), crotalin significantly inhibited
thromboxane B2 formation of platelets caused by thrombin
(0.03 U/mL), whereas it did not affect thromboxane B2
formation stimulated by collagen.
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Table 1.
Effects of Crotalin on Thromboxane B2
Formation of Washed Platelets Stimulated by Thrombin and Other
Agonist
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Characterization of the binding of 125I-crotalin to human
platelets.
125I-crotalin bound to platelets in a dose-dependent
manner, reaching a saturated binding at 10 µg/mL (0.33 µmol/L) (Fig
3). The Scatchard analysis of
125I-crotalin binding data showed that the binding sites of
crotalin were 58,632 ± 3,152 per platelet with a kd value of 3.2 ± 0.1 × 107 mol/L (Fig 4).125I-crotalin binding to platelets was blocked either by
unlabeled crotalin (100 µg/mL, >85%) or AP1 (20 µg/mL, >90%),
but not by the MoAb against GPIIb/IIIa, 7E3 (40 µg/mL), or 5 mmol/L
EDTA (data not shown).
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| Fig 3.
Binding isotherm of 125I-crotalin on human
platelet suspension. Platelets were incubated with various
concentrations of 125I-crotalin. Total binding () and
nonspecific binding () in the presence of unlabeled crotalin (200 µg/mL) were determined, respectively. Specific binding ( ) was
calculated by subtracting the nonspecific binding from total binding.
This is a representative one of four similar experiments.
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| Fig 4.
Scatchard plot of the 125I-crotalin binding
to human washed platelets. This plot is a representative one of four
experiments.
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Effect of crotalin on bleeding time of mice.
IV administration of crotalin to mice significantly prolonged the
bleeding time in a dose-dependent manner (Fig
5). Crotalin pronouncedly prolonged the
bleeding time (>10 minutes) as measured 1 hour after a bolus
injection of crotalin (300 µg/kg), and this effect slowly ran down
thereafter (Fig 6). The bleeding time
almost returned to the baseline level within 4 hours after the
administration of crotalin. However, the platelet count, as measured at
10 minutes after the administration of crotalin (300 µg/kg), did not
show a major change, although about 20% decreasement was observed
(128 ± 10 × 104, n = 4, control v
104 ± 13 × 104 platelets/µL, n = 4,
experimental; P > .05). Even when the dose of crotalin was
increased to 600 µg/kg, a transient slight decrease of platelet count
(about 20%) was observed at the first 5 minutes after the
administration of crotalin, and the platelet count returned to control
level within 20 minutes.
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| Fig 5.
Effects of crotalin on tail bleeding time in mice.
Bleeding time was measured 10 minutes after the IV administration of
saline or various doses of crotalin. Bleeding time longer
than 10 minutes was expressed as >10 min. Bar represents the mean
value (n = 6). Each different symbol represents the bleeding time of
the individual mouse.
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| Fig 6.
The effects of crotalin on the bleeding time after the IV
administration of crotalin (300 µg/kg). This study was performed as
described in Fig 5 and bleeding time was measured at 10 minutes, 1 hour, 2 hours or 4 hours after the administration of crotalin. Bar
represents the mean value (n = 6). Each different
symbol represents the bleeding time of the individual mouse.
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Platelet-rich thrombus formation in the microvessels.
As the given dose of fluorescein sodium was increased, the latent
period in inducing platelet plug formation was shortened (Table 2). IV
administration of crotalin at 300 µg/kg pronouncedly delayed
platelet-rich thrombus formation and significantly prolonged the
occlusion time in mice receiving different doses of fluoresein sodium
(Table 2). The occlusion time was
lengthened from 100 ± 14 to 311 ± 25 seconds in fluorescein sodium
(150 µg per mouse)-treated mice after IV administration of crotalin.
Crotalin dose-dependently prolonged the occlusion time in causing
platelet plug formation, and administration of 300 µg/kg of crotalin
resulted in the maximal lengthening of occlusion time (Table
3). This antithrombotic effect lasted at
least for 2 hours (Fig 7). On the other
hand, a continuous infusion of prostaglandin I2
(PGI2) at 0.5 µg/kg/min, as in our previous
study,29 showed a maximal lengthening effect on occlusion
time. Higher dose (2 µg/kg/min) of PGI2 did not further increase its antithrombotic activity. Halysin, an RGD-containing peptide purified from venom of A halys, inhibited platelet
aggregation via a competitive inhibition of fibrinogen binding to
platelet GPIIb/IIIa.30 It completely inhibited ex vivo
platelet aggregation of platelet-rich plasma induced by collagen (15 µg/mL) 20 minutes after the IV administration of halysin at the
dose of 10 mg/kg (data not shown). In comparing the maximal effect of
PGI2, halysin, ancrod,29 and crotalin on the
occlusion time in the same in vivo model, crotalin appears to be the
most efficacious agent in prolonging the occlusion time (Table
4).
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Table 2.
Effect of Fluorescein Sodium and Crotalin on the
Occlusion Time in Causing Platelet-Rich Thrombus Formation in
Mesenteric Venules of Mice
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Table 3.
Dose-Response Relationship of Crotalin on the Elapsed
Time of Inducing Thrombus Formation on Light Irradiation of Venules of
Mice Pretreated With Fluorescein Sodium (150 µg per mouse)
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| Fig 7.
The effects of crotalin on the elapsed time in causing
platelet plug formation upon the irradiation of venules in mice.
Fluorescein sodium (150 µg per mouse) was intravenously injected 10 minutes before the irradiation, and the irradiation was then started
for inducing the formation of thrombus at the indicated time intervals after the IV administration of crotalin (300 µg/kg). Values are presented as mean ± SEM (n = 5-6). BL indicates baseline
value. ( ) Represents the occlusion time of each mouse
measured at the indicated time. *P < .05, **P < .01 as compared with basal value.
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Table 4.
Effect of Crotalin, PGI2, Halysin and
Ancrod on the Lapsed Time in Inducing Thrombus Formation Caused by
Irradiation of Mesenteric Venules of Fluorescein Sodium-Treated
Mice
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DISCUSSION |
Crotalin, a newly purified protein from the venom of C atrox,
specifically inhibited ristocetin-induced platelet aggregation in vitro
and exhibited the antithrombotic activity in vivo. Crotalin specifically inhibited platelet aggregation induced by ristocetin either in platelet suspension supplemented with vWF or in platelet-rich plasma with an IC50 of 2.4 and 6.3 µg/mL, respectively.
In contrast to RGD-containing peptides, crotalin at 40 µg/mL did not
affect collagen- and U46619-induced platelet aggregation.
Ristocetin-induced platelet aggregation was mediated through the
initial binding of vWF to platelet GPIb, and subsequently resulted in
the exposure of the fibriongen receptor.31,32 The
125I-crotalin binding site was 58,632 per platelet with a
kd value of 3.2 × 107 mol/L. 125I-crotalin
binding to platelets was selectively inhibited by AP1, an MoAb raised
against platelet GPIb, but not by 7E3, an MoAb raised against
GPIIb/IIIa. The binding was not affected by EDTA, indicating that the
binding process is divalent-cation independent. Through the analysis of
binding data and its inhibitory activity on ristocetin-induced platelet
aggregation, crotalin appears to be a selective antagonist of platelet
membrane GPIb. Regarding the binding sites of crotalin, the estimation
of 58,632 per platelet is higher than that estimated with GPIb MoAb
(around 30,000). It has been reported that the binding sites of other
venom GPIb antagonists ranged from 21,500 to 47,440.20 We
also obtained a similar value of 60,000 per platelet as probed with
another GPIb antagonist purified from A acutus (unpublished
data, December 1996). Therefore, the varied number of
binding sites among GPIb antagonists and MoAbs may be explained as
following: the smaller molecular size of venom GPIb antagonists may get
a better access to the surface canalicular system of platelets as
compared to the bulky, bivalent structure of the MoAb. Earlier studies
indicate that low concentration of thrombin exerts its effect through
the binding of platelet membrane GPIb, a high-affinity binding site of
thrombin.42 Kroll et al33
suggested that the binding of vWF to GPIb leads to the activation of
phospholipase A2 and subsequent formation of thromboxane.
In this study, crotalin as well as AP1 prolonged the latent period in
triggering platelet aggregation caused by low concentrations of
thrombin (0.03 U/mL), with a slight inhibition on platelet aggregation.
Crotalin blocked thromboxane B2 formation of platelets
challenged by ristocetin plus vWF or low concentration of thrombin,
confirming the hypothesis that the ligation of GPIb may lead to the
activation of endogenous phospholipase A2. Regarding the
molecular characterization of crotalin, a platelet GPIb antagonist, the
preliminary data show that crotalin is unique as a single chain
polypeptide that is quite different from the known venom GPIb-binding
proteins because they are heterodimer in nature, sharing a highly
homologous sequence with C-type lectins.20 Whether it
exists as a monomer or dimer under physiological condition is unknown.
However, the detailed characterization of the physiocochemical
properties of crotalin is in progress.
An IV infusion of crotalin lengthened bleeding time of mice in a
dose-dependent manner. Maximal prolongation was observed during a
period of 10 to 60 minutes after injection of crotalin (300 µg/kg),
and bleeding time progressively returned to control values over a
4-hour period. However, the platelet count after the administration of
crotalin did not show a major change. It has been reported that
echicetin and jararaca GPIb-BP caused a transient
thrombocytopenia in mice,18,19 and therefore it may be an
advantage of crotalin when considering its potential use as
antithrombotic agent. From its in vivo experiment, we suggest that
crotalin may inhibit platelet aggregation as well as platelet adhesion to subendothelium in vivo through blocking the interaction of
vWF with platelet GPIb.
As the first step in hemostasis or thrombosis, the binding of vWF to
platelet GPIb is essential for platelet adhesion at high-shear blood
flow.34 The platelets from patients with Bernard-Soulier Syndrome were defective in expression of functional GPIb-V-IX complex,
and poorly adhered to subendothelium at all shear rates.2 Much effort has recently been devoted to characterize the interaction of vWF and platelet GPIb at the molecular level with an aim of developing inhibitors that could be useful in the prevention of thrombosis.35-38 Recently, it has been indicated that
inhibition of vWF-platelet GPIb interaction is effective in preventing
acute restenosis after thrombolytic therapy.39
In the present study we evaluated the antithrombotic effect of crotalin
in a mouse model. Surprisingly, crotalin apparently delayed
platelet-rich thrombus formation in mesenteric microvessels and its
antithrombotic activity was dose dependent. The time course of its
antithrombotic effect was consistent with that of its effect on
bleeding time in mice (Figs 6 and 7).
Table 4 shows the minimal dose of PGI2, halysin, ancrod,
and crotalin in causing the maximal prolongation of occlusion time in
this platelet-rich thrombus animal model. Halysin, an RGD-containing venom peptide, completely inhibited ex vivo platelet aggregation for 20 minutes after the administration of halysin at 10 mg/kg. Ancrod (1 U/kg), a thrombin-like enzyme, caused defibrinogenation and exhibited
antiplatelet activity for 60 minutes.29 Of the compounds
tested, crotalin showed the most pronounced effect in prolonging the
occlusion time of the irradiated vessels in inducing platelet-rich
thrombus formation as compared with PGI2, halysin, and
ancrod, indicating that blockade of the interaction between vWF and
platelet GPIb may be a potential strategy in causing a marked
antithrombotic effect. In addition, crotalin exhibits a antithrombotic
activity with a longer duration as compared with short duration of
PGI2, halysin or another disintegrin,
triflavin.40
Considering the pharmacokinetic of crotalin, crotalin may be more
active as an antithrombotic agent in mice than in human beings because
the effective dosage of crotalin in mice ranged from 100 to 300 µg/kg, equivalent to 1.3 to 3.8 µg/mL (assuming 2.0 mL plasma per
mouse), even if protein binding is neglected. However, the
IC50 of crotalin was about 6.3 µg/mL (in human
platelet-rich plasma), five times higher than the effective dosage of
100 µg/kg in mice. On the other hand, the in vivo antithrombotic
effect of crotalin in mice may result from both the antiplatelet and anticoagulant activities because the preliminary results show that
crotalin prolonged the whole blood clotting time and activated partial
thromboplastin time but not the prothrombin time as crotalin was
administered IV (unpublished data, December 1996).
However, its anticoagulant activity is under investigation.
In conclusion, crotalin specifically inhibited ristocetin-induced
platelet agglutination in the presence of vWF through a selective
binding of platelet membrane GPIb, resulting in a blockade of
interaction between vWF and GPIb. Furthermore, crotalin markedly prolonged the bleeding time when administered IV into mice and was
efficious in blocking platelet plug formation in vivo experimental model. Therefore, crotalin may be a valuable tool for developing a new
class of antithrombotic drugs for clinic use through the study of its
structure-activity relationship.
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FOOTNOTES |
Submitted March 10, 1997;
accepted October 20, 1997.
Supported by Grant No. NSC84-2331-B-002-217 from the National Science
Council of Taiwan.
Address reprint requests to Tur-Fu Huang, PhD, Pharmacological
Institute, College of Medicine, National Taiwan University, No 1, Jen-Ai Rd, 1st Section, Taipei, 10018, Taiwan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We appreciate the secretarial work of I.S. Peng.
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REFERENCES |
1.
Huang TF,
Chang MC,
Teng CM:
Antiplatelet protease, kistomin, selectively cleaves human platelet glycoprotein Ib.
Biochim Biophys Acta
1158:293,
1993[Medline]
[Order article via Infotrieve]
2.
Clemetson JK,
Clemetson JM:
Platelet GPIb-V-IX complex: Structure, function, physiology and pathology.
Semin Thromb Hemost
21:130,
1995[Medline]
[Order article via Infotrieve]
3.
de Groot PG:
Platelet adhesion.
Br J Haematol
75:308,
1990[Medline]
[Order article via Infotrieve]
4.
Roth GJ:
Platelet and blood vessels; the adhesion event.
Immunol Today
13:100,
1992[Medline]
[Order article via Infotrieve]
5.
Sakariassen KS,
Fressinaud E,
Girmo J-P,
Mayer D,
Baumgartner HR:
Role of platelet membrane glycoproteins and von Willebrand factor in adhesion of platelets to subendothelium and collagen.
Ann NY Acad Sci
516:52,
1987[Medline]
[Order article via Infotrieve]
6.
Sixma JJ,
Nievelstein FEM,
Zwaginga J-J,
de Groot PG:
Adhesion of blood platelets to the extracellular matrix of cultured human endothelial cells.
Ann NY Acad Sci
516:39,
1987[Medline]
[Order article via Infotrieve]
7.
Coller BS,
Anderson K,
Weisman HF:
New antiplatelet agents: Platelet GPIIb/IIIa antagonists.
Thromb Haemost
74:302,
1995[Medline]
[Order article via Infotrieve]
8.
Golino P,
Ashton JH,
McNatt L,
Glas-Greenwalt P,
Sheng-Yun Y,
O'Brein RA,
Buja LM,
Willerson JT:
Simultaneous administration of thromboxane A2 and serotonin S2-receptor antagonists markedly enhances thormbolysis and prevents or delays reocclusion after tissue-type plasminogen activator in a canine model of coronary throbosis.
Circulation
79:911,
1989[Abstract/Free Full Text]
9.
Nichols AJ,
Ruffolo RR,
Huffman WF,
Poste G,
Samanen J:
Development of GPIIb/IIIa antagonists as antithrombotic drugs.
Trends Pharmacol
13:413,
1992 [Medline]
[Order article via Infotrieve]
10.
Fenton JW:
Thrombin.
Ann NY Acad Sci
485:5,
1984[Medline]
[Order article via Infotrieve]
11.
Heras M,
Chesebro JH,
Webster NIW,
Mruk JS,
Grill DE,
Penny WJ,
Bowie EJW,
Badimon L,
Fuster V:
Hirudin heparin, and placebo during deep arterial injury in the pig. The in vivo role of thrombin in platelet-mediated thrombosis.
Circulation
82:1476,
1990[Abstract/Free Full Text]
12.
Jang IK,
Gold HK,
Ziskind AA,
Leinbach RC,
Fallon JT,
Collen D:
Prevention of platelet-rich arterial thrombosis by selective thrombin inhibition.
Circulation
81:219,
1990[Abstract/Free Full Text]
13.
Eidt JF,
Allison P,
Noble S,
Ashton J,
Golino P,
McNatt J,
Buja LM,
Willerson JT:
Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury.
J Clin Invest
84:18,
1989
14.
Teng CM,
Huang TF:
Inventory of exogenous inhibitors of platelet aggregation.
Thromb Haemost
65:624,
1991[Medline]
[Order article via Infotrieve]
15. Huang TF, Niewiarowski S: Disintegrins: The naturally-occurring
antagonists of platelet fibrinogen receptor. J Toxicol-Toxin Rev
13:253, 1994
16.
Read MS,
Shermer RW,
Brinkhous KM:
Venom coagglutinin: An activator of platelet aggregation dependent on von Willebrand factor.
Proc Natl Acad Sci USA
75:4514,
1978[Abstract/Free Full Text]
17.
Brinkhous KM,
Read MS,
Fricke WA,
Wagner RH:
Botrocetin (venom coagulatinin): Reaction with a broad spectrum of multimeric forms of factor VIII macromolecular complex.
Proc Natl Acad Sci USA
80:1463,
1983[Abstract/Free Full Text]
18.
Peng M,
Lu W,
Beviglia L,
Niewiarowski S,
Kirby EP:
Echicetin: A snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib.
Blood
81:2321,
1993[Abstract/Free Full Text]
19.
Fujimura Y,
Miura IS,
Yoshida E,
Shima H,
Nishida S,
Suzuki M,
Titani K,
Taniuchi Y,
Kawasaki T:
Isolation and characterization of jararaca GPIb-BP, a snake venom antagonist specific to platelet glycoprotein Ib.
Thromb Haemost
74:743,
1995[Medline]
[Order article via Infotrieve]
20.
Fujimura Y,
Kawasaki T,
Titani K:
Snake venom proteins modulating the interaction between von Willebrand factor and platelet glycoprotein Ib.
Thromb Haemost
76:633,
1996[Medline]
[Order article via Infotrieve]
21.
Sheu JR,
Chao SH,
Yen MH,
Huang TF:
In vivo antithrombotic effect of triflavin, an Arg-Gly-Asp containing peptide on platelet plug formation in mesenteric microvessels of mice.
Thromb Haemost
72:617,
1994[Medline]
[Order article via Infotrieve]
22.
Newmans J,
Johnson AJ,
Karpatkin MH,
Puszkin S:
Methods for the production of clinically effective intermediate and high-purity factor-VIII concentrates.
Br J Haematol
21:1,
1971[Medline]
[Order article via Infotrieve]
23.
Born GVR,
Cross MJ:
The aggregation of blood platelets.
J Physiol
168:178,
1963
24.
Kornecki E,
Niewiarowski S,
Morinelli TA,
Kloczewialc M:
Effect of chymotrypsin and adenosine diphosphate on the exposure of fibrinogen receptors on the normal human and Glanzmann's thrombathenic platelets.
J Biol Chem
256:5696,
1981[Free Full Text]
25.
Niewiarowski S,
Budzynsti AZ,
Morinelli TA,
Brudzynski TM,
Stewart GJ:
Exposure of fibrinogen receptor on human platelets by proteolytic enzymes.
J Biol Chem
256:917,
1981[Free Full Text]
26.
Scatchard G:
The attractions of proteins for small molecules and ions.
Ann NY Acad Sci
51:660,
1949
27.
Dejana E,
Villa S,
de Gaetano G:
Bleeding time in rats: A comparison of different experimental conditions.
Thromb Haemost
48:108,
1982[Medline]
[Order article via Infotrieve]
28.
Sato M,
Ohshima N:
Platelet thrombus induced in vivo by filtered light and fluorescent dye in mesenteric microvessels of the rat.
Thromb Res
35:319,
1984[Medline]
[Order article via Infotrieve]
29.
Chang MC,
Huang TF:
In vivo effect of a thrombin-like enzyme on platelet plug formation induced in mesenteric microvessels of mice.
Thromb Res
73:31,
1994[Medline]
[Order article via Infotrieve]
30.
Huang TF,
Liu CZ,
Ouyang C,
Teng CM:
Halysin, an antiplatelet Arg-Gly-Asp containing snake venom peptide, as fibrinogen receptor antagonist.
Biochem Pharmacol
42:1209,
1991[Medline]
[Order article via Infotrieve]
31.
Gralnick HR,
Williams SB,
Coller BS:
Asial von Willebrand factor interactions with platelets: Interdependence of glycoprotein Ib and IIb/IIIa for binding and aggregation.
J Clin Invest
75:19,
1985
32.
Tanoue K,
Jung SM,
Yamamoto N,
Yamazaki H:
Neutralization of the local negative charge carried by glycoprotein (GP)-Ib in ristocetin-induced platelet agglutination.
Thromb Haemost
51:79,
1984[Medline]
[Order article via Infotrieve]
33.
Kroll MH,
Harris TS,
Moake JL,
Handin RI,
Schafer AS:
von Willebrand factor binding to platelet GPIb initiates signals for platelet activation.
J Clin Invest
88:1568,
1990
34. Kawakami K, Harada Y, Sakasita M, Nagai H, Handa M, Ikeda Y: A
new method for continuous measurement of platelet adhesion under flow
conditions. ASAIO J 39:M558, 1993
35.
Sadler JE:
von Willebrand factor.
J Biol Chem
266:18172,
1991[Abstract/Free Full Text]
36.
Girma JP,
Meyer D,
Verweij CL,
Pannekoek H,
Sixma JJ:
Structure-function relationship of human von Willebrand factor.
Blood
70:605,
1987[Free Full Text]
37.
Handa M,
Titani K,
Holland LZ,
Roberts JR,
Ruggeri ZM:
The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib.
Proc Natl Acad Sci USA
261:12579,
1986
38.
Cruz MA,
Peterson E,
Turci SM,
Handin RI:
Functional analysis of a recombinant glycoprotein Ib polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen.
J Biol Chem
267:1303,
1992[Abstract/Free Full Text]
39.
Strony J,
Song A,
Rusterholtz L,
Adelman B:
Aurintricarboxylic acid prevents acute rethrombosis in a canine model of arterial thrombosis.
Arterioscler Thromb Vasc Biol
15:359,
1995[Abstract/Free Full Text]
40.
Sheu JR,
Yen MH,
Peng HC,
Chang MC,
Huang TF:
Triflavin, an Arg-Gly-Asp-containing peptide, prevents platelet plug formation in in vivo experiments.
Eur J Pharmacol
294:231,
1995[Medline]
[Order article via Infotrieve]
41.
Mustard JF,
Perry DW,
Ardlie NG,
Packham MA:
Preparation of suspensions of washed platelets from humans.
Br J Haematol
22:193,
1972[Medline]
[Order article via Infotrieve]
42.
Harmon JT,
Jamieson GA:
The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites in thrombin.
J Biol Chem
261:13224,
1986[Abstract/Free Full Text]
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Abstract
Introduction
Abstract