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© 2001 American Heart Association, Inc.
Thrombosis |
Comparative Antiplatelet Efficacy of a Novel, Nonpeptide GPIIb/IIIa Antagonist (XV454) and Abciximab (c7E3) in Flow Models of Thrombosis
From the Department of Chemical Engineering (J.P.A., N.T., O.J.T.M., D.P., K.K.), Johns Hopkins University, Baltimore, Md, and DuPont Pharmaceuticals Co (S.A.M.), Wilmington, Del.
Correspondence to Konstantinos Konstantopoulos, PhD, Department of Chemical Engineering, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218-2694. E-mail konst_k{at}jhu.edu
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Abstract |
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Abstract—Glycoprotein (GP) IIb/IIIa is pivotal in homotypic platelet aggregation and may also be involved in the heterotypic adhesion of leukocytes and tumor cells to platelets. This study was primarily undertaken to compare the antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist, XV454, to that of abciximab in 2 flow models of platelet thrombus formation: (1) direct shear-induced platelet aggregation imposed by a cone-and-plate rheometer and (2) platelet adhesion onto von Willebrand factor (vWF)/collagen I followed by aggregation in a perfusion system. XV454 inhibited platelet aggregation in a concentration-dependent manner in both experimental models. Maximal inhibition of aggregation was achieved by XV454 at
70% receptor occupancy, which is lower than the 
85% previously
reported for abciximab. At similar levels of receptor blockade
(45%), XV454 appeared to be relatively more effective than
abciximab in suppressing platelet aggregation. Neither XV454 nor
abciximab inhibited platelet adhesion to collagen. Pretreatment of
surface-adherent platelets with either XV454 or abciximab inhibited
the attachment of monocytic THP-1 cells under flow. In contrast, the
rapidly reversible GPIIb/IIIa inhibitor orbofiban failed to
suppress these heterotypic interactions. These findings demonstrate
that XV454 is a potent GPIIb/IIIa antagonist with a long
receptor-bound lifetime like abciximab and may be beneficial for the
treatment/prevention of thrombotic complications.
Key Words: platelets • adhesion • aggregation • shear stress • XV454, abciximab
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Introduction |
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Intravascular thrombosis is 1 of the most frequent pathological events and a major cause of morbidity and mortality in Western countries. Critical steps in the development of acute coronary syndromes are the disruption or erosion of atherosclerotic plaque and the formation of partially or completely occlusive thrombi. Arterial platelet thrombus formation may be initiated by platelet adhesion from rapidly flowing blood onto exposed subendothelial surfaces of injured vessels containing collagen and von Willebrand factor (vWF), with subsequent platelet activation and aggregation. In particular, platelet adhesion to immobilized, exposed vWF associated with collagen I under simulated abnormal arterial flow conditions is mediated by the platelet glycoprotein (GP) Ib/IX complex.1 2 This adhesion step is followed by aggregation predominantly mediated by the binding of fibrinogen and vWF to GPIIb/IIIa in the presence of ADP.1 2 3 4 In addition, direct platelet aggregation in the bulk phase under conditions of abnormally elevated fluid shear stresses, analogous to those occurring in atherosclerotic or constricted arterial vessels,5 may be important. Shear-induced platelet aggregation is dependent on the availability of vWF and the presence of both GPIb/IX and GPIIb/IIIa on the platelet membrane. It has been postulated that at high shear stress conditions, the interaction of vWF with the GPIb/IX complex is the initial event leading to platelet activation, which also triggers the binding of vWF to GPIIb/IIIa to induce platelet aggregate formation.6 7 Platelet activation and aggregation have been implicated in a number of vascular diseases such as unstable angina, myocardial infarction, transient ischemic attack, and stroke.8 9 Thus, pharmacological modulation of platelet function is of clinical importance.
The final common step in homotypic platelet aggregation, regardless of the stimulus, involves the interaction of adhesive proteins such as fibrinogen and vWF with platelet GPIIb/IIIa. Several studies have identified the pivotal role of GPIIb/IIIa receptors in coronary thrombosis. Hence, this platelet integrin receptor has emerged as a rational therapeutic target in the management of acute coronary syndromes.10 Various large-scale phase III clinical trials have illustrated the usefulness of GPIIb/IIIa antagonists in the treatment and prevention of acute ischemic syndromes. Intravenous administration of abciximab (c7E3) in high-risk patients undergoing angioplasty has been shown to reduce the composite incidence of major ischemic events at the 30-day primary end point.10 In other clinical studies, abciximab demonstrated efficacy when given in combination with thrombolytic therapy and in refractory unstable angina patients before angioplasty.11 12 13 Additionally, several other selective GPIIb/IIIa antagonists, including eptifibatide (Integrilin, Cor Therapeutics), tirofiban, and lamifiban, are in advanced stages of clinical development and aimed primarily for intravenous use in the treatment and prevention of acute ischemic heart diseases.13 14 15 Current intravenously administered small-molecule GPIIb/IIIa antagonists, such as Integrilin or tirofiban, in clinical trials have a faster rate of dissociation from human platelets, reflecting their short duration of antiplatelet effects compared with that of abciximab.3 16 17 Sustaining antiplatelet efficacy levels with intravenous GPIIb/IIIa antagonists can be achieved with intravenous bolus and infusion regimens. Clinical studies with orally active, rapidly reversible, small-molecule GPIIb/IIIa antagonists, including orbofiban, xemilofiban, sibrafiban, and lefradafiban, have demonstrated variable antiplatelet activity in humans on administration 2 or 3 times per day.16 18 19 20 21 These factors prompted us to develop a potent, highly specific GPIIb/IIIa antagonist, XV454,22 with a relatively slow platelet dissociation rate for the treatment of different thromboembolic disorders.
The primary goal of this study was to assess the antiplatelet efficacy of XV454 in in vitro flow models of platelet thrombus formation and compare it with that of abciximab. A rheometric/flow cytometric method was used to monitor direct shear-induced platelet aggregation in the bulk phase.17 23 24 In addition, we combined a parallel-plate perfusion chamber with a computerized epifluorescence video microscopy system to visualize in real time and quantify the adhesion and subsequent aggregation of human platelets in whole blood flowing under conditions of abnormally elevated shear stress (1500 s-1) over type I fibrillar collagen.3 4
GPIIb/IIIa may also be involved in the heterotypic adhesion of leukocytes and tumor cells to platelets.25 26 27 These cell-cell interactions have been implicated in the pathogenesis and/or progression of inflammatory, metastatic, and thrombotic disorders.25 27 Consequently, we evaluated the effects of GPIIb/IIIa antagonists on the attachment of monocytic THP-1 cells to surface-adherent platelets under dynamic flow conditions (1.5 dyne/cm2), thus simulating events encountered at sites of vascular injury in postcapillary venules. In this in vitro flow assay, any unbound GPIIb/IIIa antagonist is removed by a brief washing step after platelet preincubation and before perfusion of the monocytic cells, thereby enabling us to distinguish agents with slow platelet off-rates such as XV454 and abciximab from those with relatively faster off-rates such as orbofiban.
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Methods |
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Reagents
XV454 and YZ202 (the active, free acid form of orbofiban) were synthesized at DuPont Pharmaceuticals Co. Abciximab was obtained from Centocor, Inc, and conjugated to FITC by standard techniques.24 The fluorescein-to-protein molar ratio was
2. XL086-FITC was
prepared by reaction of cyclic
[D-Lys-N2-methyl-L-arginylglycyl-L-aspartyl-3-(aminomethylbenzoic
acid)] with FITC and purified by reverse-phase
high-performance liquid
chromatography.28
XL086 has 1 fluorescein coupled at the
D-Lys
position.28 CD42a
(anti-GPIX)-FITC and CD42b (anti-GPIb)-FITC were purchased from Becton
Dickinson and Pharmigen, respectively.
Blood Collection
Venous blood was collected from healthy, aspirin-free
volunteer donors into polypropylene syringes containing either porcine
heparin (Elkins-Sinn Inc; heparin sodium, 10 U/mL final
concentration)1 29
or sodium citrate (0.38% final concentration). Specimens were
transferred to plastic tubes, stored at room temperature, and used
within 4 hours of collection in flow assays.
Rheometric/Flow Cytometric Experiments
Citrated blood in the presence or absence of
GPIIb/IIIa antagonists was subjected to a pathological
shear rate of 4000 s-1 for 60 seconds by
the use of a cone-and-plate rheometer (RS150, Haake). Controls included
blood specimens that had been introduced into the rheometer but were
not exposed to shear stress. Aliquots of control and sheared blood were
immediately fixed with 1% formaldehyde in Dulbecco’s phosphate
buffered saline (PBS, Sigma) and incubated with saturating
concentrations of CD42-FITC for 30 minutes in the dark. Subsequently,
specimens were diluted with 2 mL of 1% formaldehyde and
analyzed in a FACScan (Becton-Dickinson) flow cytometer
as described elsewhere in
detail.17 23 24
Binding of fluoresceinated CD42b (anti-GPIb), XL086 (an analogue of XV454),28 or abciximab to platelets was calculated by measuring the mean FITC fluorescence intensity of at least 5000 platelets, which were identified on the basis of their characteristic forward- and side-scatter profiles in citrated blood specimens in the presence or absence of GPIIb/IIIa antagonists. At each experimental state, the FITC fluorescence values were normalized by using as a reference the pre-XV454/preabciximab incubation (control specimen) fluorescence levels. Nonspecific XL086 binding was determined by the addition of a 5-fold excess of XV454 and accounted for <15% of the total binding.28 Prior work has demonstrated that binding of fluoresceinated monoclonal antibodies (CD42b or abciximab) or cyclic RGD peptide (XL086) detected by flow cytometry and expressed as mean fluorescence intensity is highly correlated with that detected by a radiometric method.28 Because treatment of blood specimens with XV454 or abciximab inhibits the subsequent binding of XL086-FITC or abciximab-FITC, respectively, to platelets, the decrease in FITC fluorescence values after drug incubation is correlated with the percentage of GPIIb/IIIa receptors occupied by the drug.17 28
Preparation of Collagen-Coated Surfaces
Collagen suspensions of type I acid-insoluble fibrils
(1 mg/mL final concentration) were prepared from bovine Achilles’
tendon (Sigma) in 0.5 mol/L acetic acid, pH
2.8.30 Glass coverslips
(24x50 mm, Corning) were coated with 200 µL of fibrillar
collagen (coated area=12.7x23
mm2)3 4
and placed in a humid environment for 45 minutes, and the excess
collagen was rinsed off with 10 mL of Dulbecco’s PBS before assembly
into the flow chamber. The collagen density on the glass surfaces was
estimated to be 2
µg/cm2.29
Perfusion Studies
Platelet deposition onto collagen was quantified
under dynamic flow conditions by using a parallel-plate flow chamber. A
coverslip coated with type I collagen was assembled in a chamber (205
µm channel depth, 1.26 cm channel width) and mounted on the stage of
an inverted microscope (Nikon TE300) equipped with an
epifluorescence illumination attachment, an X60 fluor
objective, an X1 projection lens, and a silicon-intensified target
video camera (VE-1000, Dage) connected to a VCR and a TV monitor.
Heparinized blood containing the fluorescent dye mepacrine
(quinacrine dihydrochloride, Sigma) at a final concentration of 10
µmol/L3 4 29
was perfused through the chamber for 1 minute at a wall shear rate of
1500 s-1, thereby simulating flow
conditions in partially constricted
arteries.5 The accumulation
of mepacrine-labeled platelets to collagen was monitored in real
time and recorded on videotape. Before the perfusion experiments,
blood was incubated with either XV454, abciximab, or vehicle (control)
at 37°C for 10 minutes. The microscope stage, flow chamber, and blood
were maintained at 37°C by an incubator heating module and incubator
enclosure during the experiment.
Evaluation of Platelet Adhesion and
Subsequent Platelet Aggregation
Images from videotapes were digitized by using a
Scion frame grabber and a personal computer and processed with the use
of OPTIMAS image processing software (Agris-Schoen Vision Systems).
Images were analyzed by computer at 5, 15, and 60 seconds for
each perfusion experiment. The number of adherent individual
platelets in the microscopic field of view
(3.2x104 µm2)
during the initial 15 seconds of flow was used as a measure of
platelet adhesion that initiates platelet thrombus
formation.3 4 The
number of platelets in each individual thrombus was calculated as
the total thrombus intensity (areaxaverage intensity) multiplied by a
value determined by dividing the number of single platelets in the
5-second images by the sum of the total intensities of these single
platelets.3 4
The extent of platelet aggregation (platelet-platelet
cohesion) that occurred subsequent to platelet adhesion was
represented by the number of platelets in each
individual thrombus over the perfusion period of 1
minute.
Preparation of Immobilized
Platelet Layers
Platelet-rich plasma was prepared by
centrifugation of citrated whole blood at
160g for 15 minutes. The
platelet count was adjusted to 2x108
per milliliter before being bound to
3-aminopropyltriethoxysilane-treated glass slides for 60
minutes.26 Under these
conditions, a confluent layer of platelets (17 500
platelets/mm2) was formed, as evaluated
by light microscopy for each
experiment.26 Nonspecific
binding was blocked with 0.1% bovine serum albumin for 10
minutes at 37°C.
THP-1–Platelet Adhesion
Assays
The monocytic THP-1 cell line was obtained from the
American Type Culture Collection (Manassas, Va) and cultured in the
recommended medium. THP-1 cells were washed once, resuspended in
serum-free media containing 0.1% bovine serum albumin at a
concentration of 106 cells/mL, and used
within 4 hours in the adhesion flow assay.
THP-1 cell adhesion to immobilized platelets
was visualized by phase-contrast video microscopy (Nikon TE200
microscope and a CCD100 camera
[Dage-MTI]).26
Surface-adherent platelets were then incubated with thrombin (1
U/mL) for 10 minutes at 37°C. After the platelet layer was washed
with Dulbecco’s PBS/0.1% bovine serum albumin for 2
minutes, THP-1 cells were perfused through the chamber for 3 minutes at
a wall shear stress of 1.5 dyne/cm2, thereby
mimicking the fluid-mechanical environment of the postcapillary
venules.5 A single field of
view (x20, 0.20 mm2) was monitored
during the 3 minutes of the experiment, and at the end, 5 fields of
view (x20) were monitored for 15 seconds each. Three
parameters were quantified in the
analysis26 : (1) the
number of total interacting cells during the entire 3-minute
experiment; (2) the number of firmly adherent cells after 3 minutes of
shear flow; and (3) the average rolling velocity. Interacting
cells were defined as those that tethered to the platelet layer for
at least 2 seconds and included both firmly adherent and rolling cells.
Their number was determined manually by reviewing the videotapes.
Firmly adherent cells were considered those that remained stationary
for at least 10 seconds at the end of the 3-minutes run, as evidenced
by image processing. Rolling velocity was computed as the distance
traveled by the centroid of the THP-1 cell divided by the time interval
by using OPTIMAS 6.5. For inhibition studies, surface-adherent
platelets were preincubated with abciximab (3.5 µg/mL), XV454
(100 nmol/L), or orbofiban (100 nmol/L) for 10 minutes during the
thrombin incubation.
Statistical Analysis
The data are expressed as mean±SEM. Statistical
significance of differences between means was determined by
single-factor ANOVA. When means were shown to be significantly
different, multiple comparisons by pairs were performed by Tukey’s
test. Probability values <0.05 were selected to indicate statistical
significance.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Shear-Induced Platelet Aggregation: Rheometric/Flow Cytometric Studies
XV454 inhibited, in a concentration-dependent manner, shear-induced (4000 s-1 for 60 seconds) platelet aggregation in citrate-anticoagulated blood specimens (Figure 1A
). Maximal inhibition of platelet aggregation
was achieved by XV454 at 50% to 75% platelet GPIIb/IIIa receptor
occupancy
(Figures 1A and 1B). High concentrations of XV454 (50 to 100
nmol/L) demonstrated similar potency in suppressing direct
shear-induced platelet aggregation to that of abciximab when used
at a concentration of 3.5 µg/mL (76 nmol/L)
(Figure 1A). This abciximab concentration corresponds to the
theoretical maximal blood level produced by injecting the recommended
dose of 0.25 mg/kg into a patient with a blood volume of 72
mL/kg31 and resulted in
97% receptor blockade in all donors
(Figure 1B). The extensive GPIIb/IIIa receptor blockade
caused by either abciximab or XV454 did not interfere with the binding
of anti-GPIb monoclonal antibody on the platelet surface
(Table 1).
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We next evaluated the efficacy of both XV454 and abciximab
in inhibiting shear-induced platelet aggregation when similar
levels of receptor blockade were achieved by both drugs in vitro. At a
45% GPIIb/IIIa receptor occupancy
(Figure 1B
), XV454 (20 nmol/L) demonstrated a relatively
higher potency than abciximab (0.6 µg/mL, or 13 nmol/L) in inhibiting
platelet aggregation induced by high shear in the absence of any
exogenously added chemical agonist
(Figure 1A).
Perfusion Experiments
Heparinized whole blood from healthy donors, treated in
vitro with XV454, abciximab, or vehicle, was perfused at 1500
s-1 for 1 minute over collagen I. The
resulting platelet adhesion, measured by the number of adherent
platelet thrombi on the collagen I surface after 15 seconds of
flow,3 4 is shown
in
Table 1. The data indicate that there was no significant
change in the extent of GPIb-mediated platelet adhesion to collagen
I/vWF between GPIIb/IIIa antagonist-treated and control
(vehicle-treated) blood specimens at all concentrations tested. This
finding is in agreement with flow cytometry results showing that the
GPIb receptors remained available for binding of vWF, as probed by an
anti-GPIb monoclonal antibody
(Table 1).
As individual platelet thrombi merge into larger clumps
in control specimens after 60 seconds of flow, the number of thrombi
decreases concomitantly (data not
shown).3 4 The
platelet aggregation that occurs subsequent to platelet
adhesion to collagen I is mediated by vWF and fibrinogen binding to
platelet
GPIIb/IIIa.1 3 4
At a shear rate of 1500 s-1, 18 444±1473
platelets were deposited on a
3.2x104-µm2
collagen I surface area after 60 seconds of blood perfusion
(Figure 2). Also at this time point, large thrombi containing
500 platelets accounted for 80% of all platelets on the
surface
(Table 2). Both GPIIb/IIIa antagonists, XV454
and abciximab, significantly reduced total platelet accumulation
and the number of platelets per thrombus (platelet aggregation)
in a concentration-dependent fashion
(Figures 2 and 3). High concentrations of either XV454 (100
nmol/L) or abciximab (3.5 µg/mL) essentially eliminated the formation
of platelet aggregates
(Table 2). At a similar GPIIb/IIIa receptor occupancy level,
XV454 (20 nmol/L) was relatively more effective than abciximab (0.6
µg/mL) in inhibiting platelet deposition onto a collagen type I
surface
(Figures 2 and 3).
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THP-1 Cell Attachment to
Immobilized Platelets Under Flow Conditions
In this flow assay, surface-anchored platelets were
pretreated with the GPIIb/IIIa antagonists for 10 minutes,
and any unbound drug was removed by a brief washing step (2 minutes)
before perfusion of the THP-1 monocytes. We therefore reasoned that
agents with slow platelet off-rates, such as XV454
(t1/2 of dissociation=110 minutes,
Kd=1
nmol/L)22 32 and
abciximab (t1/2 of dissociation=40 minutes,
Kd=9.0
nmol/L)22 32 that
are distributed predominantly as receptor-bound entities with little
unbound in the plasma,33
could effectively block these heterotypic interactions. In contrast,
agents with relatively fast platelet dissociation rates such as
orbofiban (t1/2 of dissociation=0.2 minutes,
Kd >110
nmol/L),32 whose
antiplatelet efficacy depends on the plasma concentration of the
active drug, would not exhibit any inhibitory effects.
Therefore, this assay could allow us to distinguish GPIIb/IIIa
antagonists with slow platelet off-rates from those
with relatively faster off-rates.
Our data indicate that immobilized platelets
supported extensive THP-1 cell adhesion (1525±483 interacting THP-1
cells/mm2, n=4). At a wall shear stress of
1.5 dyne/cm2, THP-1 cells tethered and
rolled stably along the platelet surface for some distance, which
was variable but sometimes quite long (the entire path of a field
of view), with an average velocity of 14.0±1.9 µm/s. A significant
number of interacting THP-1 cells became firmly adherent within a 1- to
5-second interval after tethering to the platelet substrate
(Figure 4). XV454 (100 nmol/L) and abciximab (3.5 µg/mL)
significantly reduced the stable adhesion of THP-1 cells to
immobilized platelets
(Figure 4) while simultaneously increasing the
number of rolling THP-1 cells (data not
shown).26 Furthermore,
blockade of the platelet GPIIb/IIIa receptor with either agent
significantly increased the rolling velocity of THP-1 cells (44.9±3.3
and 43.3±3.3 µm/s for XV454- and abciximab-treated platelets,
respectively). This effect is similar to that previously reported for
leukocyte rolling over stimulated endothelium in the
presence of blocking antibodies against
integrins.34 In distinct
contrast, the short-acting GPIIb/IIIa antagonist orbofiban
(100 nmol/L) failed to suppress these heterotypic adhesive interactions
(Figure 4) and to affect the average rolling velocity of
interacting THP-1 cells (17.8±2.3 µm/s). It should be noted that
orbofiban (100 nmol/L) is effective in eliminating the formation of
large platelet aggregates, defined as 300 platelets per
thrombus, onto collagen type I surfaces when this concentration (100
nmol/L) is maintained in the suspension medium during the perfusion
process (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study, we evaluated the efficacy of a novel, nonpeptide, small-molecule platelet GPIIb/IIIa antagonist, XV454, in inhibiting platelet responses under dynamic flow conditions. Two different in vitro models of thrombosis, cone-and-plate rheometry and perfusion, were tested. The rheometry model simulates the elevated levels of fluid shear stress generated in stenosed coronary arteries, which can directly cause platelet activation and aggregation in the absence of any exogenously added chemical agonist.6 17 The extent of shear-induced platelet aggregation is dependent on both the magnitude of shearing stress and shear exposure time.24 The perfusion system represents a model of blood flowing over injured, exposed arterial subendothelium and allows us to monitor and quantify both platelet adhesion and subsequent aggregation in real time in the presence of controlled levels of elevated shear stress.1 2 3 4 In both models, XV454 consistently inhibited platelet aggregation in a concentration-dependent fashion. The inhibition of aggregation was correlated with the percentage of GPIIb/IIIa receptor occupancy. Maximal inhibition was achieved by XV454 at
75%
GPIIb/IIIa receptor blockade, which appears to be lower than the 85%
occupancy required by
abciximab.4 10
Furthermore, when similar extents of GPIIb/IIIa receptor blockade were
achieved by XV454 and abciximab in vitro, XV454 appeared to be
relatively more effective than abciximab in suppressing platelet
aggregate formation. This finding is in concert with previous data
showing that XV459, a nonpeptide, small molecule similar to XV454, had
a relatively lower IC50 than abciximab in
reducing platelet aggregation in response to chemical agonist
stimulation.35
The 75% receptor occupancy achieved by 100 nmol/L XV454
was sufficient to eliminate platelet aggregate formation under
flowing conditions regardless of the choice of anticoagulant. Previous
work has demonstrated comparable IC50 values for
both XV459 and abciximab in inhibiting platelet aggregation
irrespective of the heparin or citrate
choice.22 This result is in
clear contrast to the significant shift in the
IC50 values of
Integrilin13 and orbofiban
in inhibiting platelet aggregation to a greater extent in citrate
(relatively lower IC50) than in heparin
(relatively higher IC50) collected blood, which
is due to the partial calcium-chelating effect of citrate that results
in artificial enhancement of ex vivo or in vitro Integrilin
antiplatelet efficacy.
Despite the extensive GPIIb/IIIa receptor blockade caused by high concentrations of either XV454 or abciximab, the GPIb receptors remained available for binding ligand, as evidenced by flow cytometry. This result is in accord with our perfusion experiments that showed that treatment of whole-blood specimens with either drug did not affect platelet adhesion onto a collagen I surface under conditions of abnormally high arterial flow, a situation that is entirely dependent on GPIb-vWF interaction.1 2
Evidence suggests that GPIIb/IIIa may also mediate leukocyte
attachment to immobilized platelets under
flow,27 although other
reports have failed to confirm this
finding.36 37
Nevertheless, GPIIb/IIIa has been consistently reported to play
a pivotal role in the adhesive interactions of platelets with a
variety of tumor cell lines, a process that appears to be critical to
the hematogenous dissemination of tumor
cells.25 26 Along
these lines, our data clearly demonstrate the involvement of GPIIb/IIIa
in the stable adhesion of monocytic THP-1 cells to surface-anchored
platelets under dynamic flow conditions. In this flow assay, any
unbound GPIIb/IIIa antagonist was removed by brief washing
(2 minutes) after platelet preincubation and before perfusion of
the monocytic THP-1 cells, thereby enabling us to distinguish agents
with markedly distinct affinities and receptor-bound lifetimes. We
reasoned that agents with slow platelet off-rates would effectively
block these heterotypic interactions, whereas rapidly reversible agents
would not exhibit any inhibitory effects. Our results
indeed show that XV454 and abciximab, but not orbofiban, significantly
inhibited THP-1 cell attachment to surface-bound platelets, a
finding that correlates with their respective platelet
off-rates.32 These
observations suggest a possible extended duration of the in vivo
antiplatelet efficacy of XV454 compared with that of other
short-acting, small-molecule GPIIb/IIIa
antagonists.
Nearly all GPIIb/IIIa antagonists, including abciximab, induce conformational changes within the GPIIb/IIIa complex that can be probed by using ligand-induced binding site antibody reagents.33 38 It has been suggested that ligand-induced binding site epitope expression may report a potential intrinsic activating property of certain GPIIb/IIIa antagonists and that this effect should be considered when evaluating these agents.38 39 However, the currently available clinical data do not indicate that GPIIb/IIIa antagonist–induced expression of ligand-induced binding site epitopes is associated with platelet stimulatory events in vivo,33 possibly because receptor-activating properties of GPIIb/IIIa antagonists may be masked by the sustained blockade of the receptor.
GPIIb/IIIa inhibitors, when administered at suboptimal doses, may transiently bind to the GPIIb/IIIa complex, induce the active conformational change, and allow increased fibrinogen binding to occur after their dissociation from the receptor.19 38 It was recently shown that oral administration of orbofiban enhanced platelet reactivity with respect to fibrinogen binding and P-selectin expression.19 Although in vitro studies suggest that low concentrations of certain GPIIb/IIIa antagonists with either slow or fast off-rates induce platelet-stimulatory events,38 it is likely that these responses are more pronounced for short-acting inhibitors whose antiplatelet efficacy depends on the plasma concentration of the active drug and rapidly declines after discontinuation of infusion. In contrast, drugs with slow off-rates such as XV454 and abciximab are distributed predominantly as receptor-bound entities, and their platelet-inhibitory effects can be measured for several days after drug administration has been terminated.17 Therefore, the number of GPIIb/IIIa receptors per platelet that are left in an activated state after drug dissociation occurs is likely to be lower for GPIIb/IIIa antagonists with slow dissociation rates than those with relatively faster off-rates. Consequently, an oral version of a high-affinity antagonist (low Kd) like XV454 might be expected to have a higher chance of success than rapidly reversible GPIIb/IIIa antagonists, although this aspect still lies in the future. However, the clinical benefits demonstrated by the long-acting agent abciximab and the short-acting inhibitors eptifibatide and tirofiban, which are known to induce ligand-induced binding site epitope expression, suggest that these agents, when administered at optimal doses and for the appropriate period of time, could effectively block platelet-stimulatory responses and mask any potential intrinsic receptor-activating properties. Taken altogether, we conclude that XV454 is a potent, small-molecule GPIIb/IIIa antagonist with a long receptor-bound lifetime like abciximab, and it may have a beneficial effect on the treatment and/or prevention of thrombotic disorders.
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Acknowledgments |
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This work was supported by a United Engineering Foundation Inc grant, the DuPont Educational fund, and a DuPont Young Professor Grant (K.K.). The authors wish to thank Dr Larry V. McIntire and Nancy Turner (Rice University, Houston, Tex) for the 3-dimensional representations of platelet adhesion and subsequent aggregation onto collagen I/vWF.
Received June 15, 2000; accepted October 4, 2000.
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