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Review article
The growing complexity of platelet aggregation
Shaun P. Jackson
Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
Platelet aggregation, the process by which
platelets adhere to each other at sites of
vascular injury, has long been recognized
as critical for hemostatic plug formation
and thrombosis. Until relatively recently,
platelet aggregation was considered a
straightforward process involving the
noncovalent bridging of integrin ␣IIb␤3
receptors on the platelet surface by the
dimeric adhesive protein fibrinogen. However, with recent technical advances enabling real-time analysis of platelet aggre-
gation in vivo, it has become apparent
that this process is much more complex
and dynamic than previously anticipated.
Over the last decade, it has become clear
that platelet aggregation represents a multistep adhesion process involving distinct receptors and adhesive ligands, with
the contribution of individual receptorligand interactions to the aggregation process dependent on the prevailing blood
flow conditions. It now appears that at
least 3 distinct mechanisms can initiate
platelet aggregation, with each of these
mechanisms operating over a specific
shear range in vivo. The identification of
shear-dependent mechanisms of platelet
aggregation has raised the possibility that
vascular-bed–specific inhibitors of platelet aggregation may be developed in the
future that are safer and more effective
than existing antiplatelet agents. (Blood.
2007;109:5087-5095)
© 2007 by The American Society of Hematology
Introduction
Historical aspects
The propensity of platelets to clump together at sites of vascular
injury was first recognized more than 100 years ago.1-4 This
phenomenon, most accurately described as platelet cohesion
although more commonly referred to as platelet aggregation,
was quickly identified as important for hemostatic plug formation.5 It was also recognized at the time that platelets played a
key role in the development of thrombosis,6 but, it was not until
almost a century later that it became widely accepted that
platelets played a pivotal role in development of cardiovascular
disease.7 As a consequence, inhibitors of platelet aggregation
have become increasingly important parts of the armamentarium
for the prevention and treatment of many atherothrombotic
disorders.8,9
For more than 3 decades, the factors mediating platelet
aggregation appeared conceptually straightforward, requiring a
platelet stimulus (agonist), a soluble adhesive protein (fibrinogen), and a membrane-bound platelet receptor (integrin ␣IIb␤3 or
GPIIb-IIIa), leading to a simple unified model of platelet
aggregation (Figure 1). Although these core elements remain
fundamental, recent technical advances allowing real-time
analysis of platelet aggregation in vivo have demonstrated a
much more complex and dynamic process than previously
anticipated. It is now widely accepted that one of the key
elements influencing the platelet aggregation process is blood
flow, with evidence that distinct aggregation mechanisms operate under different shear conditions.10-13 This has raised the
interesting possibility that vascular bed-specific inhibitors of
platelet aggregation may be developed in the future and
has stimulated a reevaluation of the temporal sequence of
events underlying the aggregation process. In this review, I
discuss recent developments in the study of platelet aggregation
with particular focus on the mechanisms operating under rapid
blood flow.
Platelet aggregation was first recognized in the late 1800s after a
series of pioneering studies by Zahn (1872), Hayem (1878),
Bizzozero (1881 and 1882), Osler (1886), and Eberth and Schimmelbusch (1885-1888).1-4,6 Using intravital imaging techniques,
Bizzozero clearly established that small cell elements (in which he
originated the term “Blut Plattchen” or blood platelets) clumped
together at sites of vessel damage,1,14 a process subsequently
described as viscous metamorphosis.2 Through a series of insightful observations, Bizzozero described the changes platelets undergo after exposure to foreign surfaces, including the formation of
aggregates and subsequent “white thrombi.” He also clearly
described the plugging of small vascular punctures by platelet
thrombi, heralded as a major discovery at the time.5 It was soon
appreciated that quantitative or qualitative defects in platelets lead
to a bleeding disorder; and in 1918, Glanzmann15 described a
familial bleeding diathesis (“hereditary hemorrhagic thrombasthenia”) that was later shown to be a primary defect in platelet
aggregation. Progress in the understanding of platelet aggregation
was slow to develop until the advent of the platelet aggregometer in
1962 by Born16 and independently by O’Brien.17 This relatively
simple technique involved stirring a suspension of platelets in the
presence of a platelet activating substance and, by monitoring
changes in light transmission, the device could accurately monitor
platelet clumping (aggregation) in suspension (Figure 1). Aggregometry revolutionized the study of platelet function, providing fundamental insights into the molecular mechanisms underlying plateletplatelet adhesive interactions. The identification and characterization
of all major physiological agonists, antagonists, surface receptors,
and intracellular signaling pathways in platelets has involved
platelet aggregation studies, and in many situations there is a good
correlation between defects in aggregation and bleeding disorders
Submitted December 27, 2006; accepted February 9, 2007. Prepublished
online as Blood First Edition Paper, February 20, 2007; DOI 10.1182/blood-
2006-12-027698.
© 2007 by The American Society of Hematology
BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
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JACKSON
BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
providing insight into the role of thrombin generation in regulating
platelet aggregation at different flow rates. These assays have been
instrumental in defining the adhesive steps mediating platelet
adhesion to extracellular matrices, including a key role for the von
Willebrand factor (VWF)–GPIb interaction in promoting initial
platelet tethering to the vessel wall22,23 and a major role for
collagen through engagement of GPVI and integrin ␣2␤1 in
promoting platelet arrest.21,24-26 These experimental systems have
also confirmed an essential role for integrin ␣IIb␤3 (GPIIb-IIIa) in
mediating platelet aggregation over the full range of shear conditions operating in vivo. Thus, flow-based studies were pivotal in the
development of the concept that platelet adhesion (mediated by
VWF and collagen) is distinct from platelet aggregation (mediated
by fibrinogen).
Conceptual advances in the understanding
of platelet aggregation
Figure 1. Traditional model of platelet aggregation. (A) Based on studies with the
platelet aggregometer, 3 key elements have been identified as important for platelet
aggregation: an activating stimulus (typically a soluble agonist), a plasma protein
(predominantly fibrinogen), and a platelet surface receptor (integrin ␣IIb␤3 or GPIIbIIIa). Agonist-induced activation of integrin ␣IIb␤3 is essential for the binding of
fluid-phase fibrinogen, which as a consequence of its dimeric structure, can
physically bridge 2 adjacent platelets. (B) The platelet aggregometer is a relatively
simple technique that involves stirring a suspension of platelets in the presence of a
platelet activating substance and by monitoring changes in light transmission, the
device can accurately monitor platelet clumping (aggregation) in suspension.
in affected individuals. Similarly, antiplatelet drugs that increase
bleeding risk in vivo can be detected using standard aggregation
assays, further enhancing the clinical use of the assay. Thus, for
many years, the aggregometer has provided an accurate readout of
the fundamental processes regulating aggregation; as recognized in
the early 1970s, however, this experimental system does not take
into consideration the important influence of blood flow, a key
variable increasing the complexity of the aggregation process.
Importance of blood flow in the regulation
of platelet aggregation
An important consideration for platelet aggregation, and for
platelet adhesion mechanisms more generally, is the rheological
(blood flow) conditions operating at sites of vascular injury.
Platelets are exposed to a broad range of hemodynamic conditions
in vivo, ranging from relatively low flow situations in venules and
large veins (typical wall shear rates ⬍ 500 s⫺1) to small arterioles
(shear rates up to 5000 s⫺1) to stenosed arteries with shear rates as
high as 40 000 s⫺1.18 Platelets have the unique capacity to form
stable adhesion contacts over all shear conditions operating in vivo
and are indispensable for hemostatic plug formation and thrombosis at elevated shear rates.19 In an aggregometer, platelet clumping
occurs under low, nonlaminar shear conditions, experimental
conditions that do not adequately simulate the flow-dependent
recruitment (cohesion) of platelets onto thrombogenic surfaces.
Experimental systems designed to study platelet adhesion and
aggregation on thrombogenic substrates were initially developed in
the 1970s.20,21 Such systems enable assessment of platelet aggregation in flowing whole blood (with or without anticoagulant)
A number of studies in the 1980s suggested that the processes
underlying platelet aggregation under flow conditions may be more
complicated than those operating in the aggregometer. Thus, at
high shear rates, VWF27,28 and fibronectin29 were demonstrated to
not only promote primary adhesion, but also subsequent platelet
aggregation. However, widespread recognition of the growing
complexity of the platelet aggregation process was slow to develop.
A number of technical advances in the late 1990s, including the
development of genetically engineered mouse models and improved live cell imaging techniques enabling high-resolution
visualization of the platelet aggregation process in vitro30 and in
vivo,31,32 have profoundly impacted the study of platelet aggregation, heralding a renaissance in the field. In combination, these
techniques have been instrumental in clarifying many aspects of
the platelet aggregation process, including (a) the demonstration of
a role for multiple adhesive ligands and receptors in the aggregation process; (b) identifying the importance of membrane tethers in
initiating platelet aggregation; (c) improved insight into the role of
individual adhesion and soluble agonist receptors in regulating the
dynamics of platelet aggregation; and (d) the identification of
surface ligands and receptors involved in stabilizing and sustaining
formed aggregates. In the remainder of this review, I focus on
recent developments in each of these areas.
Involvement of multiple adhesion receptors and ligands in
platelet aggregation
One of the most dramatic conceptual changes in the understanding
of platelet aggregation has been the demonstration that multiple
adhesive ligands, such as VWF, fibrinogen, and fibronectin,
regulate platelet-platelet interactions, with each ligand having
distinct roles in the thrombotic process (Figure 2). In vitro
perfusion studies on blood obtained from individuals with afibrinogenemia or type III von Willebrand’s disease,12,13,27,28,33,34 as well
as in vivo studies on mice with a targeted deletion of VWF or
fibrinogen,31,35 have demonstrated that VWF plays a major role in
initiating aggregation under high shear with fibrinogen (and fibrin)
playing a secondary role in stabilizing formed aggregates. Strikingly, mice lacking both VWF and fibrinogen still retained the
capacity to form aggregates in vivo35 through an adhesion process
dependent on plasma fibronectin36 and possibly other ligands. Why
multiple integrin ␣IIb␤3 ligands are required for platelet aggregation
and thrombus growth remains unclear.
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
PLATELET AGGREGATION
5089
Figure 2. Involvement of multiple adhesion receptorligand interactions in platelet aggregation under high
shear flow. Under conditions of rapid blood flow (typically
wall shear rates ⬎ 1000 s⫺1), the initial tethering of
platelets to the surface of immobilized platelets involves
VWF-GPIb interaction. This adhesive interaction is rapidly reversible and at shear rates up to 10 000 s⫺1 does
not readily support stable platelet-platelet adhesion, resulting in platelet movement (translocation) across the thrombus surface. Platelet stimulation by one or more soluble
agonists during translocation promotes the binding of
VWF and fibronectin to integrin ␣IIb␤3, leading to sustained platelet-platelet adhesion. At elevated shear rates,
the principal role of the fibrin(ogen)-integrin ␣IIb␤3 interaction is to stabilize formed aggregates.
Fibrinogen
The importance of the fibrinogen-integrin ␣IIb␤3 interaction in
supporting platelet aggregation has been reviewed in detail elsewhere.37 Fibrinogen, VWF, and fibronectin all have similar binding
affinities to the activated form of integrin ␣IIb␤3; under low shear
conditions, however, fibrinogen is thought to be the dominant
ligand supporting platelet aggregation, principally attributable to
its high molar ratio in plasma relative to other integrin ␣IIb␤3
ligands.38 Furthermore, with increasing shear (⬎ 1000 s⫺1), the
initiation of aggregation becomes progressively more dependent on
VWF and fibronectin, with in vivo studies of fibrinogen-deficient
mice demonstrating normal or even increased rates of initial
thrombus growth.35 These thrombi are invariably unstable, and
studies on mice expressing mutants forms of fibrinogen that have a
selective defect in their ability to engage integrin ␣IIb␤3 have
confirmed that part of the thrombus-stabilizing effects of fibrinogen
are linked to its capacity to bridge integrin ␣IIb␤3 receptors on the
surface of adjacent platelets,39 whereas fibrin formation anchors the
thrombus to the vessel wall.35
von Willebrand factor
The importance of VWF in initiating platelet-platelet adhesion
contacts under conditions of rapid blood flow is well established,40
with evidence that it participates over a much broader (and lower)
shear range than previously anticipated.12,28,33,41 There are a range
of factors that lead to the preferential binding of platelets to VWF
under flow, including (a) VWF’s very large multimeric structure,
which provides an array of binding sites for platelet receptors; (b)
VWF’s efficiency at capturing platelets from bulk flow (through
engagement of GPIb), thereby increasing its spatial proximity to
integrins30; (c) potential shear-dependent changes in VWF’s globular conformation that increases the availability of receptor binding
sites; and (d) the ability of the VWF-GPIb interaction to transmit
signals that initiate integrin ␣IIb␤3 activation, thereby increasing the
affinity of integrin ␣IIb␤3-VWF bonds.42,43 These factors, combined
with the fact that VWF-GPIb bonds are positively modulated by
shear, provides a mechanistic explanation for the dominant role of
VWF in promoting platelet aggregation with progressive increases
in blood flow. The importance of VWF multimer size in regulating
platelet thrombus formation has recently been highlighted through
the study of mice lacking the VWF cleaving metalloproteinase
ADAMTS13 whose deficiency predisposes to thrombotic thrombocytopenic purpura.44 Mice lacking ADAMTS13 have a much faster
rate of thrombus formation leading to premature vasculature
occlusion. Furthermore, infusion of recombinant ADAMTS13
promotes thrombus dissolution in injured mouse arterioles, raising
the possibility that cleavage of VWF multimers may represent an
effective antithrombotic approach.
Fibronectin
For many years, fibronectin was thought to play a minor role in
platelet aggregation. For example, deficiency of plasma fibronectin
in mice is not associated with a defect in platelet aggregation using
standard aggregometer assays, nor is it associated with a prolonged
bleeding time.45 The demonstration more than 20 years ago that
depleting fibronectin from plasma reduced thrombus formation
under flow raised the possibility for its involvement in sheardependent platelet aggregation.29 Recent in vivo studies have
supported these findings,35,36 with Wagner et al demonstrating
persistent thrombus formation in arterioles of mice lacking VWF
and fibrinogen, suggesting the involvement of a third adhesive
ligand in platelet aggregation.35 Follow-up studies on mice expressing low levels of plasma and platelet fibronectin have confirmed an
important role for fibronectin in promoting platelet aggregation and
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JACKSON
thrombus growth.36,46 Interestingly, all stages of thrombus development appear to be affected by fibronectin deficiency, including
thrombus initiation, growth, and stability. Fibronectin influences
both platelet adhesion and aggregation as crosslinking fibronectin
with polymerized fibrin in combination with added plasma fibronectin, synergistically enhanced platelet adhesion, aggregation, and
thrombus growth.47 Fibronectin’s role in shear-dependent platelet
aggregation is incompletely understood and is likely to involve
multiple factors. Fibronectin is known to bind other proteins
involved in aggregation, such as fibrinogen and thrombospondin, a
process that may increase the stability of platelet-platelet interactions.48 As with VWF, fibronectin has been demonstrated to
undergo conformational changes in response to mechanical stress,49
a finding that may partially explain the preferential role for
fibronectin in promoting thrombus formation under high shear.
Fibronectin has also been noted to exhibit a distinct pattern of
matrix assembly on the surface of platelets50 and have distinct
binding characteristics from fibrinogen to the activated form of
integrin ␣IIb␤3.50,51 How these distinct binding interactions with
platelets facilitate platelet aggregation remains unknown.
Although considerable progress has been made in recent years
in defining the relative roles of VWF, fibrin(ogen), and fibronectin
in promoting shear-dependent platelet aggregation, many important issues remain, not least, the mechanisms controlling deposition
of these proteins on the platelet surface. It also remains to be
determined what the relative contributions of platelet-derived and
plasma VWF, fibrinogen, and fibronectin are in platelet aggregation. Confocal imaging of fibrinogen and VWF distribution during
thrombus development reveal distinct spatial localization of the
different protein pools with evidence that platelet stores of
fibrinogen are important for consolidating the thrombus core.52 It is
also unclear why a combination of ligands (including VWF/
fibrinogen or fibronectin/fibrin) are far more reactive to platelets
than in isolation, a finding that may have direct clinical relevance
because elevated plasma levels of VWF, fibrinogen, and possibly
fibronectin independently increase the risk of atherothrombotic
complications.
Unraveling the complex dynamics of
platelet aggregation
One of the most striking findings from intravital studies of
thrombosis is the dynamic nature of platelet aggregation in vivo. A
characteristic feature of initial thrombus formation is the high
proportion of platelets that translocate (movement from the point of
initial attachment) on the injured vessel wall and on the surface of
thrombi before forming firm adhesion contacts.12,39 This adhesive
behavior is similar to the rolling interactions of leukocytes with
postcapillary endothelial cells at sites of inflammation, where
initial tethering and rolling is mediated by the P-selectin-PSGL-1
interaction and firm adhesion is mediated by leukocyte ␤2integrins.53 The initial tethering of platelets is principally mediated
by the VWF-GPIb interaction, whereas firm adhesion depends on
ligand engagement of ␤1 and ␤3-integrins.19 Platelet translocation is
typically stop-start in nature and, depending on the type and extent
of vascular injury, a considerable proportion of tethering platelets
will only interact transiently with the thrombus surface.12 Defining
the factors regulating the stop-start nature of platelet translocation
is a critical issue, because it is one of the key variables regulating
the rate and extent of thrombus growth.
BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
Platelet shape and translocation dynamics
An important factor influencing cell rolling behavior is morphology.53 In the case of leukocytes, their round morphology is well
suited to rotational motion (rolling); the flat discoid morphology of
platelets, however, is less ideal for smooth rolling interactions.
Platelets have been demonstrated to assume different morphologies
in vitro after prolonged exposure to adhesive surfaces such as
VWF,54-56 raising the possibility that signals generated during
surface translocation may induce morphological changes that
facilitate a rolling phenotype. It has also been demonstrated that
platelets can translocate as flat discs through a rotational side-toside flipping mechanism57 or through a sliding, rotational movement of the cell body.58 Recent high-resolution imaging of platelets
in vivo have revealed that the majority of platelets translocate on
the vessel wall and on the surface of thrombi in vivo as flat, sliding
discs.59 Most platelets slide in a stop-start manner, although
rotational flipping of discoid platelets can also be observed.
Relative to rotating discs or rolling spheres, sliding platelets
experience reduced drag force and have much lower tensile stresses
on adhesive bonds. A flat disc also enables maximal surface contact
area with the adhesive surface, increasing the potential for multivalent adhesive interactions. Thus, by adopting a sliding, adhesive
behavior, platelets appear to have evolved a unique translocation
mechanism that minimizes drag forces on adhesive bonds. Combined with their small dimensions, this translocation mechanism
provides an efficient means of limiting the detaching effect of
blood flow on translocating platelets. As discussed subsequently,
this sliding mechanism appears to be greatly facilitated by the
development of small membrane protrusions (tethers), which
further reduce tensile stress on adhesive bonds.
Importance of membrane tethers in the initiation of
platelet aggregation
A significant recent development in the study of platelet adhesive
interactions under flow has been the finding that membrane tethers
play an important role in the initiation of platelet-matrix and
platelet-platelet adhesive interactions56,58-60 (Figure 3A). Membrane tethers are smooth cylinders of lipid bilayer that are pulled
from the surface of platelets under the influence of hemodynamic
drag force. These structures are dynamic and extend from localized
adhesion contacts and appear to play a role in regulating platelet
translocation behavior.60 Similar structures have been demonstrated in a variety of cell types, including red blood cells,61
neutrophils,62 neurons,63 fibroblasts,64 and endothelial cells,65 and
similar to platelets, membrane tethers are thought to play an
important role in regulating leukocyte rolling behavior.62
One of the key features of membrane tethers is their ability to
reduce the pulling forces imposed on adhesive bonds66 and, as a
consequence, increase the probability of sustaining adhesion in a
shear field. Strikingly, tethers of up to 30 ␮m have been observed
extending from the surface of platelets,58,60 a massive extension of
membrane from a cell that is only 1 to 3 ␮m in diameter. Because
surface membranes can only be normally stretched by approximately 4% before causing cell lysis,67 the creation of such tethers
must involve utilization of membrane reserves from the surfaceconnecting canalicular system. These membrane reserves can also
buffer changes in surface membrane tension, helping to dampen the
platelet activating effects of rapid fluctuations in blood flow.64
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Role of adhesion receptor signals and soluble agonists
in coordinately regulating platelet aggregation
dynamics under flow
Figure 3. Membrane tethers regulate platelet-matrix and platelet-platelet adhesive interactions. (A) (Left) Scanning electron microscopy of a single discoid platelet
forming membrane tethers during adhesion to immobilized VWF (1800 s⫺1). Note that
tethers can be readily distinguished from filopodia in that the latter is inhibited by
pretreating platelets with the actin polymerization inhibitor, cytochalasin D. (Right)
Shear-dependent formation of adhesion contacts between discoid platelets involves
the development of membrane tethers (arrows). (B) (Left) Reversible aggregation of
discoid platelets. Note that in this image, all platelets cluster around a central
activated platelet through the development of membrane tethers. (Right) The
development of stable platelet aggregates is associated with classic platelet shape
change characterized by sphering of the platelet body and the extension of multiple
filopodial projections. (Modified from Brass et al68; used with permission.) (This
research was originally published in Maxwell et al.59 and was adapted with
permission. Platelets were imaged on a Hitachi S570 scanning electron microscope
[Tokyo, Japan] at 20 kV accelerating voltage, 3 mm working distance.)
Role of membrane tethers in a 2-stage platelet
aggregation process
What is the role of membrane tethers in promoting the formation of
stable platelet aggregates? Recent high-resolution imaging of
platelets during thrombus development has provided new insights
into this process59 (Figure 3B). These studies have revealed the
existence of a 2-phase platelet aggregation process, an initial
reversible phase of aggregation occurring between discoid platelets
and a subsequent stable aggregation phase associated with platelet
shape change and granule release. The formation of discoid platelet
aggregates is shear-dependent and primarily mediated through the
development of membrane tethers. This initial phase of aggregation
involves platelet activation and the adhesive function of both GPIb
and integrin ␣IIb␤3; conspicuously, however, it does not require
soluble agonists such as adenosine diphosphate (ADP), TXA2, or
thrombin. Nonetheless, these aggregates are always reversible with
conversion to stable aggregates dependent on soluble agonist
generation, most notably ADP. Thus, tethers appear to play an
important role in maintaining close physical proximity between
platelets, which may provide a mechanism of facilitating autocrine/
paracrine stimulation by locally generated agonists. Such a mechanism may help limit the “washout” effects of blood flow on
released soluble agonists, providing a means of maintaining a high
local concentration of activating signals within the confines of a
developing aggregate.68 One of the key features of membrane
tethers is that they provide a mechanism of sustaining platelet
interactions with the thrombus surface without the requirement for
global platelet activation.
One of the more vexing issues with respect to platelet aggregation
is the significance of GPIb and integrin ␣IIb␤3-derived activating
signals. In general, there is limited insight into the spatiotemporalsignaling relationship operating between adhesion receptors and
soluble agonists in regulating platelet or leukocyte activation under
flow. For example, leukocyte rolling velocity is critically influenced by the level of cell stimulation by specific chemokines69;
these input signals, however, do not occur in isolation and are likely
to be modified (and amplified) by signals emanating from the
adhesion receptors themselves.70 Based on perfusion studies on
immobilized VWF, GPIb and integrin ␣IIb␤3 appear to elicit
distinct, cooperative activating signals71,72 that can regulate platelet
translocation dynamics partially through the reversible activation
of integrin ␣IIb␤3.71 In general, GPIb and integrin ␣IIb␤3 signals are
inefficient at inducing global platelet activation in the absence of a
costimulus such as ADP, thrombin, TXA2, or collagen.73 Most
current models of thrombus development propose a key role for
collagen (and possibly vessel wall-derived thrombin) in initiating
platelet activation in primary adherent platelets, whereas subsequent propagation of thrombi (platelet aggregation) is primarily
driven by agonists released or generated from the platelet surface,
including ADP, TXA2, and thrombin. Soluble agonists do not
recruit platelets into forming thrombi, however, adhesion receptors
do, which begs the question whether activating signals downstream
of GPIb and integrin ␣IIb␤3 are important to promote initial platelet
activation and thrombus development. The answer to this, at least
in the context of initial thrombus growth, remains uncertain and
controversial.73-76 There is currently limited evidence that signals
downstream of GPIb are essential for the initiation of hemostatic
plug formation or thrombus development. Similarly, the role of
integrin ␣IIb␤3-derived signals in initiating platelet aggregation and
thrombus development remains unclear. It is possible that activating signals downstream of GPIb play an important role in initiating
localized integrin ␣IIb␤3 activation necessary for membrane tether
formation and reversible aggregation. Similarly, there is evidence
that integrin ␣IIb␤3-derived signals, in combination with soluble
agonists, synergistically enhance platelet activation under flow.73,77
In isolation, GPIb and integrin ␣IIb␤3 signals are weak; nonetheless,
it is conceivable that localized signals from these receptors may
play an important role in facilitating the initial formation of
reversible aggregates and, in combination with soluble agonists,
Figure 4. Factors stabilizing formed platelet aggregates. The intercellular space
between aggregating platelets facilitates interaction between integrins and their
ligands and other adhesion molecules, and promotes activation of Eph receptor
kinases by cell surface ephrins. This narrow space also provides a protective
environment for the accumulation of soluble agonists (ADP, thrombin, and TXA2),
Gas-6, and the proteolytically shed exodomains of platelet surface proteins (GPIb,
P-selectin, sCD40L).
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
Figure 5. Distinct mechanisms initiating platelet aggregation at various shear rates. (A) Platelet aggregation at shear rates ⬍ 1000 s⫺1 is predominantly mediated by the
interaction of fibrinogen with integrin ␣IIb␤3. Under these conditions, stable aggregation typically occurs between shape-changed platelets. (B) At shear rates between 1000 and
10 000 s⫺1, a distinct 2-stage aggregation process can be identified. The initial formation of aggregates occurs between discoid platelets, is mediated by membrane tethers,
and is dependent on the adhesive function of both GPIb and integrin ␣IIb␤3. Conversion of reversible aggregates to stable aggregates is associated with platelet shape change
and is dependent on the generation of soluble agonists, particularly ADP. (C) At shear rates ⬎ 10 000 s⫺1, platelet aggregation can be initiated independent of integrin ␣IIb␤3 or
platelet activation and is exclusively mediated by the VWF-GPIb interaction. Although the initial aggregates form between discoid platelets, at such high shears, the platelets
adopt a smooth spherical morphology and roll (translocate in a rotational manner) across the VWF surface (unpublished observations, Erik Westein and Shaun P. Jackson).
(Figure was adapted with permission from Maxwell et al.59)
enhance the incorporation of these aggregates into stable
thrombi. Definitive resolution of this issue will require the
development of mouse models expressing mutant forms of GPIb
and integrin ␣IIb␤3 that have preserved adhesive function but a
selective signaling defect.
Sustaining platelet aggregation and
thrombus stability
Platelets must not only be able to develop adhesive contacts under
rapid blood flow, but must also sustain them to prevent thrombus
detachment (embolization) from the site of vascular injury. Recent
studies have revealed that a complex repertoire of adhesion and
signaling receptors are present on the platelet surface that may
serve to regulate the stability of forming aggregates (Figure 4). The
details of this system have recently been reviewed68 and only the
core elements are briefly presented here. Central to thrombus
stability is the maintenance of high-affinity/avidity integrin ␣IIb␤3
adhesion bonds. Initiation of integrin ␣IIb␤3 activation is controlled
by well-characterized signaling events operating downstream of
soluble agonist (Gq and G12/13) and adhesion (nonreceptor tyrosine
kinase) coupled receptors.9 Sustaining integrin ␣IIb␤3 activation is
critically dependent on signals operating downstream of Gicoupled receptors, principally the purinergic P2Y12 receptor.78
Thus, ADP plays a key role in both initiating (through the Gq-linked
P2Y1 receptor) and sustaining integrin ␣IIb␤3 activation necessary
for the development of stable platelet-platelet adhesion contacts.
There is growing evidence that the P2Y12 signals do not operate in
isolation. For example, once engaged by ligand, integrin ␣IIb␤3
initiates the formation of membrane-proximal signaling complexes
that not only serve to sustain platelet activation, but also induce
cytoskeletal changes that promote integrin ␣IIb␤3 clustering and
increased receptor avidity.79 Recent evidence suggests that members of the tetraspanin family, CD15169 and TSSC6,80 play an
important role in regulating integrin ␣IIb␤3 outside-in signaling,
with a deficiency in TSSC6 leading to a defect in thrombus
stability.80 The development of close platelet-platelet contacts also
enables the juxtaposition of ligands on one platelet with receptors
on adjacent platelets. Examples of this include various members of
the immunoglobulin superfamily (PECAM-1,81 JAM-A,82 JAMC,83 ESAM ,84 and CD22685), Eph kinases/ephrins,86 and Gas6 and
its receptors, Axl-Tyro3-Mer.87 The role of these individual components in regulating the stability of platelet aggregates is only
beginning to be addressed, although there is evidence that Eph
kinases/ephrins88 and Gas687 and its receptors play an important
role in this process. The exodomains of various platelet surface
proteins, including P-selectin,89 CD40L,90 GPIb,91 GPV,92 GPVI,93
and Sema4D,68 are also shed from the surface of platelets with
evidence that the soluble form of CD40L promotes thrombus
stability by engaging integrin ␣IIb␤3.94
Multiple adhesion mechanisms initiating
platelet aggregation
Based on findings from in vitro perfusion systems12,28,33,34,95 and in vivo
thrombosis models,35,36,39,59 at least 3 distinct mechanisms of platelet
aggregation can be identified with the relative contribution of each
mechanism dependent on the prevailing shear conditions. As demonstrated in Figure 5, the key components of the traditional model of
platelet aggregation, in which aggregation is mediated exclusively by
fibrinogen and integrin ␣IIb␤3, is likely to be the predominant mechanism operating under relatively low shear conditions (⬍ 1000 s⫺1).
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
Under such conditions, integrin ␣IIb␤3 on the surface of free-flowing
platelets can engage fibrinogen adsorbed onto the surface of thrombi.
Note that this adhesive interaction can occur independent of VWFGPIb, although even at these shear rates, the latter adhesive interaction
may have a role.41 Subsequent stimulation of platelets by locally
generated soluble agonists induces platelet shape change and an increase
in integrin ␣IIb␤3 affinity, which helps stabilize/sustain integrin ␣IIb␤3fibrinogen bonds. At shear rates between 1000 and 10 000 s⫺1, the
processes initiating aggregation are quite distinct involving additional
receptors, ligands, and membrane tethers. At these shear rates, plateletplatelet interactions become progressively more VWF-dependent with
an important role for both GPIb and integrin ␣IIb␤3 in promoting the
initial formation of discoid platelet aggregates. Whether fibronectin is
also involved in these earliest stages remains to be seen; nonetheless, at
these shear rates, there are essential contributions of VWF, fibrin(ogen),
and fibronectin to the formation of stable platelet aggregates. Recently, a
third mechanism initiating platelet aggregation has been identified that is
operative at very high shear rates (⬎ 10 000 s⫺1).95 Strikingly, this
aggregation mechanism does not require platelet activation or the
adhesive function of integrin ␣IIb␤3 and is exclusively mediated by
VWF-GPIb adhesive bonds. Aggregate formation is dependent on both
immobilized and soluble forms of VWF, and at such high shear, marked
deformation to the membrane of nucleating platelets (the nidus for
aggregate formation) is a hallmark feature of the aggregation process.
How important these membrane changes are to the formation/
maintenance of adhesive bonds remains to be established; nonetheless,
the findings that nonactivated platelets can form large aggregates under
very high shear have potentially important implications for the mechanisms promoting occlusive thrombus formation in stenosed arteries. It is
important to recognize that none of these models are identical to the
traditional model of aggregation developed from studies in the aggregometer. In the latter situation, regardless of the physiological agonist,
the platelet response is essentially the same; platelets undergo shape
change, become “sticky,” form aggregates, and release granule contents,
a temporal sequence of events distinct from those occurring under flow.
Translating basic insights into clinical benefit
The identification of discrete shear-specific mechanisms initiating
platelet aggregation, coupled with a clearer understanding of the
key elements propagating and sustaining thrombi, raises the
possibility that antiplatelet therapies may be developed that preferentially operate in certain vascular territories and at distinct stages
of the thrombotic process. Evidence supporting such a possibility
has been derived from in vivo studies targeting specific platelet
adhesion events. For example, deficiency of plasma fibronectin
produces a platelet aggregation defect that is distinct from that
observed with VWF or fibrinogen deficiency35,36 without producing
a marked prolongation in bleeding time. Given the preferential role
for fibronectin in promoting thrombus growth and stability under
elevated shear,46 selective blockade of the fibronectin-integrin
␣IIb␤3 interaction may be an attractive approach to prevent
thrombotic vascular occlusion while maintaining mural thrombus
formation. Similarly, blockade of the integrin ␣IIb␤3-binding sequence of fibrinogen (C-terminal ␥-chain dodecapeptide sequence)
also produces a marked defect in thrombus formation,39,96 raising
the possibility that selective inhibition of specific integrin ␣IIb␤3
ligands, rather than global inhibition of integrin ␣IIb␤3 itself, may
represent a more targeted antithrombotic approach. Furthermore,
given the well-defined role for the GPIb-VWF interaction in
supporting platelet adhesive interactions under high shear, inhibi-
PLATELET AGGREGATION
5093
tion of this adhesive event has been considered an attractive
approach to limit thrombus formation in high shear flow situations.
Inhibitors of the VWF-GPIb interaction are highly effective in
preventing thrombotic vascular occlusion,97 although it remains to
be established whether such approaches will ultimately have an
improved therapeutic window relative to conventional integrin
␣IIb␤3 antagonists.8
Similar concepts are beginning to emerge in the context of
soluble agonists. For example, there is a considerable body of
evidence supporting a major role for ADP in not only potentiating
platelet aggregation, but also in sustaining formed aggregates
necessary for the development of stable thrombi. Genetic deletion
or pharmacological blockade of the P2Y12 receptor leads to a major
defect in thrombus growth and stability regardless of the primary
thrombogenic stimulus.98 These amplifying effects of ADP are
likely to be important for thrombus development/stability in most
areas of the circulation, producing a significant hemostatic defect at
high levels of receptor blockade. Several signaling components
acting downstream of the P2Y12 receptor appear to also play an
important role in thrombus development/stability but are less
critical for hemostasis. These include 2 members of the PI 3-kinase
family, p110␤ and p110␥, which play an important role in
sustaining integrin ␣IIb␤3 activation and stable platelet aggregation.99,100 Other platelet components that have been demonstrated
to be important for thrombus stability, but less important for initial
thrombus growth, include sCD40L94 and Gas687 and its receptors.
Whether other agonist receptors and their downstream signaling
components regulate platelet aggregation under specific rheological conditions and at discrete stages in the thrombotic process
remains to be seen.
It should be acknowledged that a great deal of the recent
progress in the understanding of platelet aggregation has been
based on experimental animal models, particularly the mouse,
which inevitably raises issues of relevance to humans. In this
context, none of the commonly used in vivo thrombosis models
(involving ferric chloride, photochemical or laser injury of healthy
vessels) accurately reproduce the conditions of arterial thrombosis
in humans, in which thrombi typically form on ruptured or eroded
atherosclerotic plaques. Moreover, the rheological conditions in the
mouse microcirculation used for intravital studies are likely to be
significantly different from those operating in stenosed human
arteries. It also remains unclear whether observations from in vivo
thrombosis models can be extrapolated to platelet aggregation
processes relevant to hemostatic platelet plug formation. However,
as with any experimental model, the biological plausibility of
findings can only be established when carefully analyzed in the
context of other experimental data and clinical observations. Thus,
in the same way that findings in the aggregometer helped validate
the importance of various adhesive proteins, soluble agonists/
receptors, and signaling cascades in hemostasis, insights from ex
vivo perfusion studies on human platelets, combined with data
from carefully performed clinical studies, will be required to more
precisely validate the role of the growing list of “new players” in
the platelet aggregation/thrombosis process.
Conclusions
Great progress has been made over the last decade in unraveling the
complex and dynamic processes regulating platelet aggregation,
particularly in the context of arterial thrombosis. Although most of
the key elements have probably been identified, some unexpected
From www.bloodjournal.org by guest on September 7, 2015. For personal use only.
5094
BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
JACKSON
findings continue to surface that challenge many long-held beliefs
in the field, not least the recent demonstration that under certain
experimental conditions, platelet aggregation can occur independent of platelet activation and integrin ␣IIb␤3 engagement.95
These new insights, although currently of primary interest to the
academic community, are likely to be rapidly translated into new
experimental approaches that may ultimately hold great promise
in the clinic. What was once considered a straightforward
phenomenon, platelet aggregation has evolved into a complex
field of investigation with many of its most jealously guarded
secrets only now beginning to emerge.
Acknowledgments
I thank Drs Denisa Wagner and Zaverio Ruggeri, members of my
laboratory and the Department of Haematology, Alfred Hospital,
for constructive advice on the review. The assistance with preparation of figures by Dr. Simone Schoenwaelder is also gratefully
acknowledged.
This work was supported by grants from the National Health
and Medical Research Council of Australia, the Australian Research Council, and the National Heart Foundation of Australia.
Authorship
Conflict-of-interest disclosure: The author declares no competing
financial interests.
Correspondence: Shaun P. Jackson, Australian Centre for Blood
Diseases, Monash University, 6th Floor, Burnet Tower, 89 Commercial Rd., Melbourne, Australia, 3004; e-mail: shaun.jackson@med.
monash.edu.au.
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2007 109: 5087-5095
doi:10.1182/blood-2006-12-027698 originally published
online February 20, 2007
The growing complexity of platelet aggregation
Shaun P. Jackson
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