|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1693-1700
Unique Antiplatelet Effects of a Novel S-Nitrosoderivative of a
Recombinant Fragment of von Willebrand Factor, AR545C: In Vitro and Ex
Vivo Inhibition of Platelet Function
By
Aida Inbal,
Osnat Gurevitz,
Ilia Tamarin,
Regina Eskaraev,
Angela Chetrit,
Ilia Novicov,
Monica Feldman,
David Varon,
Michael Eldar, and
Joseph Loscalzo
From the Institutes of Thrombosis and Hemostasis, Neufeld Cardiac
Research Institute, and Clinical Epidemiology, Sackler School of
Medical, Sheba Medical Center, Tel-Hashomer, Israel; and the Whitaker
Cardiovascular Institute and Evans Department of Medicine, Boston
University School of Medicine, Boston, MA.
 |
ABSTRACT |
The recombinant fragment of von Willebrand factor (vWF) spanning
Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C)
has antithrombotic properties that are principally a consequence of its
ability to inhibit platelet adhesion to subendothelial matrix.
Endothelial-derived nitric oxide (NO) can also inhibit platelet
function, both as a consequence of inhibiting adhesion as well as
activation and aggregation. Nitric oxide can react with thiol
functional groups in the presence of oxygen to form S-nitrosothiols,
which are naturally occurring NO derivatives that prolong the
biological actions of NO. Because AR545C has a single free cysteine
(Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C
and to characterize its antiplatelet effects. We successfully
synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO
per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent
at inhibiting platelet agglutination than was the unmodified peptide
(IC50 = 0.02 ± 0.006 µmol/L v 0.1 ± 0.03 µmol/L, P = .001). In addition and by contrast,
S-nitroso-AR545C was a powerful inhibitor of adenosine
diphosphate-induced platelet aggregation (IC50 = 0.018 ± 0.002 µmol/L), while AR545C had no effect on aggregation. These
effects were confirmed in studies of adhesion to and aggregation on
extracellular matrix under conditions of shear stress in a cone-plate
viscometer, where 1.5 µmol/L S-nitroso-AR545C inhibited platelet
adhesion by 83% and essentially completely inhibited aggregate
formation, while the same concentration of AR545C inhibited platelet
adhesion by 74% and had significantly lesser effect on aggregate
formation on matrix (P  .004 for each parameter by
ANOVA). In an ex vivo rabbit model, we also found that
S-nitroso-AR545C had a more marked and more durable inhibitory effect
on botrocetin-induced platelet aggregation than did AR545C, and these
differences were also reflected in the extent and duration of effect on
the prolongation of the bleeding time in these animals. These data show
that S-nitroso-AR545C has significant and unique antiplatelet effects,
inhibiting both adhesion and aggregation, by blocking platelet GPIb
receptor through the AR545C moiety and elevating platelet cyclic
3',5'-guanosine monophosphate through the -SNO
moiety. These observations suggest that this NO-modified fragment of
vWF may have potential therapeutic benefits as a unique antithrombotic agent.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
VON WILLEBRAND FACTOR (vWF) is a
multimeric glycoprotein synthesized by megakaryocytes and endothelial
cells, and is released both into the circulation and the subendothelial
space.1 At sites of vascular injury, platelet activation is
initiated by interactions of vWF and a specific receptor on platelets,
glycoprotein Ib (GPIb). The interaction of vWF with GPIb is critical
for the initiation of platelet deposition, both during normal
hemostasis2 and in the setting of arterial
thrombosis.3-5 This initial hemostatic effect is triggered
by vWF associated with subendothelial matrix, and is modulated by shear
stress evoked by flowing blood in the vasculature. Platelet aggregation
is then further promoted by activation of another platelet receptor
complex, glycoprotein IIb/IIIa (GPIIb/IIIa), leading to the binding of
fibrinogen or vWF to the platelet surface.6
The A1 domain of the vWF molecule is known to contain the GPIb-binding
site, first assigned to a tryptic fragment spanning amino acids 449 to
728 that contains a large intrachain disulfide-linked (Cys509-Cys695)
loop.7,8 The pharmacological inhibition of the high shear
stress-induced platelet adhesion to vWF with monoclonal anti-vWF
antibodies,5,9 aurin tricarboxylic acid,10,11 or the recombinant A1 domain fragment VCL12 reduces
thrombus formation in various animal models, emphasizing the crucial
role vWF plays in arterial thrombogenesis.
We recently reported that the recombinant vWF fragment spanning Ala444
to Asp730 and containing an Arg545Cys mutation, denoted AR545C, has
antithrombotic properties in vitro and in vivo.13 R545C is
a gain of function mutation that results in an increased and also
spontaneous binding of the fragment to platelet GPIb,13 thereby blocking the initial interaction between native vWF and platelet GPIb, preventing any further process of platelet activation. Indeed, the mutated AR545C fragment inhibited ristocetin- and botrocetin-induced platelet agglutination of human and rabbit platelets, respectively, and enhanced the thrombolytic effect of
recombinant tissue-type plasminogen activator in a rabbit thrombosis model.13
Endothelium-derived nitric oxide (NO) inhibits platelet
aggregation14,15 and prevents adhesion of platelets to the
subendothelium,16 and does so in association with elevating
intracellular cyclic 3',5'-guanosine monophosphate (cGMP).
NO is stabilized by reacting with sulfhydryl groups in the presence of
oxygen to form S-nitrosothiols, thereby prolonging its half-life and
preserving its biological activity.17 The AR545C molecule
contains 3 cysteine residues involved in interchain bonds (residues
459, 461, and 464), 2 pairs of intrachain disulfide bonds (residues
471-474 and 509-695), and 1 apparently free cysteine (residue
545).18 S-nitrosation of AR545C (S-nitroso-AR545C) at
Cys545 should, therefore, endow the molecule with potent and
long-lasting NO-like effects. This compound may be of potential
clinical interest because 2 independent antiplatelet activities are
combined in the same molecule, viz, antiadhesive and antiaggregatory
effects. The aim of the present study is to synthesize and characterize
the antiplatelet effects of S-nitroso-AR545C in vitro and ex vivo, and
to compare these effects to the parent peptide AR545C.
| |
MATERIALS AND METHODS |
Construction, synthesis, and purification of the peptide AR545C.
The sequence encoding alanine 444-aspargine 730 and containing the
arginine-to-cysteine substitution at amino acid residue 545 (AR545C)
was derived from a full-length cDNA for human vWF.13 The
coding sequence was inserted into a pZEM229 expression vector and
expressed in a thymidine kinase-deficient BHK cell line, BHK-570, using methotrexate for growth selection as previously
described.13 The AR545C peptide was purified from the BHK
media by heparin-Sepharose CL-6B affinity chromatography (Pharmacia
Biotech, Piscataway, NJ), yielding essentially pure material. The
purity of the peptide was verified by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing
or reducing conditions, as well as by reverse-phase high-performance
liquid chromatography with a Vydac C8 column (Vydac,
Hesperia, CA).13 The amount of the peptide was quantified
by a sandwich enzyme-linked immunosorbent assay (ELISA) using 1:100
rabbit anti-human vWF (Dakopatts A082; Dako, Glostrup,
Denmark) as the coating antibody and 1:1,000
peroxidase-conjugated anti-vWF antibody (Dakopatts P226) as the
detecting antibody. The standard was a human pool of platelet-poor
plasma (30 volunteers) that was assumed to contain 10 µg/mL of vWF.
ELISAs were developed with o-phenylenediamine as the colorimetric
substrate and quantified at A490 on an ELISA reader
(Molecular Devices, USA), as described previously.13
Synthesis of S-nitroso-AR545C peptide.
S-nitrosation of AR545C was performed by 2 methods: (1) direct
nitrosation by acidified NaNO2,17 and (2)
transnitrosation by the NO congener S-nitrosoglutathione
(SNO-Glu).19,20 SNO-Glu was prepared within 5 minutes of
use, kept at 4°C, and incubated with the purified AR545C fragment
for 1 hour at an approximate 28:1 molar ratio (SNO-Glu:AR545C). The
acidic pH of the mixture was then neutralized to pH 7.4 with NaOH and
the unbound SNO-Glu was separated from the nitrosated AR545C
(S-nitroso-AR545C) fragment by Sephadex G-25M (Pharmacia Biotech)
column chromatography. The protein concentration of S-nitroso-AR545C
was determined by the Coomassie Plus Protein Assay reagent (Pierce,
Rockford, IL). The formation of the S-nitroso-AR545C peptide was
confirmed by the Saville method21 and UV-visible
spectroscopy.19 In the Saville method, NO is displaced from
the S-nitrosothiol group with Hg2+ and assayed by
diazotization of sulfanilamide with subsequent coupling to the
chromophore N-(1-naph)-ethylenediamine; absorbance at 540 nm is
measured to determine S-NO concentration.21 In this method,
the sample containing S-nitroso-AR545C was first mixed with 0.5%
ammonium sulfamate in 0.4 N HCl for 1 minute to remove free
NO2 or HNO2 from the sample.
The S-nitroso content was calculated according to a standard curve
constructed with 2.5 to 20 µmol/L NaNO2.
UV-visible absorbance spectroscopy.
Spectra were recorded at room temperature on a Cary 4E UV Visible
spectrophotometer (Varian, Inc, Australia Pty, Ltd).
Recorded spectra represent the absorbance of S-nitrosated AR545C
compared with nonnitrosated AR545C.
Preparation of platelet-rich plasma (PRP).
After obtaining informed consent, 9 vol of peripheral blood from
healthy adult volunteers was drawn into 1 vol of 0.129 mol/L trisodium
citrate. After centrifugation (150g, 15 minutes, 22°C), the
supernatant PRP was separated by removing the top two thirds of the
plasma layer. Platelet-poor plasma (PPP) was prepared by centrifugation
of PRP at 1,200g for 10 minutes and used immediately.
Preparation of gel-filtered platelets (GFP).
GFP were obtained by passing PRP over a Sepharose-2B column (Pharmacia
Biotech) in Tyrode's-HEPES-buffered saline as previously described.22 Platelet counts were determined using a
Coulter Counter, model Technicon H2 (Bayer Diagnostics, Tarrytown, NY).
Cyclic nucleotide assay.
Measurements of cGMP were performed using an ELISA methodology using
anti-cGMP antiserum (Amersham Pharmacia Biotech, Uppsala, Sweden). cGMP
was extracted from platelets as previously described.23 Briefly, 10% trichloroacetic acid (TCA) was added to PRP before (negative control) and 5 minutes after the addition of SNO-Glu (positive control), AR545C, or S-nitroso-AR545C. Samples were vortexed,
placed on ice, and centrifuged (8,000g, 4 minutes) at room
temperature. The supernatant was extracted 4 times with diethyl ether
and assayed for cGMP as above. Acetylation of samples with acetic
anhydride was used to increase the sensitivity of the cGMP assay.
Effect of AR545C or S-nitroso-AR545C on ristocetin-induced platelet
agglutination.
Ristocetin-induced platelet agglutination was performed using
lyophilized, formalin-fixed platelets (Helena Hemostasis, Beaumont, TX)
as described previously.13 Various concentrations of either AR545C or S-nitroso-AR545C were incubated with the platelets (2 × 108 platelets/mL) for 10 minutes in a platelet PACK-4
aggregometer (Helena Laboratories, Beaumont, TX) at 37°C before the
addition of PPP as a source of vWF and 1.5 mg/mL of ristocetin (Sigma
Chemical Co, St Louis, MO). The extent of agglutination was monitored
to quantify the agglutination response.
Effect of AR545C or S-nitroso-AR545C on platelet aggregation.
Platelet aggregation experiments were conducted using human PRP or GFP.
Various concentrations of AR545C or S-nitroso-AR545C were incubated for
10 minutes with stirring at 37°C with PRP or GFP, and aggregation
was induced with 5 µmol/L adenosine diphosphate (ADP). In some of the
experiments, methylene blue (final concentration, 5 µmol/L) was added
to the reactions containing S-nitroso-AR545C. Extent of aggregation was
recorded in a PACK-4 aggregometer (Helena Laboratories).
Effect of AR545C or S-nitroso-AR545C on platelet interaction with
extracellular matrix (ECM).
Platelet adhesion and aggregation on ECM was tested in the cone-plate
viscometer analysis system (Galai, Beit Ha'emek, Israel), as described
previously.24 In brief, 0.23 mL of citrated whole blood was
placed on an ECM-covered plate under a shear rate of 1,300 s1 for 2 minutes. The sample was then washed and
stained with May-Grünwald-Giemsa stain. Platelet adhesion and
aggregation were determined using an image analysis system. The degree
of adhesion was assessed by calculating the percentage of total area
covered by platelets, and quantified as the percentage of surface
coverage (SC); the normal value of SC is 19% ± 5.9% at this shear
rate. The extent of platelet aggregation on the surface was estimated
by measuring the frequency distribution of platelet aggregates of
different size and the average size of ECM-bound platelet aggregates;
the latter parameter is expressed as average size (AS) of the
aggregates with the normal value of AS being 47.5 ± 15.2 µm2 at this shear rate. To evaluate the effect of
AR545C or S-nitroso-AR545C on the above-described parameters, the blood
samples were preincubated at room temperature for 10 minutes with
various concentrations of each of the peptides and the extent of
adhesion and aggregation was recorded.
Effect of AR545C or S-nitroso-AR545C on botrocetin-induced
aggregation of rabbit platelets ex vivo.
All animals used in this study were approved by the Institutional
Animal Care and Use Committee at the Neufeld Cardiac Research Institute
(Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel).
New Zealand white female rabbits, each weighing 2.5 to 3.0 kg, were
anesthetized with intravenous sodium pentobarbital (Nembutal, 30 mg/kg, followed by 10 mg at 30- to 60-minute
intervals) administered through the marginal ear vein. The
auricular artery was cannulated for blood sampling. The rabbits were
injected intravenously with 1 mg/kg AR545C (n = 3) or 0.5 mg/kg
S-nitroso-AR545C (n = 3). In each animal, 3 mL blood was drawn before,
and at various time intervals after, administration of the peptides.
Eight parts of rabbit blood were drawn into 1 part of 0.129 mol/L
trisodium citrate and rabbit PRP prepared. The effect of bolus
injection of either AR545C or S-nitroso-AR545C on rabbit
botrocetin-induced platelet aggregation was evaluated in an
aggregometer (Helena Laboratories) as in the ristocetin-induced
agglutination assay: 1 µg/mL botrocetin (Sigma Chemical Co) was added
to rabbit PRP containing 2 × 108 platelets/mL and 5 mmol/L EDTA at 37°C with stirring as described previously,13 and the extent of aggregation was then
recorded. ADP-induced platelet aggregation experiments were performed
using rabbit PRP and 5 µmol/L ADP, and the extent of aggregation was recorded in a PACK-4 aggregometer (Helena Laboratories) as described above.
Coagulation tests and bleeding time.
Prothrombin time (PT) and activated partial thromboplastin time (PTT)
assays of rabbit plasma were performed using standard techniques.
Innovin reagent (Dade, Miami, FL) was used for PT and Thrombosil I
(Hemoliance Ortho Diagnostic Systems Inc, Raritan, NJ) or
Actin FS (Dade) reagents were used for PTT. Bleeding time was measured
as described previously.25 Briefly, a shaved rabbit ear was
placed into a 37°C saline bath for 5 minutes. A full-thickness standardized incision was made with a Surgicutt pediatric device (International Technique Corp, Edison, NJ). The ear was then returned to the saline bath, observed until all blood flow ceased, and the time recorded.
Statistical analysis.
Statistical comparisons were performed using the 2-tailed Student's
t-test for means and 2-way analysis of variance for
dose-response. P values <.05 were considered significant. In
the experiments with ECM comparison between AR545C and nitroso-AR535C,
fragments for each dose were evaluated by 1-way analysis of variance
(ANOVA). Ex vivo dose effect of the 2 fragments was evaluated by 2-way analysis of variance.
The synergism of the 2 agents was tested using isobole method for
mutually exclusive and nonexclusive compounds as described previously.26 For mutually exclusive compounds the isobole
(I) representing the effect of the combination is calculated using the
following formula: I = (A/Ae) + (B/Be) = 1, where
Ae and Be are the corresponding
doses of the individual compounds producing the same quantitative
effect, and A and B are the doses of the compounds used
in combination showing the same effect. For mutually nonexclusive
drugs, the equation is changed to: I = (A/Am) + (B/Bm) + (A/Am) (B/Bm) = 1, where
Am and Bm represent the
concentrations yielding the median effect. I is less than 1 when the compounds interact to produce synergistic effect.
| |
RESULTS |
Chemical analysis of S-nitroso-AR545C.
Analysis of the formation of S-nitroso-AR535C performed by the method
of Saville21 showed that the S-nitrosothiol content was
0.96 mol of S-NO/1 mol of AR545C. UV-visible spectroscopy of
S-nitroso-AR545C compared with AR545C is presented in
Fig 1. The characteristic 350-nm absorption
peak of the S-nitrosothiol group of nitrosated AR545C is illustrated,
confirming that the peptide was nitrosated. The additional absorption
peak at 280 nm represents the protein content of the S-nitroso-AR545C.
Absorption spectra of AR545C shows only the 280-nm peak characteristic
for the peptide absorbance.
View larger version (14K):
[in this window]
[in a new window]
| Fig 1.
UV-Vis absorption spectra of S-nitroso-AR545C and AR545C.
Spectra were recorded against a blank containing phosphate-buffered
saline. Solid line indicates S-nitroso-AR545C; dashed
line indicates AR545C.
|
|
The putative structure of the S-nitrosated AR545C monomer is presented
in Fig 2. It seems reasonable that the
apparently free cysteine at residue 545 will be nitrosated; however,
additional cysteines of the molecule may be involved in the nitrosation
process as well.
View larger version (12K):
[in this window]
[in a new window]
| Fig 2.
Structure of S-nitroso-AR545C. Note that the first 3 cysteines are depicted as disulfide-linked to their corresponding
cysteines in a second monomer, only the partial structure of which is
shown, as S-nitrosation was performed with the intact dimer.
|
|
Effect of AR545C or S-nitroso-AR545C on ristocetin-induced platelet
agglutination.
Preincubation of human platelets with either AR545C or S-nitroso-AR545C
resulted in inhibition of ristocetin-induced platelet agglutination in
a dose-dependent manner. As shown in Fig
3, whereas 0.2 µmol/L AR545C decreased agglutination by
40%, the same concentration of S-nitroso-AR545C completely abolished
it. The concentration of AR545C required to inhibit ristocetin-induced
agglutination by 50% (IC50) was 0.1 ± 0.03 µmol/L,
whereas the IC50 for S-nitroso-AR545C was 0.02 ± 0.006 µmol/L (P = .001). Thus, S-nitrosation of AR545C inhibited
platelet agglutination approximately 5-fold.
View larger version (17K):
[in this window]
[in a new window]
| Fig 3.
Dose-dependent ristocetin-induced inhibition of human
platelet agglutination by S-nitroso-AR545C and AR545C. Formalin-fixed
platelets (2 × 108/mL) were incubated with either AR545C
( ) or S-nitroso-AR545C ( ) for 10 minutes at various
concentrations followed by addition of normal PPP and 1.5 mg/mL
ristocetin.
|
|
To test the effect of synergistic interactions between NO and AR545C,
subthreshold concentrations of NO congener (SNO-Glu) and AR545C used
alone or in combination with one another were added to PRP, and
ristocetin-induced platelet aggregation was recorded in aggregometer.
As shown in Fig 4, subthreshold
concentrations of SNO-Glu or AR545C alone did not inhibit platelet
aggregation. However, when the subthreshold concentrations of these
agents were used in combination, inhibition of aggregation was
obtained. Using an isobole method to establish synergy,26
the isobole index for mutually exclusive and nonexclusive compounds was
calculated for each combination. When combination of 0.08 µmol/L
AR545C with 0.2 µmol/L or 0.5 µmol/L of SNO-Glu was analyzed,
isobole indices of 0.57 and 0.81, respectively, were obtained.
Similarly, combination of 0.2 µmol/L AR545C with 0.2 µmol/L or 0.5 µmol/L SNO-Glu revealed isobole indices of 0.41 and 0.53, respectively. Thus, the result of this analysis showed that all the
isobole indices were below 1, indicating synergy.
View larger version (14K):
[in this window]
[in a new window]
| Fig 4.
Inhibition of human ristocetin-induced platelet
aggregation: Dose-response to combination of SNO-Glu and AR545C. Human
PRP (2.5 × 108/mL) was incubated with increasing
concentrations of AR545C followed by addition of 1.5 mg/mL ristocetin
in the absence () or presence of 2 concentrations of SNO-Glu: ( ),
0.2 µmol/L SNO-Glu; ( ), 0.5 µmol/L SNO-Glu. ( ), Increasing
concentrations of SNO-Glu in the absence of AR545C. Each point
represents mean ± SEM of 3 experiments.
|
|
Effect of AR545C or S-nitroso-AR545C on ADP-induced platelet
aggregation.
The effects of AR545C or S-nitroso-AR545C were first studied in
gel-filtered platelets and confirmed in PRP experiments. Results of
aggregation experiments are provided in Fig
5. AR545C did not affect platelet aggregation. In contrast,
dose-dependent inhibition of ADP-induced platelet aggregation was
observed with S-nitroso-AR545C, with an IC50 = 0.018 ± 0.002 µmol/L. Platelet aggregation was completely inhibited by
S-nitroso-AR545C at concentrations above 0.5 µmol/L. This inhibition
was reversed by addition of 5 µmol/L methylene blue, cGMP inhibitor
(Fig 5, ). NO congener SNO-Glu inhibited ADP-induced platelet
aggregation only at concentrations above 1 µmol/L (Fig 5, ).
View larger version (18K):
[in this window]
[in a new window]
| Fig 5.
Dose-dependent inhibition of human ADP-induced platelet
aggregation by S-nitroso-AR545C. Human PRP (2.5 × 108/mL)
were incubated with increasing concentrations of S-nitroso-AR545C
(), AR545C (), or SNO-Glu (), and aggregation was induced with
5 µmol/L ADP. (), Aggregations obtained after addition of
methylene blue (final concentration, 5 µmol/L) to the mixture of PRP
and S-nitroso-AR545C.
|
|
Effect of AR545C or S-nitroso-AR545C on platelet cGMP.
Normal PRP was incubated with 1.5 µmol/L SNO-Glu (positive control),
0.5 µmol/L AR545C, or 0.5 µmol/L S-nitroso-AR545C, the platelet
proteins were precipitated with TCA, and the protein-free supernatant
was assayed for cGMP content as described in Materials and Methods.
Compared with PRP alone (1.7 ± 0.4 pmol/109 platelets,
negative control), addition of AR545C did not significantly alter basal
cGMP level (2.4 ± 0.3 pmol/109 platelets) (P = .19) (Table 1). In contrast, there was a
significant increase in cGMP levels with the addition of SNO-Glu or
S-nitroso-AR545C (P < .0001 for each compound compared to
AR545C) (Table 1). Thus, the increases in platelet cGMP levels after
platelet exposure to SNO-Glu or S-nitroso-AR545C correlate with the
inhibition of platelet aggregation by the effect of NO molecules
provided by these compounds.
Effect of AR545C or S-nitroso-AR545C on platelet interaction with
ECM.
Normal whole blood tested in the cone-plate viscometer analysis system
exhibited a typical adhesion and aggregation pattern with surface
coverage of 28.4% ± 5.9% and an average size of the aggregates of
47.2 ± 15.2 µm2 (Table
2). The normal blood sample was then preincubated for 10 minutes at
room temperature with increasing concentrations of AR545C or
S-nitroso-AR545C. As shown in Table 2, a dose-dependent inhibition of adhesion (represented by surface coverage) and
aggregation (represented by average size of the aggregates) was
observed with either fragment compared to control. However, the
inhibitory effect of S-nitroso-AR545C was significantly more pronounced
at concentration above 1.0 µmol/L. A representative picture shown in
Fig 6 demonstrates that incubation of
normal blood (Fig 6A) with 1.5 µmol/L AR545C resulted in a 74%
decrease in adhesion and 64% inhibition in aggregate formation (Fig
6B). Similar concentration of S-nitroso-AR545C resulted in complete
inhibition of both adhesion and aggregate formation (Fig 6C). Analysis
of the frequency distribution of aggregate sizes shows that
S-nitroso-AR545C significantly shifted the size distribution leftward
compared with control blood and with AR545C (Fig 6D through F). Thus, a
more marked antiplatelet effect of S-nitroso-AR545C compared with
AR545C was observed in these experiments, both with respect to
inhibition of adhesion and aggregation under conditions of high shear.
View larger version (469K):
[in this window]
[in a new window]
| Fig 6.
The effect of the S-nitroso-AR545C or AR545C on platelet
interaction with ECM under flow conditions (shear rate of 1,300 s1). Citrated whole blood (0.25 mL) was tested in the
cone-plate viscometer analysis system after a 10-minute preincubation
with either control buffer, 1.5 µmol/L AR545C, or 1.5 µmol/L
S-nitroso-AR545C, and shown in representative field of the video screen
(A, B, and C, respectively). The corresponding frequency histograms (D,
E, and F, respectively) for the size distribution of the adhered
platelets and platelet aggregates are also depicted.
|
|
Effect of AR545C or S-nitroso-AR545C on botrocetin or ADP-induced
aggregation of rabbit platelets ex vivo.
Rabbits were injected with 1 mg/kg AR545C or 0.5 mg/kg S-nitroso-AR545C
(3 in each group), and the effect on ex vivo botrocetin or ADP-induced
rabbit platelet aggregation was monitored, as shown in
Figs 7 and 8.
In animals injected with AR545C, the aggregation induced by botrocetin
was significantly inhibited in a time-dependent manner, reaching a
maximal effect 45 minutes after injection. The inhibitory effect was
completely reversed by 2 hours after the injection. By contrast, in
animals injected with S-nitroso-AR545C at one half the dose of AR545C,
botrocetin-induced aggregation was completely abolished 45 minutes
after the injection and the inhibitory effect persisted, showing 60%
inhibition 2 hours after injection (Fig 7) (P < .0001 by
2-way ANOVA). AR545C had no effect on ADP-induced platelet aggregation
(data not shown), but S-nitroso-AR545C exhibited time-dependent
inhibition of platelet aggregation, reaching a maximal effect (almost
60% inhibition) at 1 hour after the injection and persisting for 1 additional hour (Fig 8).
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
Effect of S-nitroso-AR545C or AR545C on rabbit platelet
aggregation ex vivo. Female rabbits were injected with 0.5 mg/kg of
S-nitroso-AR545C or 1 mg/kg AR545C intravenously (3 in each group).
Blood samples were drawn before and at different time intervals after
the injection. Platelet aggregation was induced ex vivo with 1 µg/mL
botrocetin added to PRP prepared from animals treated with AR545C ()
or S-nitroso-AR545C () and plotted relative to the pretreatment
values. Results are expressed as mean ± SEM values for n = 3 animals in each group.
|
|
View larger version (57K):
[in this window]
[in a new window]
| Fig 8.
Effect of S-nitroso-AR545C on rabbit platelet aggregation
ex vivo. Female rabbits were injected with 0.5 mg/kg S-nitroso-AR545C
and blood samples were collected before and at different time intervals
after the injection. Platelet aggregation was induced in PRP with 5 µmol/L ADP. Results are expressed as mean ± SEM for n = 3 animals.
|
|
Effect of AR545C or S-nitroso-AR545C on hemostatic parameters in
rabbits.
The hemostatic parameters measured before and at different time
intervals after the injections of AR545C or S-nitroso-AR545C are
presented in Table 3. No change in the
platelet count, PT, or PTT values was observed. However, prolongation
of the bleeding time was noted in both groups. Twofold prolongation of
bleeding time was observed 1 hour after injection in the group treated with AR545C that normalized after 2 hours. The prolongation of bleeding
time was significantly greater in the group treated with S-nitroso-AR545C, increasing almost 8-fold compared with the
pretreatment value. The bleeding time shortened by the end of the
experiment but was still prolonged at 2 hours after injection.
| |
DISCUSSION |
Several agents have been shown to block the interaction between vWF and
its platelet receptor GPIb.8-12 Some of these compounds, such as monoclonal antibodies,5,9 synthetic
peptides,27 and recombinant vWF fragments,12,13
have been tested in various models of experimental thrombosis. In
addition, it has previously been shown that NO or its
S-nitroso-congeners exhibit antiplatelet properties by inhibiting
platelet aggregation14,15 and adhesion.16
In our previous study,13 we showed that recombinant vWF
fragment AR545C inhibited ristocetin- and botrocetin-induced platelet aggregation of human and rabbit platelets, respectively. AR545C also
enhanced the thrombolytic effect of recombinant tissue-type plasminogen
activator in a rabbit thrombosis model. In the present study, we
evaluated the antiplatelet properties of the S-nitroso-derivative of
AR545C, which should combine both antiadhesive actions with antiaggregating actions in the same molecule and target the delivery of
NO to the site of vascular injury.
Our data show that S-nitroso-AR545C potentiated the antiplatelet
effects of AR545C in the 3 systems studied: ristocetin- or botrocetin-induced platelet aggregation in vitro or ex vivo,
ADP-induced platelet aggregation, and interaction of platelets with ECM
under conditions of high shear. The superior antiplatelet effect of S-nitroso-AR545C can be attributed to the independent action of 2 moieties of the molecule: blocking platelet GPIb receptor through the
AR545C moiety and elevating platelet cGMP through the -SNO moiety.
Moreover, the data showed that these 2 effects are synergistic.
S-nitroso-AR545C inhibited ristocetin-induced platelet agglutination in
a dose-dependent manner with an average IC50 = 0.02 ± 0.006 µmol/L; this concentration is one fifth of that for
AR545C.13 Similarly, in the cone-plate viscometer analysis
system, a dose-dependent inhibition of adhesion and aggregation on ECM
was more notable at each concentration of S-nitroso-AR545C compared
with AR545C, reaching a statistically significant difference at
concentrations greater than 1.0 µmol/L (P < .05 by ANOVA).
Moreover, 1.0 µmol/L of S-nitroso-AR545C resulted in complete
inhibition of aggregate formation, whereas the same concentration of
R545C had only a slight effect on aggregate formation.
As expected, AR545C showed no effect on ADP-induced platelet
aggregation, because its effects are a consequence of competition with
vWF for binding to GPIb. By contrast, a significant dose-dependent inhibition of ADP-induced platelet aggregation was observed with S-nitroso-AR545C. S-nitroso-AR545C abolished platelet aggregation completely at concentrations greater than 0.5 µmol/L, and the IC50 was 0.018 ± 0.002 µmol/L. Finally, the ex vivo
efficacy of S-nitroso-AR545C was examined in rabbits. At concentrations
one half that of AR545C, S-nitroso-AR545C was able to inhibit
significantly botrocetin-induced platelet aggregation to a much greater
extent and for a longer period of time than AR545C itself, probably
reflecting the synergistic effect of NO with the vWF fragment. In
addition, ADP-induced platelet aggregation was inhibited by
S-nitroso-AR545C by almost 60%. Taken together, the potencies of the
S-nitroso-AR545C in vitro and ex vivo appear to be significantly
greater (3- to 10-fold and 4-fold, respectively) than those of AR545C.
The doses of AR545C or S-nitroso-AR545C used in this study had no
effect on platelet count or plasma coagulation tests (PT, PTT);
however, bleeding time was significantly prolonged, especially when
rabbits were injected with the S-nitroso-AR545C, apparently because of
its dual inhibitory effects on platelet function. Despite the
prolongation in bleeding time, no bleeding was observed during the
experiment or at necropsy of the animals. The prolongation of bleeding
time observed in our study contrasts with the lack of effect on
bleeding time reported by Azzam et al12 after injection of
VCL in guinea pigs. This difference may stem from differences in the
model or the compound used.
Most drugs interfering with platelet function exhibit a single
antiplatelet action, such as inhibition of adhesion or of aggregation. In contrast, S-nitroso-AR545C manifests 2 independent but synergistic antiplatelet activities combined in the same molecule. This compound likely has 2 pharmacologically relevant mechanisms in target cells: it
may act through interference of platelet binding to vWF and through
NO-mediated intracellular soluble guanylyl-cyclase activation. The data
from this study show that S-nitroso-AR545C exhibits significantly more
potent antiplatelet activity than AR545C, and this effect seems to be
attributed to its independent actions via GPIb- and NO-dependent pathways.
| |
ACKNOWLEDGMENT |
We thank Stephanie Tribuna and Ann Ward Scribner for excellent
technical assistance. We are indebted to David Castel, DVM, for his
assistance in experiments with rabbits.
| |
FOOTNOTES |
Submitted October 26, 1998; accepted May 10, 1999.
Supported in part by National Institutes of Health (NIH) Grants No.
HL53919, HL48743, HL53993, and by a Merit Review Award from the US
Veterans Administration.
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.
Address reprint requests to Aida Inbal, MD, Institute of Thrombosis & Hemostasis, Sheba Medical Center, Tel-Hashomer 52621, Israel.
| |
REFERENCES |
1.
Sadler JE:
von Willebrand factor.
J Biol Chem
266:22777, 1991[Free Full Text]
2.
Ruggeri ZM, Zimmerman TS:
von Willebrand factor and von Willebrand disease.
Blood
70:895, 1987[Abstract/Free Full Text]
3.
Fuster V, Bowie EW, Lewis JC, Fass DN, Owen CA Jr, Brown AL:
Resistance to arteriosclerosis in pigs with von Willebrand disease. Spontaneous and high cholesterol diet-induced arteriosclerosis.
J Clin Invest
61:722, 1978
4.
Nichols TC, Bellinger DA, Johnson TA, Lamb MA, Griggs TR:
von Willebrand's disease prevents occlusive thrombosis in stenosed and injured porcine coronary arteries.
Circ Res
59:15, 1986[Abstract/Free Full Text]
5.
Bellinger DA, Nichols TC, Read MS, Reddick RL, Lamb MA, Brinkhous KM, Evatt BL, Griggs TR:
Prevention of occlusive coronary artery thrombosis by a murine monoclonal antibody to porcine von Willebrand factor.
Proc Natl Acad Sci USA
84:8100, 1987[Abstract/Free Full Text]
6.
Ikeda Y, Handa M, Kawano K, Kamata T, Murata M, Araki Y, Anbo H, Kawai Y, Watanabe K, Itagaki I, Sakai K, Ruggeri ZM:
The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress.
J Clin Invest
87:1234, 1991
7.
Andrews RK, Gorman JJ, Booth WJ, Corino GL, Castaldi PA, Berndt MC:
Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis (sulfosuccinimidyl) suberate.
Biochemistry
28:8326, 1989[Medline]
[Order article via Infotrieve]
8.
Fujimura Y, Titani K, Holland LZ, Russell SR, Roberts JR, Elder JH, Ruggeri ZM, Zimmerman TS:
von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib.
J Biol Chem
261:381, 1986[Abstract/Free Full Text]
9.
Cadroy Y, Hanson SR, Kelly AB, Marzec UM, Evatt BL, Kunicki TJ, Montgomery RR, Harker LA:
Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates.
Blood
83:3218, 1994[Abstract/Free Full Text]
10.
Golino P, Ragni M, Cirillo P, Pascucci I, Ezekowitz MD, Pawashe A, Scognamiglio A, Pace L, Guarino A, Chiariello M:
Aurintricarboxylic acid reduces platelet deposition in stenosed and endothelially injured rabbit carotid arteries more effectively than other antiplatelet interventions.
Thromb Haemost
74:974, 1995[Medline]
[Order article via Infotrieve]
11.
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]
12.
Azzam K, Garfinkel LI, Bal dit-Sollier C, Cisse Thiam M, Drouet L:
Antithrombotic effect of a recombinant von Willebrand factor, VCL, on nitrogen laser-induced thrombus formation in guinea pig mesenteric arteries.
Thromb Haemost
73:318, 1995[Medline]
[Order article via Infotrieve]
13.
Gurevitz O, Goldfarb A, Hod H, Feldman M, Shenkman B, Varon D, Eldar M, Inbal A:
Recombinant von Willebrand factor fragment AR545C inhibits platelet aggregation and enhances thrombolysis with rtPA in a rabbit thrombosis model.
Arterioscler Thromb Vasc Biol
18:200, 1998[Abstract/Free Full Text]
14.
Stamler J, Mendelsohn ME, Amarante P, Smick D, Andon N, Davies PF, Cooke JP, Loscalzo J:
N-acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor.
Circ Res
65:789, 1989[Abstract/Free Full Text]
15.
Cooke JP, Stamler J, Andon N, Davies PF, McKinley G, Loscalzo J:
Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol.
Am J Physiol
259:H804, 1990[Abstract/Free Full Text]
16.
de Graaf JC, Banga JD, Moncada S, Palmer RMJ, de Groot PG, Sixma JJ:
Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions.
Circulation
85:2284, 1992[Abstract/Free Full Text]
17.
Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, Singel DJ, Loscalzo J:
S-nitrosylation of proteins with nitric oxide: Synthesis and characterization of biologically active compounds.
Proc Natl Acad Sci USA
89:444, 1992[Abstract/Free Full Text]
18.
Ruggeri ZM:
von Willebrand factor as a target for antithrombotic intervention.
Circulation
86:26, 1992 (suppl 3)
19.
Zhang Y-Y, Xu A-M, Nomen M, Walsh M, Keaney JF Jr, Loscalzo J:
Nitrosation of tryptophan residue(s) in serum albumin and model dipeptides. Biochemical characterization and bioactivity.
J Biol Chem
271:14271, 1996[Abstract/Free Full Text]
20.
Mendelsohn ME, O'Neill S, George D, Loscalzo J:
Inhibition of fibrinogen binding to human platelets by S-nitroso-N-acetylcysteine.
J Biol Chem
265:19028, 1990[Abstract/Free Full Text]
21.
Saville B:
A scheme for the colorimetric determination of microgram amounts of thiols.
Analyst
83:670, 1958
22.
Hawiger J, Parkinson S, Timmons S:
Prostacyclin inhibits mobilisation of fibrinogen-binding sites on human ADP- and thrombin-treated platelets.
Nature
283:195, 1980[Medline]
[Order article via Infotrieve]
23.
Butt E, Walter U:
Cyclic nucleotides: Measurement and function, in
Watson SP,
Authi KS
(eds):
Platelets. A Practical Approach. Oxford, UK, Oxford University Press, 1996, p 259.
24.
Varon D, Dardik R, Shenkman B, Kotev-Emeth S, Farzame N, Tamarin I, Savion N:
A new method for quantitative analysis of whole blood platelet interaction with extracellular matrix under flow conditions.
Thromb Res
85:283, 1997[Medline]
[Order article via Infotrieve]
25.
Johnstone MT, Andrews T, Ware JA, Rudd MA, George D, Weinstein M, Loscalzo J:
Bleeding time prolongation with streptokinase and its reversal with 1-desamino-8-D-arginine vasopressin.
Circulation
82:2142, 1990[Abstract/Free Full Text]
26.
Collen D:
Synergism of thrombolytic agents: Investigational procedures and clinical potential.
Circulation
77:731, 1988[Free Full Text]
27.
Mohri H, Zimmerman TS, Ruggeri ZM:
Synthetic peptides inhibit the interaction of von Willebrand factor-platelet membrane proteins.
Peptides
14:125, 1993[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
J. Loscalzo
Nitric Oxide Insufficiency, Platelet Activation, and Arterial Thrombosis
Circ. Res.,
April 27, 2001;
88(8):
756 - 762.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION