Excessive accumulation of platelets at sites of athero-

sclerotic plaque rupture is a crucial pathogenic event

that is responsible for the development of the acute
coronary syndromes, stroke and the ischaemic compli-
cations of peripheral vascular disease
1,2
. Progress in the
understanding of the central importance of platelets in
cardiovascular disease, combined with the development
of new classes of antiplatelet agents, has dramatically
altered the landscape in terms of the clinical manage-
ment of vascular diseases. Until recently, aspirin was the
only antiplatelet agent in widespread clinical use for the
prevention and treatment of cardiovascular disease.
Although it continues to remain the gold-standard
antiplatelet therapy, ADP-receptor antagonists and
phosphodiesterase (PDE) inhibitors are increasingly
recognized as important therapeutic options in high-
risk patient populations
3
. The recent emergence of
antagonists of the glycoprotein (GP) GPIIbIIIa (also
known as integrin IIb3) has heralded a new era in
antiplatelet therapy and provided clinical confirmation
of the importance of platelets in the acute and chronic
complications of various cardiovascular diseases. The
enthusiasm with which the GPIIbIIIa antagonists have
been embraced by the medical, pharmaceutical and
academic communities is indicative of their market
potential, and of the clinical desire for more effective
antithrombotic approaches. Despite the progress that
has been made in the development of antiplatelet thera-
pies, there is still a large unmet clinical need for new
treatments. On the basis of current estimates from the
American Heart Association, more than 60 million peo-
ple in the United States alone have one or more forms
of cardiovascular disease, and a high proportion of
these individuals are at increased risk of arterial throm-
bosis. There is, however, some cause for optimism,
particularly in light of the rapidly evolving under-
standing of the molecular events governing haemo-
stasis and thrombosis, and the plethora of potential
new therapeutic targets that are emerging.
An excellent review on the current status of anti-
platelet therapy has recently been published in this
journal
3
, and several extensive reviews on aspirin
4,5
,
thienopyridine antagonists
3,610
, PDE
1113
and GPIIbIIIa
antagonists
3,1416
have also been published. In the current
review, we will discuss the clinical requirement for more
effective antithrombotic therapies, and highlight limita-
tions of existing therapeutic strategies. We will also pro-
vide an update on recent advances in the understanding
of the role of platelets in thrombosis and the molecular
mechanisms involved in thrombus formation. Finally,
we will discuss important differences between physio-
logical haemostatic plug formation and pathological
ANTIPLATELET THERAPY:
IN SEARCH OF THE MAGIC BULLET
Shaun P. Jackson and Simone M. Schoenwaelder
The central importance of platelets in the development of arterial thrombosis and cardiovascular
disease is well established. No other single cell type is responsible for as much morbidity and
mortality as the platelet and, as a consequence, it represents a major target for therapeutic
intervention. The growing awareness of the importance of platelets is reflected in the increasing
number of patients receiving antiplatelet therapy, a trend that is likely to continue in the future.
There are, however, significant drawbacks with existing therapies, including issues related to
limited efficacy and safety. The discovery of a magic bullet that selectively targets pathological
thrombus formation without undermining haemostasis remains elusive, although recent progress
in unravelling the molecular events regulating thrombosis has provided promising new avenues
to solve this long-standing problem.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | OCTOBER 2003 | 1
The Australian Centre for
Blood Diseases,
Department of Medicine,
Monash University,
Arnold Street, Box Hill
Hospital, Box Hill,
Victoria 3128, Australia.
Correspondence to S.P.J.
e-mail: Shaun.Jackson@
med.monash.edu.au
doi:10.1038/nrd1198
BLEEDING TIME
The time taken for cessation of
bleeding from a standardized
injury, which is used to test for
platelet abnormalities (acquired
and congenital) and also for
von Willebrands disease.
THERAPEUTIC WINDOW
The dose (or concentration)
range between the minimum
effective dose (concentration)
and the minimum toxic dose
(concentration).
2 | OCTOBER 2003 | VOLUME 2 www.nature.com/reviews/drugdisc
R E V I E WS
Efficacy versus safety
The two most widely used oral antiplatelet drugs,
aspirin and clopidogrel, are generally well tolerated and
produce significant clinical benefit in a broad range of
patient populations. The major limitation of these drugs
is their relatively weak antithrombotic effect. For exam-
ple, extensive clinical studies have demonstrated that
despite reducing the incidence of acute myocardial
infarction and stroke by 34% and 25%, respectively,
aspirin only prevents vascular death in ~15% of
patients
17
. Clopidogrel is only slightly better, producing
a 9% risk reduction in serious vascular events relative to
aspirin
17,19
. It should be noted that in experimental
models, clopidogrel has considerably more potent
antithrombotic activity than aspirin when administered
at high concentrations. However, in most clinical stud-
ies, clopidogrel has been used at concentrations that
inhibit ADP-induced platelet aggregation by ~4050%,
as higher levels of receptor blockade lead to a marked
increase in BLEEDING TIME
20
.
The limited efficacy of aspirin and ADP-receptor
antagonists reflects the existence of alternative path-
ways for platelet activation (FIGS 2 and 3). Platelets are
exposed to a diverse range of activating stimuli at sites
of vascular injury, including adhesive proteins (for
example, fibrillar collagens, von Willebrand factor
(vWf ) and fibronectin) and soluble agonists (for
example, thrombin, ADP, thromboxane A
2
(TXA
2
) and
serotonin) that induce platelet activation through
well-defined receptor-coupled signalling pathways
21
(FIG. 3). Ultimately, these pathways converge on the
activation of GPIIbIIIa, the major platelet adhesion
receptor, which has a pivotal role in promoting
platelet aggregation and thrombus growth
2224
. This
major platelet receptor represents the final common
pathway for platelet aggregation, regardless of the pri-
mary thrombogenic stimulus, thereby providing the
rationale for the development of GPIIbIIIa inhibi-
tors. In extensive clinical studies, GPIIbIIIa inhibitors
have proven to be a powerful class of antiplatelet
agents and provide significant clinical benefit in
patients undergoing coronary interventions
25
. How-
ever, similarly to the thienopyridines (ticlopidine and
clopidogrel), the high level of receptor blockade
required to achieve a potent antithrombotic effect sig-
nificantly undermines haemostasis, resulting in an
increased risk of bleeding
26
.
The issue of improving efficacy at the expense of
safety is a significant problem that plagues all forms
of presently available antithrombotic therapy. In the
majority of patients with cardiovascular disease, com-
bination antithrombotic therapy is not appropriate
for chronic use. In particular, the combined use of
antiplatelet and anticoagulant agents has been consid-
ered taboo by many clinical centres and reserved only
for patients at very high risk of arterial thrombosis. This
contrasts with other forms of cardiovascular disease,
such as hypertension and cardiac failure, in which
multi-component therapy is common, because of the
availability of drugs that act in a synergistic manner and
which possess a wide THERAPEUTIC WINDOW.
thrombosis, highlighting potential future approaches
that might reduce pathological thrombus formation
while minimizing the impact on haemostasis.
Growing clinical need for antithrombotic therapy
A recent meta-analysis of more than 287 antithrombotic
clinical trials has demonstrated the benefit of antiplatelet
therapy in a diverse range of cardiovascular diseases
(TABLE 1)
17
. These include patients presenting acutely with
myocardial infarction, ischaemic stroke or unstable
angina, and also patients with a past history of previous
myocardial infarction or cerebrovascular ischaemic
events. Antiplatelet therapy has also proven beneficial for
primary prevention in patients with peripheral arterial
disease and atrial fibrillation, and reduces serious vascular
events in other high-risk individuals, including diabetics,
patients with end-stage renal disease requiring fistula or
shunt placement, and individuals with symptomatic
or asymptomatic carotid stenosis
17
.
The increasing use of antithrombotic therapy is
reflected by recent trends in global sales of these products.
Antithrombotics are the most rapidly growing sector of
the cardiovascular market, and sales of around US $9.1
billion in 2001 are forecast to grow to US $22 billion by
2007 (REF. 18). One of the principal factors underpinning
this growth is the rapid increase in sales of the antiplatelet
drug clopidogrel, which are presently increasing by an
average of 65% per year (FIG. 1). This dramatic growth
reflects the expansion of the clinical indications for which
the drug is approvedand a favourable safety profile rela-
tive to other antiplatelet agents, including ticlopidine.
Several factors are likely to result in further expan-
sion of the antithrombotic market, including the ageing
population with its attendant increase in vascular
pathology; the global increase in the incidence of
cardiovascular disease due to alterations in diet and
lifestyle; the increased recognition by clinicians of the
importance of antiplatelet therapy in the prevention
and treatment of cardiovascular diseases; the growing
trend of combining antiplatelet therapies in patients at
high risk of arterial thrombosis; and the increasing
number of patients requiring secondary thrombopro-
phylaxis as a result of improvements in survival from
acute myocardial infarction.
Table 1 | Conditions where antiplatelet drugs are or might be beneficial
Cardiovascular disease Prevalence Estimated costs (US $)*
Coronary artery disease 12.9 million 129.9 billion
Cerebrovascular disease 4.7 million 51.2 billion
Peripheral arterial disease 812 million 26.2 billion

(1999)
Diabetes mellitus 16.6 million
(Physician diagnosed) 10.9 million
(Undiagnosed

) 5.7 million
Atrial fibrillation
||
2.0 million 2.1 billion

(1998)
Congestive heart failure 4.9 million 24.3 billion
Statistics are for the United States only. *Estimated costs including health expenditure and lost
productivity resulting from morbidity and mortality.

Estimated hospital expenditure.

Undiagnosed
diabetes mellitus refers to a fasting blood glucose of 126 mg per dl or greater.
||
Majority of patients
receiving warfarin. Statistics taken from Heart Disease and Stroke Statistics 2003 the American
Heart Association (http://www.americanheart.org).
NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | OCTOBER 2003 | 3
R E V I E WS
paradoxically, induce a 35% relative increase in fatal
ischaemic events
2931
. Similarly, several studies of combi-
nation antiplatelet and anticoagulant therapies have
failed to demonstrate significant clinical benefit, due in
part to an increased risk of major bleeds
3234
. Bleeding
represents the most important factor influencing long-
term compliance and the overall clinical benefit of
antithrombotic therapy. The key concern regarding all
existing antithrombotic drugs relates to their therapeu-
tic window. Ideally, antithrombotic approaches should
target one or more processes that are crucial for patho-
logical thrombosis, but which are less important for
haemostasis. Despite intensive effort, major progress in
this area has been limited.
New insights into regulation of thrombus growth
Novel antithrombotic approaches are likely to evolve
from an improved understanding of the mechanisms
regulating thrombus formation. In this context, there
have been considerable recent advances, largely derived
from the development of novel experimental techniques
that enable the real-time analysis of arterial thrombus
formation in vivo
3540
. In particular, recent developments
in intravital imaging techniques have provided unprece-
dented insights into the complexity and dynamics of
thrombus formation. The application of this technology
to mouse models in which specific disruptions of one or
more haemostatic components have been engineered
has redefined our understanding of many basic aspects
of the thrombotic process (TABLES 2 and 3)
4198
.
Conceptual advances in the understanding of platelet
thrombus growth. The composition of a thrombus is
crucially dependent on the RHEOLOGICAL ENVIRONMENT,
with arterial thrombi typically being platelet-rich,
whereas venous thrombi are platelet-poor and primar-
ily consist of polymerized fibrin and red blood cells.
The central role of platelets in arterial thrombus for-
mation is a reflection of the unique ability of these cells
to form stable adhesion contacts with the damaged
vessel wall under conditions of rapid blood flow.
This capacity is largely attributable to the interaction
between vWf and its platelet receptor, the GPIb/V/IX
complex. The vWfGPIb interaction has unique bio-
mechanical properties that enable the initiation of
platelet adhesive interactions over a remarkably broad
range of haemodynamic conditions
99
. In fact, it is the
only known receptorligand interaction that can
mediate platelet interactions throughout all regions of
the vasculature, which explains its unique role in
haemostasis and thrombosis. Qualitative or quantita-
tive abnormalities of either vWf or the GPIb/V/IX
complex are associated with severe bleeding disor-
ders
47,58,59
, whereas dysregulated enhancement of this
interaction can lead to diffuse microvascular thrombosis,
resulting in life-threatening diseases such as THROMBOTIC
THROMBOCYTOPAENIC PURPURA (TTP)
100
.
Platelet adhesion to the vessel wall (primary adhe-
sion) and subsequent platelet aggregation (platelet
cohesion) have traditionally been viewed as separate
events, involving distinct adhesive ligandreceptor
Benefit/risk of combined antithrombotic therapy
Although aspirin reduces acute vascular events in
approximately 25% of patients with vascular disease, the
residual risk remains high, which justifies the need to
develop more effective antithrombotic agents or combi-
nation antiplatelet therapies. The success of short-term
combination antiplatelet therapies for patients under-
going percutaneous coronary intervention has fuelled
interest in the longer-term use of combined approaches.
The recent CURE study (Clopidogrel in Unstable
Angina to Prevent Recurrent Events)
27
showed a signifi-
cant advantage of combining aspirin with clopidogrel
versus aspirin alone (20% relative risk reduction in car-
diovascular death, nonfatal myocardial infarction and
stroke), in patients with unstable angina or suspected
myocardial infarction. As with most combination thera-
pies, this benefit was offset by a corresponding increase
in major bleeding (3.7% incidence in the clopidogrel
and aspirin group, versus 2.7% in the aspirin-alone
group; relative increase 38%). When the antithrombotic
benefit of combined therapy is offset by major bleeding
events, the overall clinical benefit could be as low as 8%.
Whether this combined approach will provide long-
term clinical benefit in other cardiovascular patient
groups remains to be established, although several
major Phase III clinical trials are presently evaluating
this approach
3
.
An important lesson that has emerged from numer-
ous antithrombotic trials is that increased antithrom-
botic potency per se does not necessarily guarantee
enhanced clinical benefit and, in general, potent
antithrombotic approaches must be reserved for high-
risk patient populations. This has been most clearly
demonstrated in clinical trials of the oral GPIIbIIIa
inhibitors, chronic administration of which, particularly
at high concentrations, produces an excess of bleeding
events (up to 70% incidence of minor bleeding and
12% major bleeds)
28
. Lower levels of platelet inhibition
produce a twofold increase in bleeding risk, although,
RHEOLOGICAL ENVIRONMENT
The blood-flow characteristics
operating within defined areas
of the vasculature.
THROMBOTIC THROMBO-
CYTOPAENIC PURPURA
A rare microvascular thrombotic
disorder associated with low
platelet count, fever,
neurological and renal
impairment and micro-
angiopathic haemolytic
anaemia.
2,000
1,500
99 00 01 99 00 01 99 00 01 99 00 01 99 00 01 99 00 01 99 00 01 99 00 01
1,000
500
0
Clopidogrel Ticlopidine Abciximab Tirofiban Eptifibatide Cilostazol Epoprostenol Beraprost
66.15% 4.3% 1.75% 46.65% 118.2% 10.5% 0% 1.75%
U
S

$

m
i
l
l
i
o
n
s
Year
Annual
growth
Thienopyridines
GPIIbIIIa antagonists
PDE inhibitors
Prostacyclin analogues
Figure 1 | Market for antiplatelet drugs, 19992001. Data taken from REF. 18. PDE,
phosphodiesterase.
4 | OCTOBER 2003 | VOLUME 2 www.nature.com/reviews/drugdisc
R E V I E WS
aggregation has changed dramatically during the past
few years. Platelet aggregation is now considered a com-
plex, dynamic process involving the cooperative adhe-
sive function of multiple adhesive ligands, including
vWf, fibrinogen and possibly fibronectin and soluble
CD40 ligand, as well as two adhesive receptors, GPIb
and GPIIbIIIa
36,3840,102114
(FIG. 4). The specific contribu-
tion of individual adhesive events to the aggregation
process is crucially dependent on the rheological con-
ditions, with the contribution of the vWfGPIb inter-
action becoming progressively more important with
increasing shear stress (see below). In fact, under high-
shear conditions, such as those typically found in small
arterioles or stenosed arteries, thrombus growth is
primarily mediated by vWf, with fibrinogen playing a
secondary role in stabilizing formed aggregates
39,113,114
.
Importance of fibrillar collagens in promoting thrombus
growth. Considerable progress has been made during
the past five years in unravelling the key molecular
events regulating platelet adhesion and thrombus
growth on collagen
115
. These insights are potentially
important, as fibrillar collagens type I and III are among
the most potent activators of platelets, and their high
local concentration in advanced atherosclerotic lesions
contributes to the heightened thrombogenic potential of
these plaques
116
. A considerable number of putative col-
lagen receptors have been described on the surface of
platelets, although recent evidence indicates that the
GPVI receptor is the dominant receptor supporting
platelet adhesion and thrombus growth
55
. GPVI, a
member of the immunoglobulin superfamily, is non-
covalently associated with the immunoreceptor tyro-
sine-based activating motif (ITAM)-bearing receptor,
the FcR -chain
117,118
. This latter receptor plays a funda-
mental role in transducing signals downstream of GPVI
via signalling pathways similar to those used by other
immunoreceptors
117,119,120
. A deficiency of GPVI leads to
a mild bleeding DIATHESIS in humans
121
, a finding that has
been reproduced in experimental mouse models
51,52
.
Immunodepletion of GPVI from the surface of mouse
platelets has recently been reported to lead to a pro-
found defect in occlusive thrombus formation in vivo
122
,
although other studies on GPVI
123
and FcR -chain-
deficient mice
124
have not demonstrated such a crucial
role for this receptor in promoting thrombus formation.
A second well-defined collagen receptor is GPIaIIa
(also known as integrin 21). A deficiency of this
receptor has been reported to cause a mild bleeding
diathesis in humans
56,57
and analysis of platelets derived
from these individuals in ex vivo perfusion chambers
have demonstrated a defect in adhesion and spreading
on the subendothelium
21,125
. Strikingly, in genetically
engineered
1
-deficient mice lacking 21, 51 and
61, bleeding time is unaffected, and adhesion of
these platelets to collagen is normal in extent, albeit
delayed relative to normal platelets
55
. Although the
significance of these findings for humans remains to be
established, they challenge the long-held belief that
GPIaIIa is the primary collagen receptor mediating
platelet adhesion.
pairs
101
. The initial adhesion to the damaged arterial ves-
sel wall involves a diverse array of adhesive ligands (for
example, vWf, collagen, fibronectin, vitronectin and
laminin) and receptors (for example, GPIb, GPVI, and
integrins 21(also known as GPIaIIa), IIb3(also
known as GPIIbIIIa), 51, 61 and possibly v3).
By contrast, subsequent platelet aggregation has tradi-
tionally been thought to involve a single adhesive inter-
action between soluble fibrinogen and GPIIbIIIa (FIG. 4).
However, the conceptual understanding of platelet
DIATHESIS
A permanent (hereditary or
acquired) condition that
predisposes the affected
individual to certain diseases.
Direct thrombin
inhibitors, vitamin
K-antagonists,
heparins
Beraprost,
epoprostenol,
iloprost
TXA
2
TXA
2
Thrombin
GPIIbIIIa
activation
ADP
Prostacyclin
NO
Ticlopidine,
clopidogrel
AA
COX
Aspirin
Abciximab,
tirofiban,
eptifibatide
Cilostazol
cAMP
AMP
PDE
Dipyridamole
cGMP
GMP
PDE
+
_
Fibrinogen
P2Y
12
*
Figure 2 | Molecular targets of current antithrombotic drugs. The principal factor regulating
the adhesiveness of platelets is the activation state of GPIIbIIIa. The affinity status of this receptor
is strictly regulated by a balance of activating (+ve; ADP, thrombin, TXA
2
) and inhibitory signals (-ve;
prostacyclin, NO). A number of these regulatory pathways have been successfully targeted
therapeutically, leading to the development of a diverse range of antithrombotic approaches.
These include various surface-receptor antagonists (ADP P2Y
12
receptor: ticlopidine and
clopidogrel; GPIIbIIIa: abciximab, tirofiban and eptifibatide), inhibitors of platelet signalling
enzymes (COX: aspirin; cAMP PDE: cilostazol; cGMP PDE: dipyridamole), receptor agonists
(prostacyclin: iloprost) and soluble agonist inhibitors (thrombin: heparins, direct thrombin inhibitors
or vitamin K antagonists). *Note: the antithrombotic effects of thrombin antagonists are primarily
due to the inhibition of fibrin generation, although they can also indirectly dampen platelet
activation. Also, it should be noted that although dipyridamole is primarily a cGMP PDE inhibitor, it
also indirectly affects cAMP levels and inhibits cellular uptake and metabolism of adenosine,
leading to further inhibition of platelet function. The efficacy of individual drug therapies (see BOX 1)
is influenced by a number of factors, including the clinical indication for antithrombotic therapy,
patient responsiveness to the therapeutic agent as well as drug pharmacokinetics and
pharmacodynamics. For example, although aspirin and ADP-receptor antagonists inhibit two
important pathways for platelet activation, other alternative pathways that is, downstream of
thrombin receptors can bypass the effects of these drugs and induce GPIIbIIIa activation and
thrombosis. Direct GPIIbIIIa antagonists overcome this limitation, producing potent
antithrombotic effects. AA, arachidonic acid; COX, cyclooxygenase; NO, nitric oxide; PDE,
phosphodiesterase; TXA
2
, thromboxane A
2
.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | OCTOBER 2003 | 5
R E V I E WS
role of thrombin in arterial thrombosis is further compli-
cated by the myriad effects of this protease on coagula-
tion proteins, fibrinolysis, inflammation and endothelial
cells
137,138
.
Recent insight into the role of ADP and thrombin
receptors in arterial thrombosis has been gained from
the study of transgenic mouse models
6874,139,140
. These
studies have indicated that the P2Y
1
and P2Y
12
receptors
might have distinct but complementary roles in throm-
bus development
136
: the P2Y
1
receptor promotes ini-
tial thrombus growth
7072
, whereas the P2Y
12
receptor
primarily serves to sustain the stability of formed
thrombi
73
. Recent studies have also provided evidence
for the importance of platelet thrombin receptors in
arterial thrombosis. For example, studies of mice lacking
either of the major thrombin receptors PAR3 (REF. 68) or
PAR4 (REF. 69) have demonstrated that both receptors
contribute to thrombus formation under high-shear
conditions in vivo
68,69
. Although the protease-activated
receptors on the surface of human platelets are distinct
from those on mice (PAR1/PAR4 versus PAR3/PAR4),
these findings nonetheless provide proof-of-concept
validation that thrombin receptor function, distinct
from other pro-thrombotic activities of thrombin, is
important for arterial thrombosis.
Factors promoting occlusive thrombus formation
The factors contributing to an exaggerated platelet
response at sites of atherosclerotic plaque rupture are
multifactorial: they are the net result of changes in
platelet reactivity, plaque thrombogenicity, rheological
disturbances and the breakdown of the normal control
mechanisms dampening platelet activation.
Plaque thrombogenicity. Several factors contribute to
the heightened thrombogenic potential of atheroscle-
rotic lesions, including high concentrations of fibrillar
collagens in the lesion
141
, tissue factor
142,143
, as well as the
presence of potent platelet-activating lipids, such as
lysophosphatidic acid
144,145
. The presence of collagen in
plaques represents a potential double-edged sword. On
the one hand, an elevated content of collagen is advan-
tageous as it maintains the strength of the atheroscle-
rotic fibrous cap, thereby preventing plaque rupture
146
.
On the other hand, its potent platelet-activating prop-
erties increase the likelihood of forming occlusive
thrombi. The importance of tissue factor in promot-
ing plaque thrombogenicity is well established. In
advanced lesions, tissue factor is primarily derived from
tissue macrophages and T lymphocytes as a conse-
quence of the shedding of apoptotic pro-coagulant
microparticles
147
. Studies demonstrating that direct
inhibition of tissue factor markedly reduces thrombus
growth on ruptured atherosclerotic plaques
143
provide
strong experimental support for the rationale of using
antithrombin agents in the management of the acute
coronary syndromes.
Platelet reactivity. Enhanced platelet reactivity is likely
to contribute to occlusive thrombus formation in high-
risk patient groups. Patients with hyper-reactive
Importance of soluble platelet agonists and their receptors
in promoting thrombus growth. Efficient platelet activa-
tion requires the synergistic contribution of multiple
input signals, involving adhesive and soluble stimuli
(FIG. 3). The importance of soluble platelet agonists, such
as thrombin, ADP and TXA
2
, in promoting thrombus
formation has been clearly demonstrated
8,68,69,126133
.
These agonists are generated locally at the site of vascular
injury and accelerate platelet activation through well-
defined receptor-coupled signalling mechanisms
8,126,134
.
However, the contribution of individual agonist receptors
to thrombus growth has not been so clearly defined. This
is particularly relevant to thrombin and ADP, as each of
these agonists can potentially induce platelet activation
through multiple surface receptors (ADP through the
P2Y
1
and P2Y
12
receptors; thrombin through protease-
activated receptor 1 (PAR1), PAR3 and GPIb)
134136
. The
Box 1 | Characteristic features of commonly used antiplatelet drugs
Drugs that target adhesion receptors
GPIIbIIIa antagonists; for example, abciximab
Powerful antithrombotics that are particularly effective in patients undergoing
percutaneous coronary interventions.
Used only as intravenous agents in select clinical indications. Long-term use associated
with a high incidence of nuisance bleeding.
No proven clinical benefit for orally active agents.
Drugs that target platelet agonist receptors
ADP- receptor antagonists; for example, clopidogrel
Well-tolerated oral drugs with proven efficacy in a broad population of patients with
cardiovascular disease.
Relatively weak antithrombotic effect, marginally better than aspirin.
Generally well tolerated when combined with aspirin.
Bleeding risk low as monotherapy. Increased bleeding risk with combination therapy.
Significantly more expensive than aspirin.
Drugs that target platelet signalling pathways
Cyclooxygenase inhibitors; for example, aspirin
Well-tolerated oral drug with proven clinical benefit in patients with a wide range of
cardiovascular diseases.
Highly cost effective.
Weak antithrombotic action with limited efficacy.
Gastrointestinal and renal toxicity, particularly in elderly patients.
Bleeding risk low as monotherapy.
Phosphodiesterase inhibitors; for example:
Dipyridamole
Safe oral antithrombotic drug. No increased bleeding risk when combined with aspirin.
Limited efficacy as monotherapy. Useful in the prevention of stroke when combined
with aspirin.
Headaches are a common side-effect at the onset of use. Also causes hypotension,
blood pressure lability and gastrointestinal irritation.
Cilostazol
Safe, well-tolerated drug. Used for the secondary prevention of cerebral infarction and
for intermittent claudication.
Weak antithrombotic action.
Minimal effect on bleeding time.
6 | OCTOBER 2003 | VOLUME 2 www.nature.com/reviews/drugdisc
R E V I E WS
hypercholesterolaemia
154
, and in cigarette smokers
155
and older age groups
156
. Dietary factors, such as alco-
hol
157
, dietary fats, fish and fish oils
158
, have also been
reported to regulate platelet responsiveness. There is
growing interest in the possible involvement of glyco-
protein polymorphisms in regulating the reactivity of
platelets
159
and, although controversial, there is evidence
that polymorphisms in GPIb
160162
, GPIIbIIIa
163
and
GPIaIIa
164
increase the risk of arterial thrombosis.
platelets are more likely to have acute coronary
events
148,149
, and diabetics in particular (who have
more reactive platelets) benefit from more intensive
antiplatelet therapy than do other patient groups
150,151
.
The mechanisms responsible for platelet hyper-reactiv-
ity are incompletely understood, but probably reflect the
contribution of various congenital and acquired influ-
ences. Increased platelet reactivity has been reported in
individuals with hypertension
152,153
, diabetes
153
and
Thrombin
GPIIbIIIa
GPIaIIa
GPIb/V/IX
vWf
Collagen
Ca
2+
flux
PLC-
PLC-
ADP
P2Y
1
P2Y
12
PAR1 PAR4
G
i
G
i
G
q
G
q
G
12/13
G
q
G
12/13
TXA
2
TP-
TP-
G
q
G
1
2
/
1
3
G
q
G
1
2
/
1
3
GPIIbIIIa
activation
Soluble
agonists
Adhesion
receptors
FcR
-chain
GPVI
Figure 3 | Potential antithrombotic targets. Schematic representation of the major adhesion and agonist receptors on the surface
of platelets. On the basis of mouse gene-knockout studies and platelets from patients with various haemostatic disorders, the specific
contribution of many of these receptors to haemostasis and thrombosis has been well defined. GPIb/V/IX: binds vWf expressed on
the subendothelium and on the surface of platelets. This receptor also potentiates platelet activation in response to low-dose
thrombin, and is important in mediating platelet adhesion and aggregation under high-shear conditions. GPIIbIIIa: fundamental for a
range of platelet responses, including platelet aggregation, spreading and clot retraction. Binds multiple ligands, including fibrinogen,
vWf, polymerized fibrin, fibronectin, vitronectin and soluble CD40 ligands. GPIaIIa and GPVI: the two major receptors supporting
platelet adhesion on collagen. The GPVI receptor is non-covalently associated with the ITAM-bearing receptor, FcR -chain. Analysis
of mouse platelets lacking GPVI or FcR -chain have demonstrated an important role for this receptor complex in supporting platelet
adhesion and activation on fibrillar collagens. ADP receptors: two metabotropic receptors for ADP have been identified P2Y
1
and
P2Y
12
. Each ADP receptor is linked to one or more different G-protein(s). Thrombin receptors: thrombin uses three agonist receptors
on the surface of platelets. Two of these are the classical protease-activated receptors (PAR1 and PAR4), which are linked to G
i,q,12/13
and G
q,12/13
, respectively. The other thrombin receptor is GPIb. TXA
2
receptors: two TXA
2
receptors have been identified, TP- and
TP-, each of which is linked to G
q
and G
12/13
. All of these receptors activate specific signalling cascades that are ultimately linked to
the mobilization of calcium from intracellular stores. All soluble agonist receptors utilize G-protein-coupled receptors that induce
activation of one or more PLC- isoforms. Adhesion receptors typically utilize non-receptor tyrosine kinases that regulate cytosolic
calcium flux through regulation of PLC- isoforms. For clarity, the details of specific signalling events linked to individual receptors have
been omitted. Targeting signalling processes linked to the regulation of GPIIbIIIa have the potential to produce a significant number of
novel antithrombotic approaches. GP, glycoprotein; ITAM, immunoreceptor tyrosine-based activating motif; PLC, phospholipase C;
TXA
2
, thromboxane A
2
; TP, thromboxane/prostanoid receptors; vWf, von Willebrand factor.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | OCTOBER 2003 | 7
R E V I E WS
that the haemostatic defect is not as severe as deficiency of
GPIIbIIIa
4143,46
, raising the possibility that inhibitors of
GPIb might have better long-term tolerance than GPIIb
IIIa inhibitors. It is worth noting that platelets lacking the
GPIb complex have other abnormalities beyond defective
vWf binding, including THROMBOCYTOPAENIA, an abnormal
morphology, decreased responsiveness to thrombin and a
reduced pro-coagulant function
46,47
, all of which could
contribute to the increased bleeding risk. Antibodies
against GPIb or vWf have potent antithrombotic activity
in vivo
44,45,175179
, and, in contrast to GPIIbIIIa inhibitors,
can inhibit thrombus growth at concentrations that do
not increase bleeding time
45,180
. Furthermore, given that
the GPIb-subunit is primarily expressed on platelets,
such inhibitors are likely to have minimal non-haemo-
static side-effects. There is growing evidence for the
importance of the GPIb subunit in supporting platelet
leukocyte interactions, owing to its ability to act as a
counter-receptor to one or more leukocyte and endothe-
lial cell-adhesion molecules
181183
. Given the growing
awareness of the importance of inflammation in influ-
encing the acute and chronic outcomes of cardiovascular
disease
184,185
, GPIb inhibitors that dampen platelet
leukocyte interactions might have additional therapeutic
benefits. Nonetheless, there are significant challenges
associated with the development of GPIb inhibitors, not
least their probable deleterious effects on haemostasis,
problems associated with antibody-induced thrombo-
cytopaenia
186,187
and potential pathogenic effects on
megakaryocytes
188,189
. The development of GPIb inhibi-
tors is also hampered by the poor cross-species reactivity
of anti-GPIb antibodies and by the lack of well-defined
small-molecule PHARMACOPHORES necessary for the genera-
tion of orally bioavailable compounds. Moreover, the
evaluation of these inhibitors in early-stage proof-of-
concept clinical trials will be a challenge, particularly in
patients with coronary disease, due to the number of
competing antithrombotic agents already available in the
clinic.
Inhibiting collagenplatelet interactions. Progress in
elucidating the key adhesive receptors mediating
plateletcollagen interactions, combined with the per-
ceived importance of these interactions for arterial
thrombosis, has focused considerable recent interest on
the development of antagonists of collagen receptors.
The case for developing such inhibitors is further
strengthened by clinical and experimental evidence
that a deficiency of either of the two major collagen
receptors GPVI and GPIaIIa produces only a modest
defect in haemostasis
53,57,125
. It should be noted that in
addition to supporting platelet adhesion directly, col-
lagen also facilitates plateletvessel wall interactions
indirectly by providing binding sites for vWf. The
interaction between collagen and vWf is particularly
important under conditions of rapid blood flow, such
as those typically found in the arteriolar circulation and
at sites of arterial stenosis
102
. The binding site on vWf
for collagen has been localized to the A3 domain of
vWf, and specific disruption of this interaction has been
shown to lead to reduced thrombus growth in vivo
190
.
Disturbed rheology. Rheological disturbances at sites of
arterial stenosis are important in promoting occlusive
thrombus formation
21
. Platelets are exposed to high-
shear forces and a range of abnormal flow patterns
during the atherothrombotic process, including flow
separation, eddy formation, flow reversal and turbu-
lence, which produce complex, rapid changes in the shear
environment
165167
. Despite their importance for throm-
bosis, the mechanism(s) by which shear forces induce
platelet activation remains incompletely understood.
On the basis of available experimental evidence, shear-
induced platelet activation is crucially dependent on the
adhesive interaction between vWf, GPIb and GPIIbIIIa,
with an important role for released ADP in potentiating
platelet activation
112,168171
. It is noteworthy that aspirin is
unable to inhibit the effects of shear on platelet activa-
tion, a finding that might partially explain its limited
clinical efficacy
169,172,173
.
New approaches to antiplatelet therapy
Future advances in antiplatelet therapy are likely to
derive from improvements in the understanding of the
molecular events regulating thrombogenesis. In this
context, the elucidation of factors specifically promoting
an exaggerated platelet response at sites of atheroscle-
rotic plaque rupture could provide a rational basis for
the design and development of novel antiplatelet agents
with improved efficacy/side-effect profiles. In addition, a
considerable number of potentially new antithrombotic
approaches are likely to emerge as a direct result of
knockout mouse studies (TABLES 2 and 3). The usefulness
of the latter approach for proof-of-concept validation of
pharmaceutical products is well established
174
. In the
remainder of this review, we will discuss potential novel
approaches for antiplatelet therapy, focusing on targets
for which there is experimental evidence that their inhi-
bition produces an antithrombotic effect in vivo. On the
basis of current knowledge, three distinct groups of
antiplatelet drugs are likely to emerge in the future: anti-
adhesive drugs; drugs that can directly inhibit agonist
receptors; and drugs that target one or more intracellular
signalling pathways linked to platelet activation.
Anti-adhesive drugs. There are potentially a large num-
ber of adhesive proteins and receptors contributing to
platelet thrombus growth; however, in only a few
cases specifically, fibrinogen, vWf and collagen is
there strong evidence for their involvement in occlusive
thrombus formation. The importance of blocking fib-
rinogen binding to GPIIbIIIa is well established and
will not be discussed further here.
Inhibiting the vWfGPIb interaction. The growing
recognition of the importance of the vWfGPIb interac-
tion for both primary platelet adhesion and aggregation,
particularly under high-shear conditions, has focused
considerable attention on this receptor as a potential
antithrombotic target. Deficiency of GPIb in humans
and mice leads to a severe bleeding diathesis
46,47
, under-
scoring the importance of this receptor for normal
haemostasis; however, studies in mouse models indicate
THROMBOCYTOPAENIA
A decrease in platelet count,
defined in humans as less than
150 10
9
per l, which results in
the potential for increased
bleeding.
PHARMACOPHORE
The ensemble of steric and
electronic features that are
necessary to ensure optimal
interactions with a specific
biological target structure and
to trigger (or to block) its
biological response.
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R E V I E WS
Table 2 | Phenotypes of mice with targeted disruption of haemostatic proteins: part 1
KO Anti-thrombotic effect Bleeding Related human condition
Adhesion receptor or ligand targets
b No reports of in vivo antithrombotic effect in ++++
41
Glanzmanns
mouse KO model. thrombasthenia: moderate
to severe bleeding effect.
3 Prevention of carotid artery occlusion induced by +++++
43
FeCl
3
(REF. 42).
GPIb Potent antithrombotic effect of GPIb +++
46
Bernard-Soulier syndrome:
antagonists
44,45
. No reports of in vivo anti- moderate to severe
thrombotic effect in mouse KO model. bleeding, giant platelets
47
.
GPV Conflicting evidence regarding role in -ve
48
NR
thrombosis
48,49
. Accelerated thrombus formation, N
50
vessel occlusion and frequent embolization
following FeCl
3
-induced injury
48
. Delayed
occlusion time in FeCl
3
- induced arterial injury
49
.
GPVI Potent anti-thrombotic effect with anti-GPVI N
52
Mild bleeding
53
antibodies
51
. Subtle in vivo antithrombotic
effect in mouse KO model.
FcR -chain Reduced platelet aggregation following N
54
NR
wire-induced arterial injury
54
.
1 (Ia) No reports of in vivo antithrombotic effect in N
55
Mild bleeding
56,57
mouse KO model.
vWf Delayed thrombus initiation and defective vessel ++++
39,40
. Some von Willebrand disease
occlusion in FeCl
3
model of injury
39,40
spontaneous type III: moderate to
bleeding events in severe bleeding
58,59
.
10% of neonates
40
.
Fibrinogen Unstable thrombi leading to downstream ++++
60
Afibrinogenaemia: moderate
vessel occlusion
39
. to severe bleeding.
Fibrinogen- No reports of in vivo antithrombotic effect in +++
61
QAGVD mouse KO model.
P-Selectin
1
Reduced thrombus weight in vivo in venous +
64
NR
thrombosis model
62
. Reduced accumulation of
fibrin and tissue factor during arterial thrombus
formation in vivo
63
.
CD31 In vivo thrombus formation (photo-activated +
67
NR
(PECAM-1) endothelial injury) in both venules and arterioles N
65
unaltered
65
. More rapid and larger (^0%)
thrombus growth following laser-induced
endothelial injury model. Decreased time to
occlusion in ferric chloride thrombosis model
66
.
Agonist receptor targets
PAR3 Protection against mesenteric thrombosis +++
68
NR
induced by FeCl
3
(REF. 68).
PAR4 Protection against mesenteric thrombosis +++
69
NR
induced by FeCl
3
(REF. 69).
P2Y
1
Protected against collagen-adrenaline +/++
70,71
NR
induced thromboembolism
7072
.
P2Y
12
No reports of in vivo anti-thrombotic +++
73
P2Y
12
mutation: mild
effect in mouse KO model. bleeding disorder
74
.
P2X
1
Mortality reduced in model of systemic N
75
One report of severe bleed-
thromboembolism and decreased mural ing disorder in humans
76
,
thrombi in the microcirculation following although this has not
laser injury
75
. been confirmed by others.
TP (thrombox- No reports of in vivo antithrombotic effect in +++
77
TP mutation: mild
ane/prostanoid mouse KO model bleeding disorder
78,79
.
receptors)
EP
3
(prosta- Decreased susceptibility to thromboembolism +
80
NR
glandin E induced by arachidonic acid
80
.
receptor type)
Bleeding time key: +++++ = spontaneous haemorrhage, anaemic, bleeding time >30 minutes
*
; ++++ = spontaneous haemorrhage,
normal Hb, bleeding time >30 minutes
*
; +++ = no spontaneous haemorrhage, normal Hb, bleeding time >30 minutes
*
, or > fivefold N; ++
= no spontaneous haemorrhage, normal Hb, bleeding time 1030 minutes, or twofold N; + = No spontaneous haemorrhage, bleeding
time >N<10 minutes, or < twofold N. *Whereno quantifiable bleeding time was reported, phenotype was classified as No cessation of
bleeding at the pre-determined end-point of the assay. -ve, reduced bleeding compared to wild type; KO, knockout; N, normal/no
prolongation; NR, no human pathology reported; PAR, protease-activated receptor; vWf, von Willebrand factor.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | OCTOBER 2003 | 9
R E V I E WS
the culprit lesion for up to a week before the clinical
event
191
. So the clinical utility of antagonists of collagen
receptors might be restricted to specific subsets of
patients with vascular disease.
Drugs that can directly inhibit agonist receptors. There
has been long-standing interest in attempting to
improve on the antithrombotic effect of aspirin.
Inhibition of cyclooxygenase-1 prevents the conversion
of arachidonic acid (AA) to prostaglandin endoperox-
ides and thromboxane A
2
(TXA
2
), thereby eliminating
an important autofeedback mechanism that serves to
amplify platelet activation
192,193
. The antithrombotic
effect of inhibiting TXA
2
is partially offset by inhibition
of another arachidonic acid metabolite, prostacyclin
(PGI
2
), a powerful endothelial-derived inhibitor of
platelets. Strategies to eliminate the platelet-activating
properties of TXA
2
without affecting PGI
2
function
Validation of the importance of these individual adhe-
sive interactions for thrombus growth, particularly in
diseased atherosclerotic arteries, has been hampered by
the lack of suitable experimental models. This is poten-
tially important, given that collagen antagonists would
be the first antithrombotic approach that targets pri-
mary platelet adhesion, rather than aggregation. So the
predictive power of any given preclinical thrombosis
model will be greatly influenced by the nature of the
thrombogenic surface exposed following vascular
injury. A further potential limitation of selective
inhibitors of platelet adhesion is that they might afford
limited antithrombotic protection in patients with
established arterial thrombosis, including patients with
unstable angina, myocardial infarction and ischaemic
stroke. In this context, it is relevant that a high proportion
of patients presenting with an acute myocardial infarc-
tion have evidence of sub-acute thrombus formation at
Table 3 | Phenotypes of mice with targeted disruption of haemostatic proteins: part 2
KO Anti-thrombotic effect Bleeding Related human
condition
Signalling targets
PI3K- Protection against ADP-induced N
81
NR
thromboembolic vascular occlusion
81
.
DiYF(
3
): defect No reports of in vivo antithrombotic N
82
(tendency to rebleed
82
). NR
in
3
outside-in effect in mouse KO model.
signalling
SLP-76 No reports of in vivo antithrombotic ++++
84,85
(mild anaemia, normal NR
effect in mouse KO model bleeding time; bleeding
diathesis due to defect in
vessel development
83
).
PLC-2 In vitro: ability to aggregate to ++++
86,87
NR
collagen inhibited
86,87
.
P-12LO (platelet Increased mortality in an ADP-induced Not reported. NR
type 12- mouse model of thromboembolism
88
.
lipoxygenase)
COX Decreased aggregation of COX-1- Not reported. Mild bleeding
deficient platelets in vitro
89
. No anti- defect
9092
.
thrombotic effect reported to date.
G
q
Protected against collagen/adrenaline- ++++
93
NR
induced thromboembolism
93
.
G
za
No overt platelet defect. No protection ++/+++
94
NR
against collagen/adrenaline-induced
thromboembolism
94
.
Syk No reports of in vivo antithrombotic N (tendency to rebleed)
95
. NR
effect in mouse KO model. Similar to SLP-76.
cGMP-dependent Increased platelet adhesion and intestinal Not reported NR
protein kinase aggregation in the post-ischaemic kidney
and microvasaculature in vivo
96
.
Miscellaneous targets
Gas6 Prevention of venous and arterial N
97
NR
thrombosis, and protection against fatal
collagen/adrenaline-induced
thromboembolism
97
.
sCD40L Delayed vessel occlusion and frequent N
98
NR
embolization in vivo
98
.
Bleeding time key: +++++ = spontaneous haemorrhage, anaemic, bleeding time >30 minutes
*
; ++++ = spontaneous haemorrhage,
normal Hb, bleeding time >30 minutes
*
; +++ = no spontaneous haemorrhage, normal Hb, bleeding time >30 minutes
*
, or > fivefold N; ++
= no spontaneous haemorrhage, normal Hb, bleeding time 1030 minutes, or twofold N; + = No spontaneous haemorrhage, bleeding
time >N<10 minutes, or < twofold N. *Whereno quantifiable bleeding time was reported, phenotype was classified as No cessation of
bleeding at the pre-determined end-point of the assay. -ve, reduced bleeding compared to wild type; COX, cyclooxygenase; Gas6,
growth arrest-specific 6; KO, knockout; N, normal/no prolongation; NR, no human pathology reported; P13K, phosphatidylinositol 3-
kinase; PLC, phospholipase C; SLP, SH2-domain-containing leukocyte protein.
10 | OCTOBER 2003 | VOLUME 2 www.nature.com/reviews/drugdisc
R E V I E WS
blunting the platelet responses to ADP and thrombin
leads to an increased tail-bleeding time
6871
. The clinical
significance of these findings remains uncertain due to
the paucity of patients identified with a bleeding ten-
dency linked to deficiency of these receptors. This con-
trasts with the P2Y
12
receptor, a deficiency of which in
humans leads to a mild bleeding tendency
74
. An impor-
tant outstanding issue with regards to PAR1-, PAR4- and
P2Y
1
-receptor antagonists relates to the potency of their
antithrombotic effect relative to P2Y
12
-receptor antago-
nism and whether these approaches offer an improved
benefit-to-bleeding risk ratio over existing therapies.
have involved developing combined inhibitors of TXA
2
synthase and the TXA
2
receptor
194197
. Despite showing
considerable promise in preclinical studies, these newer
approaches have been disappointing in clinical trials,
and have not demonstrated a benefit over aspirin.
The success of the ADP-receptor antagonists in
improving clinical outcomes has fuelled interest in the
possible inhibition of other agonist receptors. Confir-
mation of the role of the ADP P2Y
1
receptor, and the two
thrombin receptors PAR3 and PAR4, in promoting
thromboembolism has been gained through the study
of knockout mouse models. However, in these mice,
a Suspension
Inactive
Active
Stirring
GPIb/V/IX Soluble
agonist
GPCR GPIIbIIIa
Fibrinogen -granule
Intracellular
signalling
vWf
b Reactive substrate
Resting Tethering
Translocation
Tethering
Stable
aggregation
Thrombus
formation
Adhesion and spreading
Blood flow
Figure 4 | Distinct mechanisms regulating platelet aggregation and thrombus growth. a | Platelet aggregation has
traditionally been viewed as mediated by a single adhesive interaction between GPIIbIIIa and fibrinogen (suspension). In this model,
activation signals emanating downstream of soluble agonist receptors induce a conformational change in the extracellular domain of
GPIIbIIIa, enabling it to engage fluid-phase fibrinogen. The dimeric fibrinogen molecule crosslinks two GPIIbIIIa receptors on
adjacent platelets, thereby mediating platelet aggregation. b | Analysis of thrombus formation in vivo has demonstrated that platelet
aggregation is a dynamic process potentially involving multiple ligands and receptors (reactive substrate). Under high-shear
conditions, platelet aggregation is reliant on an initial tethering step (tethering), which is dependent on GPIb on the surface of flowing
platelets engaging surface-expressed vWf on immobilized platelets. This adhesive interaction also supports platelet rolling
(translocation) on the thrombus surface. Subsequent firm adhesion (adhesion and spreading/stable aggregation) is mediated by
GPIIbIIIa engagement of vWf and/or fibrinogen. Recent evidence has indicated that two other potential GPIIbIIIa ligands,
fibronectin and soluble CD40 ligand (not shown), might also participate in arterial thrombogenesis. GP, glycoprotein; GPCR,
G-protein-coupled receptor; vWf, von Willebrand factor.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | OCTOBER 2003 | 11
R E V I E WS
Similarly, patients undergoing coronary artery bypass
surgery or CAROTID ENDARTERECTOMY have a relatively high
incidence of thromboembolic events
200,201
, and the need
for an antiplatelet agent that does not increase surgical
bleeding remains high. In other acute situations, such as
PERCUTANEOUS CORONARY INTERVENTION and unstable angina,
bleeding concerns are less crucial (although still impor-
tant), and combination therapies with potent anti-
thrombotic properties are generally well tolerated and
provide significant clinical benefit. In the future, long-
term antiplatelet therapies will need to demonstrate
improved efficacy, cause fewer bleeding side-effects and,
ideally, not increase bleeding risk when combined with
other antithrombotic agents.
Which platelet adhesion receptors/ligands, when
inhibited, will most likely lead to improvements in
antithrombotic therapy? It is our view that GPIb is likely
to be the best adhesion-receptor target for the develop-
ment of novel antithrombotic agents, at least for the
management of acute thrombotic events. As with
GPIIb/IIIa, GPIb is the only platelet adhesion receptor
that has a clear-cut non-redundant role in haemostasis
and thrombosis, with a haemostatic defect that is less
severe than that associated with deficiency of GPIIb
IIIa. As stated earlier, this defect might be less apparent
with selective inhibition of the vWfGPIb interaction,
particularly if thrombin binding and other haemostatic
functions of GPIb are preserved. Also, the potential to
develop inhibitors that preferentially block platelet
adhesion under pathological shear conditions is also an
attractive feature of this target. In short, GPIb inhibitors
will almost certainly have a wider therapeutic index
than GPIIbIIIa inhibitors, although it is uncertain
whether this will be wide enough to truly define these
inhibitors as magic bullets. It is less likely that targeting
of any other single adhesion receptor will produce as
potent an antithrombotic effect as GPIb or GPIIbIIIa
inhibitors, primarily because of the degree of redun-
dancy among receptors involved in primary platelet
adhesion. On a more positive note, blocking platelet
collagen receptors might cause less severe bleeding com-
plications, thereby improving their overall tolerance and
clinical utility. Despite the obvious appeal of targeting
collagenvWf and collagenplatelet adhesive interac-
tions, there remains a considerable amount of work to be
done to more clearly establish the therapeutic potential
of such approaches.
What agonist receptors are likely to challenge the
TXA
2
and P2Y
12
receptors as preferred targets for novel
antithrombotics? This is a difficult question to answer
with the current level of experimental and clinical infor-
mation, although obvious candidates include the P2Y
1
,
PAR1 and PAR4 receptors. On the basis of human and
animal studies, blocking the platelet P2Y
12
receptor
causes a marked defect in haemostasis, underscoring the
importance of this receptor for normal platelet func-
tion, and, as a consequence, the need to develop drugs
with highly predictable pharmacokinetic properties. To
date, P2Y
1
, PAR1 and/or PAR4 antagonists with suffi-
cient specificity and potency for clinical evaluation have
yet to emerge; however, on the basis of the findings
Drugs that target one or more signalling pathways linked
to platelet activation. The importance of various sig-
nalling cascades in regulating platelet function is
becoming increasing well understood. Soluble agonists
induce platelet activation through distinct G-protein-
coupled receptors linked to the regulation of phospholi-
pase C (PLC), phosphatidylinositol 3-kinase (PI3K),
adenylyl cyclase and the Rho family of GTPases, whereas
platelet adhesion receptors typically initiate platelet acti-
vation through one or more non-receptor tyrosine
kinases
21
. There are a large number of knockout mouse
models available with specific signalling defects, and
detailed analyses of these mice will probably lead to the
identification of novel antithrombotic targets. Several
signalling enzymes have been identified, such as G
q
and PLC-2, which have an essential role in normal
platelet function
198
and their deletion is associated with
a marked bleeding tendency
86,93
. However, other sig-
nalling enzymes, such as the -isoform of PI3K, seem to
be involved in promoting thrombus formation but are
less crucial for normal haemostasis
81
.
Miscellaneous targets. A number of promising anti-
thrombotic targets that have an incompletely defined
role in platelet function have been discovered as a direct
result of mouse transgenic technology. In several exam-
ples, mice have a defect in thrombus formation with
minimal prolongation of tail-bleeding time (TABLES 2
and 3). These include growth arrest-specific 6 (Gas6), a
previously unrecognized platelet protein that seems to
be important in potentiating platelet activation and
thrombus growth
97
; the soluble form of CD40 ligand,
a principal mediator of inflammation that seems to be
important in regulating thrombus stability through its
ability to engage GPIIbIIIa
98,199
; and P-selectin, a
major platelet -granule protein that is crucial in pro-
moting plateletleukocyte interactions and fibrin
accumulation
63,64
.
A universal antithrombotic myth or reality?
So, what are some of the likely targets for the develop-
ment of the magic bullet? Before attempting to answer
this question, some consideration is required as to the
clinical context of antithrombotic therapy. In general, it
has proven difficult to develop drugs that are universally
effective in all patients with cardiovascular disease, as
the specific requirements of antithrombotic drugs are
partially dependent on the clinical circumstances of
their use. For example, the antithrombotic needs of a
patient presenting to hospital with an acute ischaemic
stroke are markedly different from those of a patient
with stable coronary artery disease requiring long-term
antiplatelet therapy. With the former, the antithrom-
botic approach must have a rapid onset of effect, must
have minimal effect on bleeding risk (due to the risk of
intracerebral haemorrhage) and must be compatible
with thrombolytic agents. Ideally, such an agent would
also have anti-inflammatory properties, thereby reduc-
ing neuronal damage associated with ischaemia-reper-
fusion injury. Fulfilling such stringent criteria will be a
challenge, particularly for a single therapeutic agent.
CAROTID ENDARTERECTOMY
A surgical procedure that
removes atherosclerotic plaque
from the walls of the carotid
arteries in an attempt to reduce
the risk of stroke or transient
ischaemic attack.
PERCUTANEOUS CORONARY
INTERVENTION
A group of techniques that can
relieve coronary artery
narrowing. The techniques
include balloon angioplasty,
laser angioplasty and
implantation of intracoronary
stents and other catheter devices.
12 | OCTOBER 2003 | VOLUME 2 www.nature.com/reviews/drugdisc
R E V I E WS
The challenge of such an approach is to identify the
crucial signalling elements required for shear activa-
tion of platelets and thrombosis, but which are less
important for haemostasis. Ideally, these targets would
have limited tissue distribution, or alternatively,
inhibitors with reduced systemic effects would be
employed, that is, aspirin-like drugs (irreversible
inhibitors with a short plasma half-life).
Conclusions
The discovery of a magic bullet that selectively targets
pathological thrombi has proven elusive, primarily
because the molecular events regulating thrombosis are
in large part identical to those underlying haemostasis.
In fact, it remains to be proven whether there are indeed
true pathogenic mechanisms operating during throm-
bus formation that are not involved in haemostasis. This
said, there are clearly important differences between the
two processes, and future strategies aimed at exploiting
these differences might lead to novel therapies with
improved safety and efficacy profiles. It remains uncer-
tain whether it will ever be possible to develop a single
drug that will be universally effective at inhibiting
pathological thrombi. It is perhaps more probable that
future advances will involve the development of combi-
nation therapies targeting one or more steps in the
thrombotic process. Such multi-component therapy
will need to take into consideration the complex inter-
play between platelets and proteins of the coagulation,
fibrinolytic and inflammation systems, although
undoubtedly platelet inhibitors will continue to take
centre stage in the antithrombotic arena.
from knockout mice, it seems that these inhibitors
might have less of an impact on haemostasis. It is note-
worthy that the platelet inhibitors that presently enjoy
the greatest clinical success aspirin and thienopy-
ridines primarily inhibit pathways that amplify
platelet activation. In this context, the finding that
Gas6-deficient platelets are less responsive to stimula-
tion by conventional stimuli and have defective throm-
bus growth in vivo makes it a target of considerable
therapeutic interest.
Unravelling the key signalling mechanisms regulat-
ing the adhesive function of GPIIbIIIa remains a
major research objective that could ultimately lead to
the development of entirely new classes of antithrom-
botic agents. In this regard, developments in mouse
transgenic technology are likely to feature promi-
nently, and several interesting platelet phenotypes have
already begun to emerge as a consequence of defective
bi-directional GPIIbIIIa signalling. A potential bene-
fit of inhibiting signalling targets, particularly those
that converge from multiple input signals, is the ability
to control overall platelet reactivity, regardless of the
nature of the primary thrombogenic stimulus. Further-
more, signalling pathways that are shared by one or
more haemopoietic cell types offer the opportunity of
developing drugs that have both antiplatelet and anti-
inflammatory activity. A novel approach that has yet to
be exploited therapeutically is the targeting of shear-
sensitive (that is, mechanosensory) signalling path-
ways in platelets. These pathways are likely to operate,
at least in part, downstream of the two major shear-
regulated adhesion receptors, GPIb and GPIIbIIIa.
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Acknowledgements
The authors wish to thank Drs Zaverio Ruggeri, Christian Gachet,
Hatem Salem and Francois Lanza, as well as other members of the
authors laboratory for valuable comments.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
Cyclooxygenase-1 | Factor II | fibronectin | GPIb | GPIIb | GPIIIa |
GPV | GPVI | GPIX | P2Y
1
| P2Y
12
| PAR1 | PAR3 | PAR 4 | PLC-2 |
tissue factor | von Willebrand factor
Online Mendelian Inheritance in Man:
http://www.ncbi.nlm.nih.gov/Omim/
Ischaemic stroke
FURTHER INFORMATION
American Heart Association: http://www.americanheart.org
Access to this interactive links box is free online.