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Platelet aggregation, the process by which gation in vivo, it has become apparent platelet aggregation, with each of these
platelets adhere to each other at sites of that this process is much more complex mechanisms operating over a specific
vascular injury, has long been recognized and dynamic than previously anticipated. shear range in vivo. The identification of
as critical for hemostatic plug formation Over the last decade, it has become clear shear-dependent mechanisms of platelet
and thrombosis. Until relatively recently, that platelet aggregation represents a mul- aggregation has raised the possibility that
platelet aggregation was considered a tistep adhesion process involving dis- vascular-bed–specific inhibitors of plate-
straightforward process involving the tinct receptors and adhesive ligands, with let aggregation may be developed in the
Introduction
The propensity of platelets to clump together at sites of vascular Historical aspects
injury was first recognized more than 100 years ago.1-4 This
phenomenon, most accurately described as platelet cohesion Platelet aggregation was first recognized in the late 1800s after a
although more commonly referred to as platelet aggregation, series of pioneering studies by Zahn (1872), Hayem (1878),
was quickly identified as important for hemostatic plug forma- Bizzozero (1881 and 1882), Osler (1886), and Eberth and Schim-
tion.5 It was also recognized at the time that platelets played a melbusch (1885-1888).1-4,6 Using intravital imaging techniques,
key role in the development of thrombosis,6 but, it was not until Bizzozero clearly established that small cell elements (in which he
almost a century later that it became widely accepted that originated the term “Blut Plattchen” or blood platelets) clumped
platelets played a pivotal role in development of cardiovascular together at sites of vessel damage,1,14 a process subsequently
disease.7 As a consequence, inhibitors of platelet aggregation described as viscous metamorphosis.2 Through a series of insight-
have become increasingly important parts of the armamentarium ful observations, Bizzozero described the changes platelets un-
for the prevention and treatment of many atherothrombotic dergo after exposure to foreign surfaces, including the formation of
disorders.8,9 aggregates and subsequent “white thrombi.” He also clearly
For more than 3 decades, the factors mediating platelet described the plugging of small vascular punctures by platelet
aggregation appeared conceptually straightforward, requiring a thrombi, heralded as a major discovery at the time.5 It was soon
platelet stimulus (agonist), a soluble adhesive protein (fibrino- appreciated that quantitative or qualitative defects in platelets lead
gen), and a membrane-bound platelet receptor (integrin ␣IIb3 or to a bleeding disorder; and in 1918, Glanzmann15 described a
GPIIb-IIIa), leading to a simple unified model of platelet familial bleeding diathesis (“hereditary hemorrhagic thrombasthe-
aggregation (Figure 1). Although these core elements remain nia”) that was later shown to be a primary defect in platelet
fundamental, recent technical advances allowing real-time aggregation. Progress in the understanding of platelet aggregation
analysis of platelet aggregation in vivo have demonstrated a was slow to develop until the advent of the platelet aggregometer in
much more complex and dynamic process than previously 1962 by Born16 and independently by O’Brien.17 This relatively
anticipated. It is now widely accepted that one of the key simple technique involved stirring a suspension of platelets in the
elements influencing the platelet aggregation process is blood presence of a platelet activating substance and, by monitoring
flow, with evidence that distinct aggregation mechanisms oper- changes in light transmission, the device could accurately monitor
ate under different shear conditions.10-13 This has raised the platelet clumping (aggregation) in suspension (Figure 1). Aggregom-
interesting possibility that vascular bed-specific inhibitors of etry revolutionized the study of platelet function, providing funda-
platelet aggregation may be developed in the future and mental insights into the molecular mechanisms underlying platelet-
has stimulated a reevaluation of the temporal sequence of platelet adhesive interactions. The identification and characterization
events underlying the aggregation process. In this review, I of all major physiological agonists, antagonists, surface receptors,
discuss recent developments in the study of platelet aggregation and intracellular signaling pathways in platelets has involved
with particular focus on the mechanisms operating under rapid platelet aggregation studies, and in many situations there is a good
blood flow. correlation between defects in aggregation and bleeding disorders
thrombus growth.36,46 Interestingly, all stages of thrombus develop- Platelet shape and translocation dynamics
ment appear to be affected by fibronectin deficiency, including
thrombus initiation, growth, and stability. Fibronectin influences An important factor influencing cell rolling behavior is morphol-
both platelet adhesion and aggregation as crosslinking fibronectin ogy.53 In the case of leukocytes, their round morphology is well
with polymerized fibrin in combination with added plasma fibronec- suited to rotational motion (rolling); the flat discoid morphology of
tin, synergistically enhanced platelet adhesion, aggregation, and platelets, however, is less ideal for smooth rolling interactions.
thrombus growth.47 Fibronectin’s role in shear-dependent platelet Platelets have been demonstrated to assume different morphologies
aggregation is incompletely understood and is likely to involve in vitro after prolonged exposure to adhesive surfaces such as
multiple factors. Fibronectin is known to bind other proteins VWF,54-56 raising the possibility that signals generated during
involved in aggregation, such as fibrinogen and thrombospondin, a surface translocation may induce morphological changes that
process that may increase the stability of platelet-platelet interac- facilitate a rolling phenotype. It has also been demonstrated that
tions.48 As with VWF, fibronectin has been demonstrated to platelets can translocate as flat discs through a rotational side-to-
undergo conformational changes in response to mechanical stress,49 side flipping mechanism57 or through a sliding, rotational move-
a finding that may partially explain the preferential role for ment of the cell body.58 Recent high-resolution imaging of platelets
enhance the incorporation of these aggregates into stable cytoskeletal changes that promote integrin ␣IIb3 clustering and
thrombi. Definitive resolution of this issue will require the increased receptor avidity.79 Recent evidence suggests that mem-
development of mouse models expressing mutant forms of GPIb bers of the tetraspanin family, CD15169 and TSSC6,80 play an
and integrin ␣IIb3 that have preserved adhesive function but a important role in regulating integrin ␣IIb3 outside-in signaling,
selective signaling defect. 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
Sustaining platelet aggregation and on adjacent platelets. Examples of this include various members of
thrombus stability the immunoglobulin superfamily (PECAM-1,81 JAM-A,82 JAM-
C,83 ESAM ,84 and CD22685), Eph kinases/ephrins,86 and Gas6 and
Platelets must not only be able to develop adhesive contacts under its receptors, Axl-Tyro3-Mer.87 The role of these individual compo-
rapid blood flow, but must also sustain them to prevent thrombus nents in regulating the stability of platelet aggregates is only
detachment (embolization) from the site of vascular injury. Recent beginning to be addressed, although there is evidence that Eph
studies have revealed that a complex repertoire of adhesion and kinases/ephrins88 and Gas687 and its receptors play an important
signaling receptors are present on the platelet surface that may role in this process. The exodomains of various platelet surface
serve to regulate the stability of forming aggregates (Figure 4). The proteins, including P-selectin,89 CD40L,90 GPIb,91 GPV,92 GPVI,93
details of this system have recently been reviewed68 and only the and Sema4D,68 are also shed from the surface of platelets with
core elements are briefly presented here. Central to thrombus evidence that the soluble form of CD40L promotes thrombus
stability is the maintenance of high-affinity/avidity integrin ␣IIb3 stability by engaging integrin ␣IIb3.94
adhesion bonds. Initiation of integrin ␣IIb3 activation is controlled
by well-characterized signaling events operating downstream of
soluble agonist (Gq and G12/13) and adhesion (nonreceptor tyrosine Multiple adhesion mechanisms initiating
kinase) coupled receptors.9 Sustaining integrin ␣IIb3 activation is platelet aggregation
critically dependent on signals operating downstream of Gi-
coupled receptors, principally the purinergic P2Y12 receptor.78 Based on findings from in vitro perfusion systems12,28,33,34,95 and in vivo
Thus, ADP plays a key role in both initiating (through the Gq-linked thrombosis models,35,36,39,59 at least 3 distinct mechanisms of platelet
P2Y1 receptor) and sustaining integrin ␣IIb3 activation necessary aggregation can be identified with the relative contribution of each
for the development of stable platelet-platelet adhesion contacts. mechanism dependent on the prevailing shear conditions. As demon-
There is growing evidence that the P2Y12 signals do not operate in strated in Figure 5, the key components of the traditional model of
isolation. For example, once engaged by ligand, integrin ␣IIb3 platelet aggregation, in which aggregation is mediated exclusively by
initiates the formation of membrane-proximal signaling complexes fibrinogen and integrin ␣IIb3, is likely to be the predominant mecha-
that not only serve to sustain platelet activation, but also induce nism operating under relatively low shear conditions (⬍ 1000 s⫺1).
BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12 PLATELET AGGREGATION 5093
Under such conditions, integrin ␣IIb3 on the surface of free-flowing tion of this adhesive event has been considered an attractive
platelets can engage fibrinogen adsorbed onto the surface of thrombi. approach to limit thrombus formation in high shear flow situations.
Note that this adhesive interaction can occur independent of VWF- Inhibitors of the VWF-GPIb interaction are highly effective in
GPIb, although even at these shear rates, the latter adhesive interaction preventing thrombotic vascular occlusion,97 although it remains to
may have a role.41 Subsequent stimulation of platelets by locally be established whether such approaches will ultimately have an
generated soluble agonists induces platelet shape change and an increase improved therapeutic window relative to conventional integrin
in integrin ␣IIb3 affinity, which helps stabilize/sustain integrin ␣IIb3- ␣IIb3 antagonists.8
fibrinogen bonds. At shear rates between 1000 and 10 000 s⫺1, the Similar concepts are beginning to emerge in the context of
processes initiating aggregation are quite distinct involving additional soluble agonists. For example, there is a considerable body of
receptors, ligands, and membrane tethers. At these shear rates, platelet- evidence supporting a major role for ADP in not only potentiating
platelet interactions become progressively more VWF-dependent with platelet aggregation, but also in sustaining formed aggregates
an important role for both GPIb and integrin ␣IIb3 in promoting the necessary for the development of stable thrombi. Genetic deletion
initial formation of discoid platelet aggregates. Whether fibronectin is or pharmacological blockade of the P2Y12 receptor leads to a major
also involved in these earliest stages remains to be seen; nonetheless, at defect in thrombus growth and stability regardless of the primary
findings continue to surface that challenge many long-held beliefs for constructive advice on the review. The assistance with prepara-
in the field, not least the recent demonstration that under certain tion of figures by Dr. Simone Schoenwaelder is also gratefully
experimental conditions, platelet aggregation can occur indepen- acknowledged.
dent of platelet activation and integrin ␣IIb3 engagement.95 This work was supported by grants from the National Health
These new insights, although currently of primary interest to the and Medical Research Council of Australia, the Australian Re-
academic community, are likely to be rapidly translated into new search Council, and the National Heart Foundation of Australia.
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 Authorship
secrets only now beginning to emerge.
Conflict-of-interest disclosure: The author declares no competing
financial interests.
Acknowledgments Correspondence: Shaun P. Jackson, Australian Centre for Blood
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