Download Beyond Hemostasis: The Role of Platelets in Inflammation and Infection Archibald McNicol

Cardiovascular & Haematological Disorders-Drug Targets, 2008, 8, 99-117
99
Beyond Hemostasis: The Role of Platelets in Inflammation, Malignancy
and Infection
Archibald McNicol1,2 and Sara J. Israels3,4,*
1
Departments of Oral Biology, 2Pharmacology & Therapeutics, 3Pediatrics & Child Health and 4the Manitoba Institute of
Cell Biology, University of Manitoba, Winnipeg, Manitoba
Abstract: Platelets play a complex role in hemostasis and thrombosis. The expression of multiple membrane receptors,
both constitutive and activation-dependent, mediates platelet adhesion and aggregation at sites of vascular lesion. Platelet
activation leads to exocytosis of granular constituents, release of newly synthesized mediators, and discharge of membrane-bound transcellular signaling molecules. Many of the same mechanisms that play a role in hemostasis and thrombosis facilitate platelet participation in other physiological or pathological processes including inflammation, malignancy
and the immune response. Platelet receptors such as GPIb/IX/V, P-selectin, P-selectin glycoprotein ligand 1, CD40 and
the IIbß3 integrin, crucial to hemostasis, have been implicated in the progression of such inflammatory conditions as
atherosclerosis, rheumatoid arthritis and inflammatory bowel disease, in the progression and metastatic spread of malignancies, and in the immune response to bacterial challenge. The release of platelet granular contents, including adhesive
proteins, growth factors and chemokines/cytokines, that serve to facilitate hemostasis and wound repair, also function in
acute and chronic inflammatory disease and in tumor cell activation and growth. Platelets contribute to host defence as
they recognise bacteria, recruit traditional immune cells to the site of infection and secrete bactericidal mediators. The
primary focus of this review is the “non-haemostatic” functions of platelets in physiological and pathological states.
Key Words: Platelets, inflammation, metastasis, angiogenesis, immunity.
INTRODUCTION
Platelets are the smallest cells in circulating blood, have a
biconvex disc structure with an equatorial diameter of 2-3
m, and are anucleate. They are derived from progenitor
megakaryocytes in the bone marrow. Following their normal
life-span of 8-10 days they are removed from the circulation
during passage through the spleen. The role of platelets in
hemostasis was recognised very early [1]; their role in
thrombosis sometime later [2]. The consequences of platelet
dysfunction, and the potential of the platelet as a therapeutic
target, have been realized and exploited [3].
In invertebrates the functions of host defence and hemostasis are shared by a single cell type. The best-characterized
examples of this are the nucleated amebocytes of the horseshoe crab--these aggregate and release their granule contents
in response to such stimuli as exposure to foreign surfaces or
bacterial endotoxin [4]. Non-mammalian vertebrate thrombocytes and mammalian platelets have retained non-hemostatic roles in host defence and inflammation. During the last
decade, advances in functional analyses, knock-out models,
microscopy and characterization of the platelet proteome
have dramatically expanded our understanding of the role of
platelets in a variety of patho-physiological states, including
inflammatory disorders, tumor progression and infection.
PLATELETS IN HEMOSTASIS AND THROMBOSIS
The dynamics of blood flow dictate that circulating platelets are found primarily along the vessel wall, well posi*Address correspondence to this author at the Manitoba Institute of Cell
Biology, Rm. ON2021A, 675 McDermot Ave. Winnipeg, Manitoba, Canada,
R3E 0V9; Tel: (204) 787-4141; Fax: (204) 786-0195;
E-mail: [email protected]
1871-529X/08 $55.00+.00
tioned for rapid response at sites of endothelial lesions. The
platelet response to vessel wall injury with exposure of subendothelial collagen is characterized by the phases of adhesion, amplification and stabilization. This response is mediated by a variety of cell surface receptors, some of which are
constitutively present in an active conformation, some of
which require structural rearrangement to gain functional
integrity, and others that translocate to the platelet surface
following platelet activation [5].
The platelet glycoprotein (GP) Ib/IX/V complex acts as
an initial adhesive receptor at the endothelial lesion by binding to collagen-associated von Willebrand Factor (vWF).
Under low shear conditions, collagen binds to platelets via
the 2ß1 integrin (GPIa/IIa); GPVI and CD36 also bind to
collagen. Interestingly, both GPIb/IX/V and CD36 are platelet receptors for thrombospondin-1, an alternative to vWF in
mediating platelet adhesion under defined conditions. Several other platelet surface integrins function as adhesive receptors, notably IIbß3 (immobilized fibrinogen), 5ß1 (fibronectin), Vß3 (vitronectin) and 6ß1 (laminin) [5].
Following adhesion, a number of soluble mediators engage specific receptors that result in platelet activation and
amplification of the initial response as additional platelets are
attracted to the site. Thrombin, a product of the coagulation
cascade, is a powerful platelet agonist, mediated by the protease-activated receptors (PAR) 1 and 4, and by GPIb/ IX/V.
Activated platelets release ADP and thromboxane (Tx) A2,
both of which promote positive feedback stimuli via P2Y 1
and P2Y12, and via TP and TPß receptors, respectively. In
addition, platelets express receptors for epinephrine, platelet
activating factor (PAF), serotonin, vasopressin and immuno© 2008 Bentham Science Publishers Ltd.
100 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2
globulin G (IgG), each contributing to, and enhancing, platelet activation [5].
Additional platelets are recruited to the site of injury
through two specific processes: First the release, by exocytosis from platelet dense granules, of serotonin and of a nonmetabolic pool of ADP- the latter, in particular, is an important secondary mediator of platelet activation [6]. Second the
synthesis and release of TxA2 a pro-aggregatory mediator
critical to the full haemostatic function of the platelet [5].
TxA2 is generated by the sequential actions of phospholipase
A2, which liberates arachidonic acid from membrane phospholipids, cyclooxygenase-1, which converts arachidonic
acid to prostaglandin endoperoxides (PGG2/PGH2), then
thromboxane synthetase for the final synthesis of TxA2. Attenuation of either of these amplification pathways inhibits
platelet function and significantly prolongs bleeding in
vivo. Therapeutic ADP receptor antagonists, such as clopidogrel, and the cyclooxygenase inhibitor aspirin are effective
anti-thrombotic agents.
Additional platelets recruited by ADP- or TxA2-mediated
mechanisms combine with the fragile layer of adherent platelets at the site of injury to form a consolidated hemostatic
plug, or aggregate. This is facilitated by an activationmediated conformational change in the IIbß3 integrin, leading to the expression of its adhesive protein binding domain.
Fibrinogen is the major physiological ligand for this domaina bivalent molecule, fibrinogen binds to IIbß3 molecules on
adjacent platelets, establishing firm cross-linking to form a
stable plug [5].
In addition to the release of dense granule constituents,
two other exocytotic processes occur during platelet activation: the release of alpha granules and of lysosomal granules.
Alpha granules contain a large number of proteins of diverse
function, including adhesive proteins, chemokines, cytokines, coagulation factors and protease inhibitors (Table 1;
note that this table provides the definitions for all alpha
granule protein abbreviations that appear in the text) [7,8].
Lysosomal granules contain a variety of acid proteases, acid
glycosidases, acid phosphatases and aryl sulfatases [5].
During the process of exocytosis, granular membranes fuse
with the plasma membrane, resulting in the translocation of
granular membrane proteins onto the platelet surface. In this
way, increased numbers of constitutively-expressed proteins,
and novel proteins, appear on the platelet surface. Proteins
up-regulated on the platelet surface in this manner include
the IIbß3 integrin, P-selectin (CD62P), CD63, and the
CD40 ligand (CD40L; CD154) [5].
The phospholipid component of platelet plasma membranes is significantly altered following activation. The increase in expression of phosphatidylserine on the external
leaflet of the plasma membrane provides a larger surface
area for the assembly of coagulation factor complexes and
generation of thrombin. Two populations of small membrane
vesicles, microparticles and exosomes, are released from
activated platelets [9]. Microparticles have a similar phosphatidylserine-enriched composition to that of the plasma
McNicol and Israels
membrane of activated platelets and serve to significantly
increase the surface area available for thrombin generation.
Thrombin activates additional platelets and cleaves fibrinogen to fibrin; polymerized fibrin stabilizes the platelet plug.
Exosomes are smaller membrane vesicles released from activated platelets following fusion of multivesicular bodies and
alpha granules with the plasma membrane [9]. Although
exosomes do not have procoagulant function, they do express CD63 and prion protein, and have been implicated in
the inter-cellular transfer of information.
PLATELETS AND INFLAMMATION
Atherosclerosis
The first suggestion that platelets have a role in inflammation came, not surprisingly, from a pair of observations
related to atherosclerosis [10,11]: that atherosclerosis was an
inflammatory lesion, and that platelets were not merely passive constituents trapped in the atherosclerotic plaque. The
etiology of atherosclerosis is highly complex [12,13] and a
detailed description is beyond the scope of this review.
Briefly, increased levels of circulating low density lipoprotein
(LDL) diffuse through endothelial cell junctions into the subendothelial matrix. The trapped LDL undergoes a variety of
structural modifications, including lipid oxidation, resulting
in the formation, and trapping, of minimally oxidized LDL.
The accumulation of minimally oxidized LDL triggers the
activation of the overlying endothelial cells. This activation
is characterized by a loss of vascular integrity, cytokine production, and the expression and shedding of leukocyte adhesive molecules [14], leading to the recruitment and binding
of monocytes and lymphocytes. These leukocytes “roll” along
the endothelium and pass through endothelial cell junctions
in a classic inflammatory response. The intensity of the response is regulated by multiple factors, including genetic factors, elevated levels of free radicals generated by smoking,
hypertension or diabetes mellitus, elevated levels of homocysteine, sex hormones and infections (e.g. herpes virus,
Chlamydophila pneumoniae). Sub-endothelial monocytes
proliferate and differentiate into macrophages that engulf
highly oxidized LDL to form foam cells - accumulation of
these cells appear as fatty streaks; they release extracellular
lipids to form a necrotic core. Under the influence of cytokines and growth factors a mature atherosclerotic fibrous
plaque is formed as the necrotic core enlarges, becomes calcified and is infiltrated by smooth muscle cells [12,13].
Although it has been long appreciated that platelets are
incorporated into the thrombus that forms at the site of mature plaque, their role in the early stages of atherosclerosis
has been recognised more recently [15; Fig. (1)]. The characterization of the platelet releasate has identified a large number of proteins with significant pro-inflammatory properties,
including PF4, interleukin (IL)-1ß and RANTES [8]. Studies
using a variety of murine knockout models established that
quiescent platelets adhere to, and roll on, stimulated endothelial cells and demonstrated that there was a delay in the progression of the disease when certain adhesive proteins were
absent [16].
Beyond Hemostasis
Table 1.
Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 101
Platelet Alpha Granule Constituents
Adhesive Proteins
Fibrinogen chain
Fibrinogen chain
Fibrinogen chain
Fibronectin
Thrombospondin 1
Vitronectin
Von Willebrand Factor (vWF)
Chemokines
Other
Connective Tissue-Activating Peptide (CTAPIII; CXCL7)
Epithelial Neutrophil Activating Peptide (ENA-78; CXCL5)
GRO- (CXCL1)
I-309 (CCL1)
Interleukin-8 (IL-8; CXCL8)
Macrophage Inflammatory Protein 1 (MIP-1; CCL3)
Monocyte Chemoattractant Protein-1 (MCP-1; CCL2)
Monocyte Chemoattractant Protein-3 (MCP-3; CCL7)
Neutrophil-Activating Peptide-2 (NAP-2; CXCL7)
Platelet Basic Protein (CXCL7)
Platelet Factor (PF4; CXCL4)
Platelet Factor 4 variant 1 (PF4alt; CXCL4L1)
Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES; CCL5)
Stromal Cell-Derived Factor (SDF-1; CXCL12)
Thymus and Activation-Regulated Chemokine (TARC; CCL17)
-Thromboglobulin (-TG; CXCL7)
Cytokines
Growth Factors
Interleukin-1 (IL-1)
High Mobility Group Box
Chromosomal Protein-1
(HMGB1)
Basic fibroblast growth factor (bFGF)
Epidermal growth factor (EGF)
Hepatocyte growth factor (HGF)
Insulin-like growth factor-1 (IGF-1)
Insulin-like growth factor-2 (IGF-2)
Platelet-derived endothelial cell growth factor (PD-ECGF)
Platelet-derived growth factor (PDGF)
Transforming growth factor (TGF-)
Vascular endothelial growth factor A (VEGF-A)
Vascular endothelial growth factor C (VEGF-C)
Albumin
Amyloid -A4 protein
Angiopoietin-1
Angiostatin
Clusterin
Endostatin
Factor V
Factor XI
Factor XIII
High-molecular weight kininogen (HMWK)
Matrix metalloproteinase-2 (MMP-2)
Metalloproteinase inhibitor-1
Multimerin
Osteonectin
Plasminogen
Plasminogen activator inhibitor (PAI-1)
Protein C inhibitor
Protein S
Secretory granule proteoglycan core protein
(SGPCP)
Thrombocidin-1 (TC-1)
Thrombocidin-2 (TC-2)
Thymosin -4
Tissue inhibitor of metalloproteinase (TIMP-4)
von Willebrand antigen-II
2 macroglobulin
2-antiplasmin
-actinin 1
-actinin 2
-actinin 4
Protein and peptide contents of alpha granules that are released upon platelet activation, in response to a variety of stimuli. From Harrison and Cramer [7] and Coppinger et al. [8].
The process underlying the adhesion of quiescent platelets to activated endothelial cells has been the subject of considerable study. In vitro studies using activated human umbilical cord endothelial cells (HUVECs) demonstrated that
antibodies to either vWF or IIbß3 attenuate platelet adhesion, supporting the role of these proteins in the process [17].
Furthermore, blocking antibodies to the primary platelet
vWF receptor GPIb/IX/V inhibited atherosclerotic plaque
formation in an apoE-/- murine model, establishing vWF as a
mediator of platelet adhesion to dysfunctional (i.e. where
endothelial cell-dependent vasodilation is impaired [14]), and
to activated endothelial cells [16,18].
P-Selectin
P-selectin has been implicated as a factor in the plateletrelated development of atherosclerosis. P-selectin is expressed
only on the surface of activated platelets and, similarly, is expressed following activation of endothelial cells. Intravital
microscopy studies in mice have demonstrated that quiescent
platelets initially bind loosely to, and then roll on, endothe-
lial cells; these processes are mediated by the expression of
P-selectin on endothelial cells, but not on the platelets
[16,17,19]. P-selectin glycoprotein ligand 1 (PSGL-1; CD15)
and GPIb/IX/V have both been implicated as P-selectin
counter-receptors on the platelet surface. Both PSGL-1 and
GPIb/IX/V are constitutively expressed on the surface of
quiescent platelets and probably function in tandem to mediate the initial interaction of platelets with endothelial cell Pselectin [16,17,19]. Regardless of the counter-receptor(s)
involved, the P-selectin-mediated adhesion is fragile and
reversible (Fig. (1b)).
During the adhesion process, activation of platelets initiates a number of events with important pro-inflammatory
implications. Activated platelets express several integrins in
their “open” conformation, of which IIbß3 is the most important [16,17,19]. The IIbß3 binds adhesive proteins such
as fibrinogen, vWF and fibronectin which tether the platelets
to ICAM-1 and Vß2 on activated endothelial cells, thereby
consolidating platelet adhesion (Fig. (1c)). Studies in an
apoE/IIb double-deficient murine model showed signifi-
102 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2
McNicol and Israels
Fig. (1). Role of platelets in the early development of atherosclerosis.
d)
e)
PGSL-1
CD40
PGSL-1
Microparticles
CD40
a-granule
exocytosis
CD40L
I
PGSL-I
IIE,03
II
II
(T1b/IN/V
I1i
aIIbb3
11
Fbgn.
I Aen.
vWF
Vbs.;
IC.AM-]
CD40L
P-selectin
P-sclectin
GP1b/IX/V
a IIE
PGSL-1
0 0
P-sclectin
Beyond Hemostasis
Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 103
0
(Fig. 1. Contd….)
Microparticles
PGSL-1
RANTES
CCRI/
CCR5
P-selectin
Fig. (1 a). The progression of platelet involvement during the early stages of atherosclerosis. Quiescent, discoid platelets bind to the dysfunctional endothelium (broken line) and become activated, losing their discoid appearance and extending filopodia. Monocytes bind to, then roll
on, the activated platelets and finally migrate through the endothelial layer. Platelet-derived transcellular mediators stimulate changes in the
endothelial cells facilitating the direct binding and rolling of monocytes on the luminal surface. This progression is seen in more detail in figures 1b-1f.
Fig. (1b). Platelet receptors (GPIb/IX/V, IIb3, PSGL-1), endothelial cell receptors/counter receptors (ICAM-1, V3, P-selectin), and
adhesive proteins (vWF) mediate the binding of quiescent platelets to dysfunctional endothelial cells.
Fig. (1c). The platelet is activated, the IIb3 integrin is in an open, fibrinogen binding conformation, and the platelets express P-selectin and
CD40L.
Fig. (1d). Monocytes, via PSGL-1 and CD40, bind to the adherent, activated platelet
Fig. (1e). Monocytes roll along the adherent platelets to an endothelial cell junction. Concomitantly, platelets release microparticles and secrete -granule contents. Microparticles serve to deliver multiple transcellular mediators to the endothelial cell including RANTES.
Fig. (1f). RANTES binds to its serpentine receptor (CCR1/CCR5) leading to an upregulation of the luminal expression of monocyte-binding
adhesive receptors.
cantly reduced atherosclerotic plaque development [17,20].
As outlined above, activated platelets also express P-selectin
on their surface, which serves multiple pro-inflammatory roles
[16,17,19,21,22]. First, P-selectin mediates the adhesion and
rolling of PSGL-1-expressing immune cells (monocytes,
neutrophils, lymphocytes) on the adherent platelets (Fig.
(1d)). Second, P-selectin-mediated signaling leads to the
clustering of IIbß3 on the platelet surface, enhancing the
anchoring capacity of the integrin and facilitating firm platelet adhesion to the endothelium (Fig. (1c,d)). Third, the engagement of P-selectin appears critical in the exocytosis of
platelet alpha granule-derived chemokines (Fig. (1e); Table
1) some of which have been shown to participate in the development of the atherosclerotic lesion. Fourth, P-selectin
facilitates the delivery of platelet-derived chemokines, notably RANTES (see below), to the endothelium (Fig. (1e)).
CD40/CD40 Ligand
Platelet adhesion/activation is associated with the upregulation of CD40L, a member of the TNF family, which is
also found on activated T lymphocytes, monocytes, endothelial cells and vascular smooth muscle cells [16,19,23,24,25].
The CD40L receptor is CD40, an integral membrane protein
expressed on a variety of cells including B cells, monocytes,
endothelial cells, fibroblasts, vascular smooth muscle cells
and platelets. A proportion of CD40L is shed from activated
platelets in a soluble form (sCD40L); it has been estimated
that 95% of circulating sCD40L is platelet-derived [24]. The
104 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2
McNicol and Israels
plasma level of sCD40L is regarded as predictive of recurrent cardiovascular disorders, such as myocardial infarction
and stroke, but not of the development of atherosclerosis
[25].
the RANTES onto the activated endothelium (Fig. (1f)). The
binding of RANTES to the endothelium enhances monocyte
recruitment and activates monocytic integrins, consolidating
their attachment and rolling at the atherosclerotic lesion [29].
In atherosclerosis, membrane-bound CD40L, but not
sCD40L, interacts with endothelial cells and induces the expression of leukocyte adhesive molecules (e.g. VCAM,
ICAM, E - selectin), pro-inflammatory cytokines (e.g. IL-6,
IL-8, MCP-1) and matrix metalloproteinases [24,25]. In addition monocytes and macrophages are activated to release a
range of pro-inflammatory mediators. However, the association between platelet-derived CD40L and the early stages of
atherosclerosis is less well defined than, for example, the
role played by T lymphocyte-derived CD40L. In contrast,
both platelet membrane-bound CD40L and platelet-derived
sCD40L play important roles in the later stages of atherothrombosis where they induce tissue factor expression on
endothelial cells and monocytes, and bind directly to IIbß3
to stabilize thrombi [23,24,25]. The engagement of platelet
CD40 by either sCD40L or CD40L-expressing T-cells leads
to the activation of platelets and the exocytosis of RANTES
which, as outlined below, appears to be an important proinflammatory mediator [26].
PF4 was the first member of the chemokine family identified in platelets. It is released from activated platelets in a
P-selectin-dependent manner and, depending on the presence
of individual co-stimuli, plays multiple pro-inflammatory
roles [19]. In the presence of tumor necrosis factor- (TNF), platelet-derived PF4 stimulates both neutrophil exocytosis and firm adhesion to the endothelium. It also facilitates
RANTES-induced adhesion of monocytes to the endothelium. The presence of PF4 and RANTES together is associated with the progression of atherosclerosis, and conversely
antagonism of RANTES receptors significantly decreases the
size of atherosclerotic lesions. The effects of PF4 are not
limited to direct interactions with leukocytes. PF4 has been
shown to inhibit the degradation of the LDL receptor, thereby
limiting lipoprotein removal with associated pro-atherogenic
consequences [17]. PF4 also enhances the uptake of oxidized
LDL by macrophages-it is found associated with macrophages
in early, and with foam cells in late, atherosclerotic lesions
[17].
Studies using either CD40L-deficient mice or anti-CD40L
blocking antibodies showed both decreased overall atherosclerotic plaque development and a more stable, collagenrich, macrophage- and T lymphocyte-poor plaque structure
[23]. Thus the CD40/CD40L system is implicated in atherosclerosis, and in a number of other inflammatory disorders
(e.g. arthritis, psoriasis, multiple sclerosis, Crohn’s disease).
Chemokines/Cytokines
Interestingly, platelets also express receptors for a variety
of chemokines, notably for MCP-1 (CCR2), IL-8 (CXCR2),
fractalkine (CX3CR1), RANTES (CCR1/CCR5), SDF-1
(CXCR4), and TARC and MDC (both CCR4) [19]. Platelet
activation has been reported to occur in response to SDF-1
and fractalkine [27]; murine platelets lacking the fractalkine
receptor CX3CR1 display a decreased atherosclerotic phenotype [28]. In contrast, the engagement of CXCR4 or CCR4
by their respective chemokine, although not leading to platelet activation per se, significantly enhances the effect of low
levels of platelet agonists, such as thrombin or ADP [19].
Thus, these chemokines may play a role in the post-adhesion
platelet activation early in the development of the atherosclerotic lesion. These platelet receptors may also serve to accumulate chemokines at the lesion, as has been postulated for
CCR2. CCR2 is a low affinity MCP-1 receptor on platelets;
the engagement of CCR2 by MCP-1, however, has no effect
on platelet function. Platelets transport MCP-1 to the lesion
where it dissociates from CCR2 and accumulates at that site
[22].
RANTES is a major contributing factor to the vascular
inflammation associated with atherosclerosis. It is stored in
platelet alpha granules and released following activation as a
component of microparticles [19]. The microparticles provide an efficient, localised transfer mechanism, depositing
Platelets store and, upon activation, release a number of
other chemokines including MIP-1 , MCP-3, TARC, GRO and ENA-78 (Table 1; Fig. (1e)). In addition, ß-TG is a
precursor of CTAPIII, PBP and NAP-2. These molecules are
likely to have an impact on the recruitment and activation of
leukocytes during the progression of atherosclerosis [19,22].
Several cytokines are secreted by activated platelets
[17,19], of which IL-1 has been the most widely studied.
Platelets store IL-1ß in its precursor pro-IL-1ß form [17,19].
Platelet activation leads to the rapid release of active IL-1ß in
both a soluble form and in association with microparticles
[19] (Fig. (1e)). Although both forms enhance endothelial
cell adhesiveness for neutrophils, microparticle-associated
IL-1ß is significantly more effective, possibly because of the
presence of additional pro-inflammatory factors, such as Pselectin, on microparticles [30]. The conversion of pro-IL-1ß
to IL-1ß, and subsequent release of IL-1ß from platelets, occurs in a ß3 integrin-mediated process and, as demonstrated in
vitro, continues for several hours [30]. This suggests that
platelets play a significant role in the chronic inflammatory
response.
Growth Factors
Several other atherogenic mediators are released by activated platelets that may have a role in the chronic stages of
atherosclerosis, although in most cases the evidence is currently circumstantial. PDGF and TGF-ß, both secreted from
-granules, lead to smooth muscle cell proliferation and biosynthesis respectively [31]. Consequently, both are likely to
be involved during the stages of plaque development where
smooth muscle cells proliferate and infiltrate the plaque [17].
The contribution of platelets to inflammation associated
with other disorders is less well delineated, however platelets
have been implicated in the etiology of conditions such as
Beyond Hemostasis
Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 105
inflammatory bowel disease (IBD), rheumatoid arthritis,
systemic lupus erythematosus, psoriasis and migraine [32].
Inflammatory Bowel Disease
Several lines of evidence support a role for platelets in
ulcerative colitis and Crohn’s disease, the two most common
forms of IBD [24]. The platelets of individuals with both
conditions express significantly elevated levels of activation
markers, such as P-selectin and CD40L, in combination with
elevated levels of plasma ß-TG, PF4, and sCD40L, consistent with platelet activation [24]. Interestingly, the P-selectin
expression on platelets has been reported to be found primarily in mesenteric rather than in peripheral venous blood.
Histopathological examination of rectal mucosal capillaries
from patients with IBD showed the presence of microthrombi, although their presence is unrelated to the severity of inflammation. Platelets readily adhere to human intestinal microvascular endothelial cells (HIMECs) incubated in
vitro with IL-1ß. The accompanying expression of platelet
CD40L serves to facilitate platelet binding to the endothelial
cells. CD40L stimulates endothelial cells to upregulate surface levels of VCAM-1 and ICAM-1, two primary leukocyte
receptors, and to release the neutrophil chemoattractant IL-8
[33]. Platelet-derived sCD40L also stimulates intestinal cells
to increase adhesive receptor expression and chemokine release. The platelets of individuals with IBD also release
RANTES, which in in vitro studies, has been shown to be
highly bound to HIMECs [24].
Based on these studies, Danese and colleagues have suggested a sequence of events whereby platelets adhere to intestinal microvascular endothelial cells activated by IBD.
The platelets release sCD40L, RANTES and potentially other
chemokines, which are localized to the endothelium. This
results in the increased expression of adhesive receptors,
leukocyte adhesion and migration into the interstitium where
inflammation occurs [24].
Rheumatoid Arthritis
It is well-documented that platelets accumulate in the
synovial fluid of individuals with rheumatoid arthritis, although the significance is not well understood. The platelets
from individuals with rheumatoid arthritis express increased
levels of IIbß3 and form increased numbers of complexes
with monocytes, neutrophils and lymphocytes. However,
their platelets do not express more P-selectin, nor does their
plasma have elevated levels of TGF-ß [34]. Studies using an
animal model of arthritis have shown that platelets adhere to,
and roll on, arthritic endothelium [35]. Thus it is unclear
whether platelets contribute to the disease progression or
respond to the pathological setting of rheumatoid arthritis.
For example, platelets are responsive to both platelet activating factor and IgG, both of which are elevated in rheumatoid
arthritis.
Psoriasis
It has been recognized for 30 years that psoriasis is a risk
factor for thrombosis. The circulating blood of patients with
psoriasis has significantly more platelet microaggregates and
elevated ß-TG levels, when compared to blood of control
individuals [36]. The platelets of individuals with psoriasis
are hypersensitive to soluble agonists such as ADP, collagen
and arachidonic acid due, at least partly, to enhanced cyclooxygenase activity [37]. It was suggested that platelet hyperactivity led to the release of mitogenic and inflammatory
mediators that contributed to the psoriasis [36]. More recent
studies in a murine model demonstrated that activated platelets could roll on skin venules in a P-selectin- and IIbß3 mediated manner [38]. Furthermore platelet P-selectin expression is increased in individuals with psoriasis [38]. Although many platelet-associated inflammatory mediators are
implicated in the progression of psoriasis (e.g. CD40,
CD40L, fractalkine) the role, if any, that platelets play in the
development of the disease remains conjecture.
Migraines
For several decades platelets have been implicated as one
of the principle etiological factors underlying migraine headache [3 9,40]. This was based largely on the hypothesis that
migraine was considered to be a disorder of vascular tone,
that serotonin was the major contributing factor, and that the
platelets of these patients abnormally handled serotonin [40].
There is now evidence that migraine can be considered as an
inflammatory disorder specifically within cephalic tissue. It
is proposed that the release of inflammatory mediators activates peripheral nocioceptors associated with sensory fibres
innervating the meninges [41]. A variety of contributing mediators have been proposed, including protons, free radicals,
complement, prostaglandins, kinins and cytokines [41], including several platelet-derived cytokines (e.g. IL-1, ß-TG).
Although there is no conclusive evidence linking the
exocytosis of cytokines from platelets with the onset or progression of migraines, it is interesting to note that platelet
hyperactivity has long been recognized as a hallmark of this
condition; indeed there is a small, but significant, increase in
the incidence of stroke associated with migraines. Thus it is
plausible that enhanced platelet release may provide inflammatory mediators that synergize with other mediators of migraine. Furthermore, in common with other inflammatory
conditions, there is now evidence that patients experiencing
migraine without aura, in contrast to either those with aura or
controls, have significantly increased formation of plateletleukocyte complexes [42]. The identity of the receptors involved has not been determined.
Inflammatory Pulmonary Disease
Finally, a role for platelets in various pulmonary conditions has been postulated. The platelets of individuals with
asthma have higher than control levels of expressed Pselectin [43], and platelet-derived P-selectin functions to recruit leukocytes to their lungs [44]. Studies in human ex vivo
asthma models demonstrated that antibodies to P-selectin
attenuate platelet-leukocyte complex formation and subsequent pulmonary recruitment [45].
O’Sullivan and Michelson have summarized reports of an
intrinsic platelet dysfunction underpinning cystic fibrosis
106 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2
McNicol and Israels
[46]. The platelets from these individuals appear hyperactive
with increased production of both TxA2 and sCD40L. The
relationship between platelets and the progression of cystic
fibrosis remains to be fully addressed [46].
number of inflammatory conditions, the specific beneficial
anti-microparticle effects of these drugs have not been addressed.
A role for platelet-derived P-selectin in the development
of inflammatory lung disease has been recently postulated
[47]. In a murine model of acute lung injury, P-selectinmediated platelet-neutrophil interactions were critical to disease progression, and pulmonary inflammation was attenuated by blocking P-selectin. Furthermore antagonism of the
platelet TxA2 receptor significantly inhibited platelet-neutrophil interactions and subsequent neutrophil adhesion to, and
activation of, endothelial cells. Thus, it appears the platelet
cyclooxygenase pathway modulates the progression of inflammatory lung disease.
Platelets play a significant role in the pathogenesis of
malignancy. There is now evidence for the contribution of
platelets to tumor cell proliferation, metastasis, and tumor
angiogenesis, in animal models, and in a variety of tumors
types including carcinomas of breast, colon, lung, ovary and
melanoma. The association between malignancy and thrombosis was first recognized in 1865 by the French clinician
Armand Trousseau who made the observation that migratory
thrombophlebitis could be an indicator of occult malignancy;
he subsequently diagnosed his own fatal pancreatic carcinoma
by this sign [50,51]. Trousseau’s syndrome, the clinical
manifestation of the hypercoagulable state, seen particularly
with advanced carcinomas, is the result of a complex interaction among tumor cells, inflammatory cells, endothelium and
platelets. A decade later, the German surgeon Theodor Billroth described tumor thrombi within the blood vessels of
patients with cancer, and surmised that tumor emboli played
a role in the development of metastatic disease [52].
Therapeutic Implications for Inflammation
Anti-platelet drugs (i.e. those agents which affect platelet
function) play a well-defined role in the primary and secondary prevention of arterial thrombotic disorders. Inhibition of
cyclooxygenase-1 by aspirin with resultant decreased TxA2
generation, and antagonism of the ADP receptor by clopidogrel or ticlopidine attenuate the amplification pathways of
platelet activation. In addition, the blockade of the active site
of IIbß3 by humanized antibodies such as abciximab significantly reduces thrombotic complications associated with
percutaneous coronary intervention. The development of
safe, effective, orally active IIbß3 antagonists as antithrombotics has proved to be more challenging [48].
The multiple effects of platelets in inflammatory diseases
suggest that anti-platelet therapy may produce clinical benefit in these disorders. Aspirin is well accepted as an antiinflammatory agent, however its anti-platelet actions are
observed at doses (81-325 mg/day), significantly lower than
those required for traditional anti-inflammatory effects (1500
mg/day). Some of the beneficial effects of aspirin in cardiovascular disease, including atherosclerosis, are clearly not a
result of an anti-platelet action. For example, aspirin inhibits
reactive oxygen formation, increases nitric oxide synthase and
reduces C-reactive protein levels, each of which is cardioprotective but platelet-independent. Thus the complexity of the
situation in atherosclerosis makes it difficult to assign specific
benefit to the anti-platelet actions of aspirin in early stages of
atherosclerosis. In contrast, platelet inhibition is of fundamental importance to aspirin’s actions in athero-throm-bosis.
Few studies have addressed traditional anti-platelet drugs
in non-atherosclerotic inflammatory conditions. At present,
there is little evidence to support a specific anti-platelet action
of aspirin in inflammatory disorders.
Interestingly, a number of drugs in addition to traditional
anti-platelet agents (e.g. aspirin, ticlopidine, clopidogrel,
abciximab) have been shown to decrease the release of microparticles specifically from platelets, including: pravastatin, eprosartan, nifedipine, digoxin, iloprost and vitamin K
[49]. Although microparticles have been implicated in a
PLATELETS AND MALIGNANCY
The interaction of platelets and tumor cells is bidirectional, with tumor cell-expressed proteins activating platelets, and activated platelets promoting tumor survival and
progression by a number of different mechanisms discussed
below. The clinical importance of understanding the details
of these interactions is to determine whether it is possible to
use targeted therapy that prevent the contribution of platelets
or their releasates without incurring significant bleeding.
Some of these potential targets are common to inflammation
and malignancy; inhibiting those targets could abrogate the
contribution of platelets in both settings.
The role of platelets in the development of experimental
pulmonary metastases was originally recognized by Gasic
and colleagues in the 1960s. Thrombocytopenic mice were
protected from development of pulmonary metastases following tail vein injection of tumor cells [53]; this benefit was
reversed by platelet transfusion [54]. Subsequent studies
have confirmed a role for platelets in the development of
metastases, the formation of tumor emboli and the arrest of
tumor cells at sites on the vessel wall [55].
Tumor Cell-Platelet Aggregate Formation
The formation of platelet-tumor aggregates confers a
survival advantage to malignant cells by protecting them
from immune surveillance [56,57], allowing their persistence
in the vascular compartment and enhancing metastatic potential [53,54,58,59]. Platelets facilitate the adhesion of tumor
cells to vessel wall and release growth factors that may enhance tumor cell growth and angiogenesis at sites of extravasation (Fig. (2)).
Activation of platelets is the result of multiple mechanisms available to tumor cells. Thrombin, cancer procoagulant or cathepsin B, and matrix metalloproteinase-2 released
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Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 107
Fig. (2). The role of platelets in tumor progression and metastases.
Fig. (2a). The progression of platelet binding to tumor cells (TC). Tumor cells activate platelets using a variety of mechanisms (e.g. tissue
factor (TF) and thrombin) leading to changes in membrane composition, including the expression of IIb3 in its open conformation and Pselectin. The binding of platelets to tumor cells to form heterotypic aggregates is mediated by integrin/adhesive protein combinations (e.g.
platelet IIb3, tumor cell v3, fibrinogen) and by complexes of tumor cell mucins and P-selectin. Tumor cells tether, roll and adhere in a
manner similar to leukocytes shown in Fig. 1.
Fig. (2b). Activated platelets promote survival of tumor emboli in the circulation by protecting tumor cells from clearance by natural killer
cells (NK), allowing their adhesion and extravasation at metastatic sites. Release of platelet-derived factors, stimulate tumor growth (e.g.
LPA) and modulate neoangiogenesis (e.g. VEGF and endostatin).
from cancer cells can all activate platelets. Tumor-generated
thrombin also has an autocrine effect on tumor cells, and
stimulates heterotypic aggregate formation. A second mechanism depends on the constitutive expression of tissue factor
on the surface of some tumor cells leading to activation of
the coagulation cascade and thrombin generation on the procoagulant platelet surface. The potential to activate platelets
varies among tumor cell lines, but the capacity of tumor cell
lines to induce platelet aggregates in vitro correlates with increased in vivo thrombosis and metastases [55,60].
Platelet-tumor cell aggregates result from the cross-linking
of platelet integrins, primarily IIb3, with integrins expressed on tumor cells, such as v3 [61], by adhesive
ligands such as fibrinogen, fibronectin and vWF. In some
tumor cell lines, including breast, prostate, colon and melanoma, ectopic expression of megakaryocytic genes results in
the expression of functional IIb3 [62-65]. Expression of
IIb3 on tumor cells also may result from its transfer by
microvesicles, released from activated platelets, to the surface of malignant cells [66,67]. GPIb/IX/V is involved, and
blocking of its interaction with vWF reduces platelet-tumor
cell interactions [68-70]. Participation by other receptors
(31, 51) and adhesive proteins (laminin, vitronectin,
thrombospondin, and collagen) also has been identified [71].
Tumor cell-platelet adhesion mimics that of inflammatory
cells, with initial tethering and rolling mediated by P-selectin
108 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2
followed by firm adhesion to 3 integrins by fibrinogen and
vWF [72](Fig. (2a)).
P-selectin, which plays a critical role in the interaction of
platelets with inflammatory cells, is also emerging as a pivotal receptor in platelet-tumor cell interaction and the formation of platelet-tumor cell aggregates. The binding of Pselectin to carcinoma mucins mediates this interaction. Although some tumor cells, such as HL-60, express PSGL-1
[21], in most cases, the selectins recognize aberrantly glycosylated mucins [73-75] or the GPI-linked surface mucin
CD24 [76], which carry sialylated, fucosylated and sulfated
carbohydrates [77,78]. Specific cleavage of these mucins
blocked platelet binding to the tumor cells [77]. Heparan
sulfate-like proteoglycans expressed on some tumors can
also function as P-selectin ligands [77,79], and this finding
has prompted the investigation of heparin therapy targeted at
the selectin-ligand association (see below).
Platelets and Tumor Metastases
The role of platelets in promoting experimental metastases in mouse models has been demonstrated using a variety
of techniques to produce thrombocytopenia. More recently,
NF-E2 mice with marked thrombocytopenia have been shown
to be relatively protected from pulmonary metastases following tail vein injection of melanoma cells [80]. Fibrinogen
deficiency also is protective [80,81]. Platelets play an active
role in these models as inhibition of platelet activation or
aggregation abrogates metastatic potential. Inhibition of IIb3-mediated fibrinogen binding by monoclonal antibodies,
pharmacological inhibitors or by the genetic loss of integrin
3 decreases metastases [82,83,84]. Inhibition of platelet
activation by prostacyclin blocks tumor-induced platelet activation and experimental metastases [85]. Genetic alterations resulting in thrombin receptor PAR-4 deficiency [80]
or G protein Gq deficiency [86], give rise to mice with significant defects in agonist-induced platelet aggregation and
thrombus formation despite normal platelet counts, and protection from hematogenous metastases in comparison to
wild-type mice. Similar protection is observed in P-selectindeficient mice when injected with mucin-producing carcinomas [87].
The contribution of activated platelets to metastatic potential is likely multifactorial (Fig. (2b)). The mechanisms
by which platelets contribute to metastases include: (1) The
arrest of tumor cells at sites of vascular damage by tethering
and rolling on adherent platelet and or endothelial cell expressed P-selectin, followed by integrin-mediated stable adhesion [72,88]. The interaction of platelets and tumor cells at
the vessel wall bears many similarities to the interaction of
platelet/leukocyte responses to inflammation. (2) The provision of a physical barrier protects tumor cells from elimination by the immune surveillance of natural killer cells.
Common to several of the knock-out murine models described above, was the observation that impaired platelet
function did not alter the initial localization of tumor cells in
the lungs, but did affect the survival and growth of the tumor
emboli [86,87]. In the Gq -/- mice this survival advantage
McNicol and Israels
was lost following depletion of NK cells using anti-asialo
GM1 antibodies [86], suggesting that activated platelets may
prevent the direct cell-cell contact required for NK cellmediated tumor lysis [56,57,89].
Platelets and Tumor Growth
Activated platelets also contribute directly to tumor cell
proliferation and survival at both primary and metastatic
sites, by contributing to thrombin generation, release of
soluble mediators, and the generation of microparticles and
exososmes that transfer other growth mediators to adjacent
tumor cells.
Thrombin has been demonstrated to promote tumor
growth and metastases in murine models of spontaneously
metastasizing breast cancer. Pretreatment of mice with hirudin decreased tumor growth by ten-fold, and metastases
more significantly [90]. Endogenous thrombin generation
results from the expression of tissue factor on the surface of
tumor cells activating coagulation factors on the procoagulant surface of activated platelets. Ectopic expression of the
thrombin receptor PAR-1 on tumor cells and its activation by
thrombin enhances tumor cell growth and metastases [71].
This cycle of tumor-induced thrombin generation by platelets
and thrombin-induced tumor cell activation and growth can be
interrupted in vitro by either direct thrombin inhibitors [71]
or PAR-1 inhibitors [91].
Lysophosphatidic acid (LPA) is a bioactive water-soluble
phospholipid that has recently been shown to have a significant role in cancer cell proliferation, migration and metastases. LPA interacts with G - protein-coupled LPA receptors
that feed into multiple effector pathways by activating three
G proteins, Gq, Gi and G12/13. In tumor cells these pathways
enhance a variety of biological activities including cell proliferation, survival, migration, invasion, and the inhibition of
differentiation [92,93]. There appear to be several sources
for the LPA found in the tumor milieu, including generation
of LPA by a tumor cell ecto-enzyme autotaxin, a lysophospholipase that cleaves lysophosphatidylcholine. LPA also is
generated and released by platelets, the product of phospholipase A2 cleavage of phosphatidic acid. Activation of platelets by tumor cells stimulates LPA production and release,
which then binds to tumor cell LPA receptors (Fig. (2b)).
Using a breast cancer cell line that over-expressed the LPA
receptor, tumor cells could be stimulated by the supernatant
from activated platelets, and this effect was abolished by
treatment of the supernatant with LPA degradation enzymes
[94]. In this model, platelet LPA also stimulated tumor cell
release of IL-6 and -8, enhancing osteoclast activity and bone
resorption at sites of bony metastases [94].
Platelets release growth factors, adhesive proteins and
chemokines that stimulate tumor activity directly, or recruit
inflammatory cells that release soluble mediators such as
TNF that inhibits tumor cell apoptosis [95], promoting tumor cell survival. A potent source of mediators that can influence tumor cell growth is platelet microvesicles, both surface-derived microparticles and exosomes, which have been
shown to increase the growth and invasive potential of lung
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Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 109
and breast cancer cells. The effects of isolated platelet microvesicles on tumor cells in vitro included activation of
proliferative signaling pathways, and enhanced proliferation
and survival of cells in serum-free cultures [66,67]. Increased numbers of circulating peripheral blood microvesicles have been correlated with poorer prognosis in patients
with gastric carcinoma [96].
Tumor Angiogenesis
Platelet granules contain both pro-angiogenic and antiangiogenic factors (Table 2). These factors are released following platelet activation and secretion (Fig. (2b)); the balance appears to favour platelets providing a pro-angiogenic
stimulus in the context of malignancy [97,98]. Recent evidence suggests that pro- and anti-angiogenic mediators may
be packaged in distinct subsets of alpha granules [99], and
released by specific stimulation of one proteinase-activated
receptor, (PAR-1 induces release of VEGF) versus another
(PAR-4 induces release of endostatin) [100].
Table 2.
Angiogenic Factors in Platelet Alpha Granules
Pro-Angiogenic Mediators
Anti-Angiogenic Mediators
VEGF-A and VEGF-C
PF4 (CXCL4)
bFGF
Thrombospondin- 1
HGF
Angiostatin
IGF-1 and -2
Endostatin
EGF
TGF-
PDGF
PAI-1
PD-ECGF
TIMP-4
Angiopoietin- 1
MMP-2
Therapeutic Implications for Malignancies
The complex interaction of platelets with tumor cells
makes the platelet and platelet-generated effector molecules
apt targets. Despite in vitro studies showing inhibition of tumor-cell -induced platelet aggregation, aspirin, ticlopidine
and prostacyclin failed to affect experimental metastases in
mouse models [54,68]. More success was seen in experimental models with agents such as the chimeric antibody abciximab and the peptide epifibatide that block integrin binding to
adhesive proteins. Integrin blockade has been shown to inhibit platelet-tumor cell adhesion, experimental metastases
and platelet-induced sprouting of endothelial cells [68,82,
106]. Inhibitors of thrombin generation, including coumarins, heparins, and direct thrombin inhibitors such as hirudin,
also decreased experimental metastases [55,71,107].
At present, the most promising therapeutic agent is heparin. Clinical trials have demonstrated a survival advantage
for unfractionated and low molecular weight heparins over
placebo in subgroups of cancer patients without thrombosis
[108,109], and over warfarin in cancer patients with venous
thrombosis [110]. The advantage of heparin may be that its
effects on tumor growth and metastases are multiple, including inhibition of thrombin generation, fibrin formation, tumor
cell stimulation through PAR-1 receptors, and platelet activation (Table 3). Heparin also blocks binding of tumor cell
glyscosaminoglycans to selectins on platelets and endothelial
cells, inhibiting formation of heterotypic aggregates and their
adhesion to the blood vessel wall [77,111].
Table 3.
Anti-Malignant Properties of Heparin
Inhibition of thrombin and fibrin formation
Inhibition of tumor cell adhesion to platelets and endothelial cells
Interference with selectin binding to tumor cell glycosaminoglycans
Inhibition of angiogenesis
The most significant link between platelets and tumor angiogenesis may be VEGF. Platelets provide a large reservoir
of VEGF that is released following tumor cell-induced activation. The concentration of platelet-derived VEGF is a better predictor of tumor progression than serum concentrations
of VEGF [101]. In vitro studies have shown that platelets
stimulate endothelial cell proliferation and tube formation
[102,103], and inhibitors of IIb3 such as abciximab block
tumor cell-induced platelet VEGF secretion and sprouting of
endothelial cells [104]. VEGF stimulates the endothelial cell
expression of tissue factor and release of vWF, enhancing
platelet adhesion to tumor vessel wall. The abnormal tumor
vasculature and resultant turbulent flow also may contribute
to platelet adhesion, degranulation and increased release of
angiogenic factors within the tumor [105].
The release of platelet-derived microparticles under these
conditions also may play a role in angiogenesis; platelet microparticles stimulate tumor cell expression of angiogenic
factors including IL-8, VEGF and HGF and membrane metalloproteinases involved in degradation of extracellular matrix and remodeling of the basement membrane [66].
Stimulation of apoptosis
Immune system modulation
The ideal therapeutic agent(s) will be one that inhibits
platelet adhesion and release without increasing the risk of
bleeding beyond a tolerable range. We know little about the
optimal timing or duration for these agents to be effective in
preventing tumor progression and metastases, as most experimental models are not true reproductions of clinical tumor progression.
PLATELETS AND IMMUNITY
The potential association of infections, both bacterial and
viral, with the development of cardiovascular disease has
long been suspected. The best characterized is infective endocarditis [112], although a variety of infectious agents also
have been implicated in the etiology of conditions such as
atherosclerosis [113], myocardial infarction [114] and stroke
[115]. Only recently have mechanistic relationships been
110 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2
partially elucidated, and platelets have been implicated as
major contributors to the development and progression of
infection-associated cardiovascular disease. It is becoming
clear, however, that in addition to contributing to adverse outcomes, platelets play a host-defence role against bacterial
pathogens in particular [19].
Platelets and Bacteria
The interaction between platelets and bacteria has recently been extensively reviewed [116]. Bacterial species
cause platelet aggregation utilizing a variety of the mechanisms; even within individual species there may be strainspecific mechanisms of platelet activation. For example
Staphylococcus aureus, the most frequent causative pathogen
responsible for infective endocarditis [117], activates platelets by differing mechanisms depending on the presence of
specific proteins on the bacterial cell wall. S. aureus can
stimulate platelets via mechanisms involving: IgG and its
associate platelet receptor Fc RIIA, fibrinogen and its receptor IIbß3, vWF and its receptor GPIb/IX/V, and complement [116]. Indeed, the specific process invoked by individual strains of S. aureus likely dictates the speed of the platelet activation response [116]. A number of other pathogens
implicated in infective endocarditis, including species of the
Staphylococcus (S. epidermidis, S. capitis) and Streptococcus (S. sanguis, S. agalactiae, S. pyogenes, S. gordonii, S.
pneumoniae, S. mitis) genii, as well as Neisseria gonnorrhoeae and Pseudomonas aeruginosa, have been shown to
activate platelets using similar strategies to those utilized by
S. aureus [116].
The relationship between bacterial infections and coronary artery disease is less clear and somewhat controversial.
Individuals with active infections are at a higher risk and
those who have received antibiotics in the previous year are
at lower risk for acute cardiovascular events [116]. Ott and
colleagues identified the presence of DNA from a number of
different bacterial species in atherosclerotic plaques [113].
Consequently, it has been suggested that coronary artery
diseases, such as atherosclerosis [113] and myocardial infarction [114], are not associated with any single pathogen
but rather with the cumulative effects of multiple organismsthe concept of total pathogenic burden [113,114,116].
Helicobacter pylori, coincidentally first reported by Giulio Bizzozero [118] who also shares credit for the original
identification of platelets [2], is now accepted as the causative organism of many gastric disorders, and may also increase the risk of myocardial infarction and atherosclerosis
[119]. Similarly, Chlamydophila pneumoniae (previously
known as Chlamydia pneumoniae) has been reported to increase the risk of myocardial infarction, although prophylactic antibiotic therapy failed to prevent acute events in a highrisk population group [120-122].
Transient bacteremias with normal orally-derived bacteria are common, not only after dental procedures (extraction,
endodontic treatment, periodontal surgery, root scaling)
[123,124] but following regular “non-invasive” procedures
such as brushing [125] (notably with the use of electronic
McNicol and Israels
toothbrushes [126]), and in individuals with periodontal disease [127]. Such transient bacteremias, which involve both
anaerobes, such as Porphyromonas gingivalis, and aerobes,
such as S. sanguis, would contribute significantly and regularly to total pathogenic burden. P. gingivalis and S. sanguis
have both been isolated from atherosclerotic plaque [128],
although only P. gingivalis is a perio-pathogen [129]. Consequently there has been considerable interest in the relationship between oral disease, primarily periodontal disease, and
cardiovascular disease.
A systematic review completed in 2003 suggests an overall
moderate association between periodontal disease and atherothrombosis, including coronary artery disease, stroke and
peripheral vascular disease [130]. Furthermore, studies in
pigs and in mice have shown that infection with P. gingivalis
is as effective as a high cholesterol diet at inducing atherosclerosis [131,132]. Invasive, perio-pathogenic strains of P.
gingivalis have been shown to upregulate the expression of
adhesive receptors on [133], and the release of chemoattractants from, cultured endothelial cells [134]. This provides a
potential mechanism for the genesis of atherosclerotic lesions, including the adhesion of platelets to the activated
endothelial cells, as outlined above. Interestingly an invasive
strain of P. gingivalis enhances tissue factor expression and
activity, but attenuates tissue factor pathway inhibitor levels
[135], suggesting multiple targets in addition to platelets.
Individual strains of H. pylori, S. sanguis and P. gingivalis have been shown to activate platelets in vitro [116].
In each case, the presence of IgG and its platelet receptor
Fc RIIA and probably, vWF and its platelet receptor
GPIb/IX/V, are essential for activation to occur. Fitzgerald
and colleagues have proposed a general scheme by which
platelet activation can be stimulated by bacterial pathogens
(Fig. (3a)). This involves the initial adhesion of the bacterium to the platelet - this may involve either the direct interaction of a bacterial wall or membrane protein (e.g. clumping
factors A and B, fibronectin binding proteins A and B, serine-rich protein, serine-aspartate repeat protein), with a platelet receptor (e.g. GPIb/IX/V, IIbß3) or via an intervening
plasma protein (e.g. vWF, fibrinogen). Subsequently, circulating antibodies to proteins on the bacterial surface activate
the adherent platelets by engaging Fc RIIA [116]. Initial
activation includes the synthesis and release of TxA2 (most
bacteria-induced platelet aggregation is aspirin-sensitive),
and the subsequent expression and occupation of fibrinogen
binding sites on IIbß3 for full aggregation to take place. It
should be noted that a role for factors excreted by bacteria in
platelet activation cannot be excluded [116] (Fig. (3)).
Platelets and Innate Immunity
The response to infection comprises a rapid, antigenindependent innate response and a delayed, prolonged antigen-specific adaptive response. The innate response to bacterial infections is multi-factorial and has been reviewed for
several specific invading organisms [136,137]. In general,
bacteria secrete and/or express ligands (e.g. gram negative
bacteria-derived LPS, gram positive-derived lipoteichoic ac-
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Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 111
Fig. (3). Association of platelets with bacteria
Fig. (3a). Schematic representation of the association of platelets with bacteria (Bact), based on the model proposed by Fitzgerald and colleagues [113]. Bacterial cell wall or membrane proteins interact directly, or via adhesive proteins (e.g. vWF), with platelet membrane proteins, notably the GPIb/IX/V complex. IgG, bound to bacterial antigens engage FcRIIa on the platelet, stimulating signaling pathways culminating in TxA2 release and full platelet activation. Activated platelets engulf the bacteria in a heterogenous aggregate.
Fig. (3b). Transmission electron micrograph of platelets and S. sanguis (B) stirred together in a platelet aggregometer, demonstrating platelet
activation and extension of filopodia (arrows) that surround the bacteria. Scale bar = 0.5m.
ids, bacterial cell wall peptidoglycans) for the toll-like receptor (TLR) family of membrane proteins [138,139]. To date
12 TLRs have been described in humans and have been localized primarily to phagocytic cells (e.g. monocytes,
macrophages), epithelial cells and, as discussed below, platelets. Several of the TLRs, including TLR-1, TLR-2, TLR-4
and TLR-6 are expressed on the cell surface. TLR-2 forms
heterodimeric complexes with TLR-1 or TLR-6 and binds
lipoproteins and lipoteichoic acids, whereas TLR-4 recognises a variety of ligands, although LPS and viral proteins
appear to be its primary agonists. In contrast, some TLRs
such as TLR-8 and TLR-9 are located intracellularly and
serve as receptors for single stranded RNA and DNA, respectively [138,139]. Engagement of TLR-expressing cells
leads to cell-specific release of transcellular mediators, pri-
marily chemokines and cytokines that mediate the immune
response to invading organisms [138,139].
Interestingly platelets have been shown to express several
TLRs, including TLR-1, TLR-2, TLR-4, TLR-6, TLR-8 and
TLR-9 [140,141] with increased surface expression of TLR-2
and TLR-9 following activation [141]. The role of platelet
TLRs remains unclear. Studies in mice demonstrated that
engagement of TLR-4 by LPS stimulated an IIbß3-mediated binding of platelets to fibrinogen in the absence of Pselectin expression [142]. Furthermore LPS binding to TLR-4
caused thrombocytopenia in mice and, in the presence of
anti-platelet antibodies, was associated with decreased TNF
production [143]. Recently Semple and colleagues demonstrated that platelet-bound LPS, presumably via TLR-4, syn-
112 Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2
ergizes with anti-platelet antibodies thereby enhancing Fcreceptor-mediated phagocytosis [144].
The effects of LPS on IIbß3-mediated fibrinogen binding
may suggest a role for TLRs in platelet activation. The LPSinduced release of sCD40L from platelets was attenuated by
blockade of TLR-4 but not of TLR-2 or TLR-9 [145], consistent with the elevated levels of sCD40L observed in the
plasma of patients with meningococcal sepsis [146]. In contrast, Ward and colleagues were unable to show an effect of
ligands for TLR-2 or TLR-4 on cytosolic calcium mobilisation, P-selectin expression or aggregation, either alone or in
combination with traditional platelet agonists such as platelet
activating factor, epinephrine or ADP[147].
Anti-Bacterial Efects of Platelets
Intriguingly platelets may serve an anti-bacterial function.
It has been proposed that, as platelets rapidly accumulate at
sites of injury/infection, they may play similar “surveillance”
roles to that of traditional immune cells such as macrophages, mast cells and dendritic cells [148]. The upregulation of CD40L on the platelets surface (in response to vascular damage, soluble agonists or bacteria) has effects on adaptive immunity, including accelerating dendritic cell maturation, stimulating IgG production by B cells and enhancing T
cell activity [149]. Bacteria-induced platelet activation results in the secretion of granular contents, including various
chemokines that, as outlined above for inflammation, serve
to attract immune cells (monocytes, basophils, NK cells,
macrophages) to the site of infection [148]. Finally, platelets
adhering to and aggregating around bacteria may promote
bacterial clearance [148]; indeed activated platelets may internalize bacteria thereby removing them from the circulation [19].
Platelet alpha granules also contain thrombocidins, members of a family of anti-bacterial proteins that are found primarily in neutrophils and play a central role in the innate
immune system [19]. Two thrombocidins have been characterized, TC-1 and TC-2, which are variants of neutrophilactivating peptide-2 (NAP-2) and connective tissue-activating peptide-III (CTAP-3) respectively. TC-1 and TC-2, but
not the parent molecules, are bactericidal for several organisms including E. coli and S. aureus, and are fungicidal for
Cryptococcus neoformans [150]. Studies in rabbits have
shown thrombocidins to be important for host defence
against Streptococci-induced endocarditis [151]. Furthermore, the thymosin- 4, PBP, RANTES and PF-4 released
from activated platelets have been reported to have bacteriocidal effects against E. coli and S. aureus when tested in
vitro [152].
Platelets and Viruses
Associations between platelets and several viral infections
have been proposed. The hepatitis B virus (HBV) will bind to
GPVI on the platelet membrane which, it has been suggested, may serve to facilitate viral transport and promote
viral survival [153]. Studies, primarily in animal models of
HBV infection, have demonstrated that platelets facilitate the
McNicol and Israels
accumulation of cytotoxic T lymphocytes in intrahepatic
sites of necrosis and inflammation It is unclear whether this
is the result of the interaction of platelets with HBV particles
or platelet participation in the inflammatory response as outlined above.
Platelets have been shown to engulf viruses, most notably
human immunodeficiency virus type 1 (HIV-1); a significant
proportion of circulating HIV-1 is associated with platelets.
The adhesion of HIV-1 to platelets is mediated by two platelet membrane proteins, dendritic cell-specific intercellular
adhesion molecule 3-grabbing non-integrin (DC-SIGN), and
C-type lectin-like receptor 2 (CLEC-2) [156]. DC-SIGN
facilitates the endocytosis of HIV-1 by platelets [157],
whereas CLEC-2 is linked to Src kinase/phospholipase C 2
pathways and is unlikely to be involved in HIV-1 uptake
[156]. The importance of these platelet receptors, viral uptake and intracellular signaling to the progression of HIV-1
disease is unknown.
Platelets and Prions
The transmissible spongiform encephalopathies (TSE)
are a family of neurodegenerative disorders that include bovine spongiform encephalopathy in cattle, scrapie in sheep,
and Creutzfeldt-Jakob Disease and Gerstmann-StrausslerSchienker syndrome in humans. Infectious forms of TSE are
due to a variant protease-resistant form (PrPres) of a normal
cellular protein, prion protein (PrP) [158]. PrP is a glycophosphatidylinositol-linked membrane protein of unknown
function. However, its conversion to PrPres renders the protein highly infectious, affecting health care and agricultural
policies worldwide. PrP is present on the membrane of platelet alpha granules [159] and is released on exosomes following
platelet activation [160]. The presence of PrPres in platelets
has not been reported, however its sequestration, and subsequent exosomal release, could have significant implications
for TSE disease transmission, notably by blood transfusions
[161].
Therapeutic Implications for Infections
The therapeutic implications of manipulating interactions
between platelets and bacteria have not been widely addressed, although several antibiotics, notably penicillin, may
have anti-platelet side-effects. The increased understanding
of the role of both bacterial pathogens and platelets in the
development of atherosclerosis has led to the identification
of specific potential targets. The IgG receptor FcRIIA is a
common mediator of platelet responses to multiple bacterial
species. Antagonism of this receptor represents a logical strategy [116], particularly in keeping with the concept of total
pathogenic burden. Furthermore, FcRIIA antagonists are
unlikely to compromise the hemostatic function of platelets,
avoiding the bleeding risks associated with traditional antiplatelet drugs. The anti-bacterial potential of platelets could
be utilized by selectively releasing, or mimicking, thrombocidins.
The role of platelet interactions with T lymphocytes in the
progression of hepatic necrosis and inflammation caused by
Beyond Hemostasis
Cardiovascular & Haematological Disorders-Drug Targets, 2008, Vol. 8, No. 2 113
HBV, has raised the possibility that anti-platelet drugs may
reduce the severity of chronic liver disease in HBV infected
individuals [154] Studies are currently underway to determine the beneficial effects of aspirin and clopidogrel in
HBV-induced liver disease.
[6]
[7]
[8]
CONCLUSION
Platelets have retained many of the functions of the
primitive multi-functional hemocyte. The predominant function of platelets is considered to be hemostasis, where they
play a complex role integrated with other soluble and cellular
participants. Platelets adhere to the exposed sub-endothelial
collagen of a damaged vessel wall, are activated, up-regulate
additional adhesive receptors and secrete transcellular mediators, contributing to the formation of a stable platelet aggregate. Defective activation of platelets compromises hemostasis and can result in excessive clinical bleeding. In
contrast inappropriate platelet activation is associated with
arterial thrombotic conditions such as myocardial infarction
and stroke. Platelets are a primary target for the prevention of
recurrent cardiovascular thrombosis.
It is now clear that platelets have a wider repertoire of
physiological roles and therefore are implicated in more
pathological conditions than thrombosis; they are active participants in the immune response and host-defence. In these
situations, platelets function like traditional immune cells
such as macrophages and mast cells, binding to bacteria,
secreting chemokines, and clearing invading organisms from
the circulation. Many of the underlying mechanisms used by
platelets in their immune response are identical to, or extensions of, the hemostatic processes. Their contributions to
pathological lesions in inflammatory disorders, and to tumor
progression, are the result of these responses at the wrong
place and time.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
The challenge for therapeutic intervention in these diseases will be to identify drugs that preferentially block specific targets involved in the complex contribution of platelets
to inflammation or tumor progression, while leaving their
hemostatic function at least partially intact.
[22]
ACKNOWLEDGEMENTS
[23]
The authors would like to thank Dr. E. D. Israels for
helpful suggestions during the preparation of this manuscript
and E.M. McMillan-Ward for preparation of Fig. (3b). SJI is
funded by a grant from Cancer Care Manitoba and AM by a
grant from the Heat and Stroke Foundation of Canada.
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Accepted: 12 November, 2007