Can J Cardiol Vol 26 No 4 April 2010e140
The involvement of circulating microparticles
in inflammation, coagulation and
Paolo Puddu MD, Giovanni M Puddu MD, Eleonora Cravero MD,
Silvia Muscari, Antonio Muscari MD
Department of Internal Medicine, Aging and Nephrological Diseases, University of Bologna and S Orsola-Malpighi Hospital, Bologna, Italy
Correspondence: Dr Antonio Muscari, Department of Internal Medicine, Aging and Nephrological Diseases, University of Bologna – S Orsola-Malpighi
Hospital, Via Albertoni, 15, 40138 Bologna, Italy. Telephone 39-051-636-2280, fax 39-051-636-2210, e-mail email@example.com
Received for publication July 31, 2008. Accepted September 24, 2009
‘dust’ consisted of small vesicles (less than 0.1 µm in diameter) capable
of promoting coagulation. Subsequently, the release of microparticles
(MPs) from endothelial cells (ECs), vascular smooth muscle cells,
leukocytes, lymphocytes and erythrocytes has also been shown in
vitro. Some of these MP populations have been found in the blood of
both patients and healthy individuals.
MPs might play a significant role in the interactions among circulat-
ing and vascular cells. Several papers (3-9) have described the possible
effects of MPs in regulating vascular function, and their potential physi-
ological and pathological involvement in cardiovascular diseases. In
addition, MPs have recently been proposed as new therapeutic targets in
the treatment of cardiovascular diseases, in consideration of their pro-
thrombogenic and proinflammatory actions (10).
The present review discusses the putative roles played by MPs in
inflammation, coagulation and endothelial/vascular function, as well
as the possible and importance of MPs in the diagnosis, prognosis and
therapy of atherosclerotic diseases.
n 1967, membrane fragments of platelet origin with procoagulant
activity were described in human plasma as ‘platelet dust’ (1,2). This
FUNCTIONAL CHARACTERISTICS OF MPs AND
MOLECULAR BASIS OF THEIR FORMATION
MPs are phospholipid- and protein-rich submicron particles. These
fragments, originating from plasma membranes of eukaryotic cells,
typically contain cell surface proteins and cytoplasmic components of
their cell of origin (11,12), ranging in size from 0.1 µm to 2 µm. More
precisely, vesicles larger than 100 nm in diameter originating from
plasma membranes are usually called MPs, while smaller vesicles origi-
nating from the endoplasmic reticulum are described as ‘exosomes’.
Finally, larger particles (greater than 1.5 µm) containing nuclear com-
ponents are called ‘apoptotic bodies’ (3,13,14).
MPs are released from cell membranes by triggers such as cytokines,
thrombin, endotoxins, hypoxia and shear stress, capable of inducing
activation or apoptosis (3). It is unclear whether the mechanisms
underlying MP formation during these two events are identical (9).
The activation of platelets by different agonists promotes platelet
aggregation and secretion, as well as membrane vesiculation and MP
release. Thrombin, collagen and adenosine diphosphate bind specific
transmembrane receptors that, through changes in second messenger
©2010 Pulsus Group Inc. All rights reserved
P Puddu, GM Puddu, E Cravero, S Muscari, A Muscari. The
involvement of circulating microparticles in inflammation,
coagulation and cardiovascular diseases. Can J Cardiol 2010;
Microparticles (MPs) are small vesicles, ranging in size from 0.1 µm to
2 µm, originating from plasma membranes of endothelial cells, platelets,
leukocytes and erythrocytes. MPs can transfer antigens and receptors to
cell types that are different from their cell of origin. Circulating MPs pro-
vide a procoagulant aminophospholipid surface for the assembly of the
specific enzymes of coagulation. Both tissue factor and phosphatidylserine
are exposed on MP outer membranes. In addition, MPs can play a signifi-
cant role in vascular function and inflammation by modulating nitric oxide
and prostacyclin production in endothelial cells, and stimulating cytokine
release and tissue factor induction in endothelial cells, as well as monocyte
chemotaxis and adherence to the endothelium. Finally, increased levels of
MPs have been found in the presence of acute coronary syndromes, isch-
emic stroke, diabetes, systemic and pulmonary hypertension, and hyper-
triglyceridemia. From a practical point of view, MPs could be considered to
be important markers of cardiovascular risk, as well as surrogate end points
for assessing the efficacy of new drugs and therapies.
Key Words: Atherosclerosis; Coagulation; Inflammation; Microparticles
La participation de microparticules circulantes
dans l’inflammation, la coagulation et les
Les microparticules (MP) sont de petites vésicules de 0,1 µm à 2 µm qui
proviennent des membranes plasmiques des cellules endothéliales, des
plaquettes, des leucocytes et des érythrocytes. Les MP peuvent transférer
des antigènes et des récepteurs à d’autres types de cellules que leur
cellule d’origine. Les MP circulantes procurent une surface
amiophospholipidique procoagulante à l’ensemble des enzymes
spécifiques de coagulation. Tant le facteur tissulaire que la
phosphatidylsérine sont exposés sur les membranes externes des MP. De
plus, les MP peuvent jouer un rôle important dans la fonction et
l’inflammation vasculaires en modulant la production de monoxyde
d’azote et de prostacycline dans les cellules endothéliales et en stimulant
la libération de cytokine et l’induction de facteur tissulaire dans les
cellules endothéliales, de même que la chimiotaxie monocytaire et
l’adhésion à l’endothélium. Enfin, on constate des taux accrus de MP en
présence de syndromes coronariens aigus, d’accidents ischémiques
cérébraux, de diabète, d’hypertension systémique et pulmonaire ainsi
que d’hypertriglycéridémie. Sur le plan pratique, les MP peuvent être
considérées comme d’importants marqueurs de risque cardiovasculaire et
comme paramètres ultimes auxiliaires pour évaluer l’efficacité de
nouveaux médicaments et de nouvelles thérapies.
Microparticles in cardiovascular diseases
Can J Cardiol Vol 26 No 4 April 2010e141
concentrations, can modulate cellular responses (15,16). Alternatively,
the intracellular concentration of the second messenger can be directly
changed by agents such as calcium ionophores. The increase in intra-
cellular levels of calcium ions leads to the activation of calpain, with
subsequent degradation of cytoskeletal proteins. This mechanism is
believed to play a putative role in MP formation (15-17).
In the apoptotic pathway, caspase-3 plays an important role, acti-
vating the rho-associated kinase, resulting in the release of apoptotic
membrane vesicles (18).
The proteinic composition of MPs reflects the composition of the
cell membrane from which they are released. This includes constitu-
tively expressed antigens, and antigens that have been induced on the
parent cell by the activating or apoptotic triggers, leading to MP
The composition and distribution of constitutive cell membrane
phospholipids are highly specific. Phosphatidylserine (PS) and phos-
phatidylethanolamine are mainly sequestered in the inner leaflet of
the plasma membrane, while phosphatidylcholine and sphingomyelin
are mainly located in the outer membrane layer (11,20). This asym-
metric distribution is essential for biomembrane function and is under
the control of a complex transmembrane enzymatic balance that
involves enzymes such as gelsolin (present only in platelets), amino-
phospholipid translocase, floppase, scramblase and calpain (7).
During cell activation and the subsequent increase of Ca2+ concen-
tration in the cytosol, plasma membranes are modified and phospho-
lipid asymmetry is compromised. In particular, the loss of phospholipid
asymmetry results in the exposure of PS on the outer cell surface.
Because PS efficiently binds coagulation factors (21), it leads to a
prothrombotic state (22-25). Furthermore, following cellular activa-
tion, the cytoskeleton undergoes several changes. Spectrin and actin
are cleft, and protein anchorage to the cytoskeleton is disrupted.
Thus, an increase in bleb formation takes place and bleb-generated
MPs are released into the circulating blood. It has been shown that
platelet MPs (PMPs) express P-selectin, the integrin glycoprotein
(GP) IIb/IIIa, platelet endothelium adhesion molecule-1 (PECAM-1
[CD31]), CD63, CD42a and CD42b (7,15,26). GP IIb/IIIa blockade
inhibits platelet PS exposure by potentiating translocase and attenuat-
ing scramblase activities (27). Endothelial cell MPs (EMPs) express
CD31, CD34, CD51, CD54, CD146, E selectin and endoglin, and
bind von Willebrand factor (28). CD4, CD3 and CD8 are present at
the surface of leukocyte MPs (29,30).
MPs bear antigens of their cell of origin and can transfer these
surface signalling molecules to other types of cells. The binding of
surface antigens to their specific counter-receptors leads to the activa-
tion of intracellular signalling pathways (6). In this way, MPs behave
as vectors disseminating biological information to the cells of the vas-
cular compartment, which expose appropriate counter-receptors for
the ligands they harbour (31).
METHODS FOR MP MEASUREMENT
MP analysis has the potential to enter the mainstream of clinical test-
ing because it may provide important data for investigating various
vascular disorders, such as acute coronary syndromes, venous thrombo-
sis and stroke. However, the wide variety of methodologies used by
different laboratories to measure circulating MPs has occasionally
provided inconsistent or conflicting results (32), making data analysis
and clinical correlations challenging (33).
The main methods of MP detection include flow cytometry,
enzyme-linked immunoassays and functional coagulative assays
(32,34,35). Lal et al (36) recently developed a new method for the
detection of plasma MPs using a fluorescence-based antibody array
system that can rapidly identify the cell origin of MPs.
MP counting, as currently performed by flow cytometry, certainly
needs to be standardized. In this respect, the preanalytical phases of
blood sampling and MP separation according to standardized centrifu-
gation steps (37) are key factors. Robert et al (38) recently developed
a strategy for PMP counting with a widely available flow cytometry
instrument (Cytomics FC500; Beckman Coulter Inc, USA), using
size-calibrated fluorescent beads in a fixed numerical ratio (Megamix;
BioCytex, France). The intra- and interinstrument reproducibility was
tested by using annexin and CD41 coexpression to count MPs in pre-
viously frozen aliquots of the same platelet-free plasma over four
months and in platelet-free plasma from 10 healthy subjects in three
independent flow cytometers. Using the three instruments, similar
PMP counts were obtained. With the use of this standardized flow
cytometry protocol, PMP levels were significantly higher in women
than in men. This strategy for PMP count standardization could repre-
sent a first step toward multicentre studies, and could also be used for
MPs derived from other cell types. However, the measurement of cir-
culating MPs still presents standardization problems and is not yet
widely available in clinical practice.
ROLE OF MPs IN COAGULATION
Because PMPs were the first species identified, the studies on the role
played by MPs in physiological and pathological conditions have, for a
long time, concerned blood coagulation and hemostasis.
Activated platelets and circulating MPs provide a procoagulant
aminophospholipid surface for the assembly of the specific enzymes of
the coagulation cascade. After activation, MPs exhibit negatively
charged phospholipids (chiefly PS) at their surface, which, once in
contact with circulating blood factors, allow the local concentrations
necessary to achieve optimal thrombin generation as well as efficient
hemostasis (39). PS increases the procoagulant activity of tissue factor
(TF). TF and PS are both exposed on MP outer membranes and are
considered to be the main initiators of the coagulation cascade (8,40).
TF is a key player in the onset of blood coagulation (40) because in
vivo coagulation is initiated when TF binds factor VIIa and catalyzes
its activation. TF circulates in plasma, largely on monocyte/
macrophage- derived MPs that can bind activated platelets through a
mechanism involving P-selectin GP ligand-1 (PSGL-1) on MPs and
P-selectin on platelets (41). TF has been identified on leukocyte MPs,
EMPs and PMPs (12,19,42-46). Del Conde et al (47) found that MPs
derived from monocyte/macrophage cholesterol-rich rafts are selec-
tively enriched in both TF and PSGL-1, and deficient in CD45, sug-
gesting that they arise from distinct membrane microdomains.
Interestingly, the shedding of MPs was significantly reduced with
depletion of membrane cholesterol. MPs not only bound the activated
platelets, but fused with them via PSGL-1, transferring lipids and pro-
teins, including TF, in the plasma membrane. The phospholipids on
the surface of MPs from platelets and ECs provide a number of binding
sites for factors Va, VIII, IXa and IIa (15,48-50). EMPs express ultra-
large von Willebrand factor multimers, which promote and stabilize
platelet aggregates (51). These findings provide a mechanism by
which blood coagulation can be initiated and propagated on the sur-
face of activated platelets (47). MPs can also contribute to the devel-
opment of platelet- and fibrin-rich thrombi at sites of vascular injury,
through the recruitment of cells and the accumulation of TF (52).
Whether MPs are procoagulant in vivo is not a completely resolved
issue, but several data suggest that MP-mediated coagulation may be
clinically significant. For instance, an association between the number
of circulating MPs and the risk of thromboembolic complications has
repeatedly been demonstrated (9).
The procoagulant activity of MPs can be quantified using the
thrombin generation test. In this system, MPs supply the procoagulant
surface, while TF and plasma provide the necessary coagulation fac-
tors. By adding calcium ions, coagulation factors bind to MPs to initi-
ate coagulation. In this assay, the generation of thrombin is dependent
on the presence and activity of MPs, and in their absence, no coagula-
tion would occur (9).
ROLE OF MPs IN INFLAMMATION AND
EMPs might directly lead to the development of endothelial dysfunc-
tion. In fact, in vitro experiments have shown that EMPs
Puddu et al
Can J Cardiol Vol 26 No 4 April 2010e142
might regulate vascular tone by modulating both nitric oxide (NO)
and prostacyclin production in ECs (5). Moreover, the oxidized phos-
pholipids in the MPs released from ECs exposed to oxidative stress
may be particularly active in causing monocyte adherence to ECs and
activation of neutrophils (53,54).
A further key feature in atherogenesis is leukocyte adhesion to
ECs, with subsequent transendothelial migration of leukocytes (55).
Specific adhesion molecules on ECs interact with ligands that are pres-
ent not only on leukocytes, but also on leukocyte-derived MPs. Mesri
and Altieri (56,57) suggested that leukocyte MPs stimulate cytokine
release and TF induction in ECs by activating a signalling pathway
involving the tyrosine phosphorylation of c-Jun NH2-terminal
kinase-1. This may lead to increased proinflammatory and procoagu-
lant activity in ECs.
High shear stress-induced activation of platelets and the addition
of PMPs may also enhance the expression of cell adhesion molecules,
and the production of cytokines in the human monocytic leukemia
cell line (THP-1) and ECs (58,59). Moreover, PMPs may deliver
arachidonic acid to ECs, with consequent upregulation of intercellular
adhesion molecule-1 and subsequent monocyte adhesion (60). PMPs
can also promote leukocyte-leukocyte aggregation (61), as well as
monocyte chemotaxis (60) through the transformation of MP arachi-
donic acid into the proinflammatory and vasoconstricting thrombox-
ane A2 (62). However, PMPs may also induce the endothelial
production of cyclooxygenase-2 and of the vasodilating prostacyclin.
Finally, PMPs contain a significant amount of RANTES (Regulated
on Activation, Normal T cell Expressed and Secreted), an inflamma-
tory chemokine, and can deposit it on activated ECs, triggering mono-
cyte adhesion on these cells (63). By means of these mechanisms,
PMPs can modulate the inflammatory and vasomotor response (64).
However, although MPs may have deleterious, as well as beneficial
effects on vascular function in vitro, there is not yet any direct evi-
dence that they play a significant role in vascular dysfunction in vivo.
Specific studies are needed to address this question.
ROLE OF MPs IN CARDIOVASCULAR DISEASES
Although in vitro studies have suggested various possible molecular
mechanisms leading to MP formation (2,7,14), the precise mecha-
nisms of in vivo MP generation remain unclear. Moreover, it is also
unclear whether increased MPs are a cause or a consequence of vascu-
lar disease states because cardiovascular disease-related factors, such as
metabolic disturbances, cytokines and, possibly, infectious agents, can
trigger MP production.
It was suggested that MPs can spread proinflammatory and proco-
agulant mediators throughout the body in response to a stimulus,
including activation and apoptosis, contributing to the severity of the
disease (9). On the other hand, Agouni et al (65) reported possible
beneficial effects of MPs in a mouse model of endothelial dysfunction.
After injection in mice, MPs from human activated/apoptotic
T-lymphocytes were able to stimulate NO production from ECs,
enhancing endothelium-dependent coronary vasodilation. Fur-
thermore, the same MPs reversed endothelial dysfunction in a model
of mouse coronary arteries subjected to ischemia/reperfusion. This
effect was mediated by the morphogen sonic hedgehog (Shh), a
modulator of NO production carried by MPs that is also involved in
embryonic and adult development. Thus, MPs might exert beneficial
or deleterious effects for the vascular wall depending on their cellular
origin, the stimuli involved in their cellular generation and the clini-
cal setting (66,67).
Nevertheless, owing to the complex procoagulant and proinflam-
matory activities of MPs, research has mainly been focused on their
possible role in cardiovascular diseases (9). There is evidence that MP
levels are increased in patients with cardiovascular diseases and risk
factors, including acute coronary syndromes, diabetes, hypertension
and hypertriglyceridemia (29,68-72) (Table 1).
Human atherosclerotic plaques contain MPs released during cell
activation or apoptosis. Plaque MPs bear TF activity and expose PS, a
major determinant of their procoagulant activity (73,74). Leroyer et al
(75) demonstrated that plaque MPs originate from macrophages,
erythrocytes and smooth muscle cells, whereas circulating MPs are
mainly derived from platelets. MPs were more abundant and thrombo-
genic in plaques than in plasma. However, the study showed that most
of the circulating MPs do not originate from ruptured plaques, but are
generated within the blood compartment or at the blood-vessel
Atherosclerosis is initiated and propagated by EC dysfunction.
Werner et al (76) recently showed that EC apoptosis is independently
involved in the pathogenesis of endothelial dysfunction, and circulat-
ing CD31+/annexin V+ apoptotic MPs positively correlated with the
impairment of coronary endothelial function, independent of classic
Elevated levels of MPs with procoagulant potential are present in
the circulating blood of patients with recent clinical signs of coronary
plaque disruption and thrombosis (29). EMPs are associated with high-
risk coronary lesions, including multiple, irregular lesions, those with
an eccentric appearance and those with thrombi (69). Thus, EMPs
may be a useful marker for the risk of acute coronary events.
High levels of circulating EMPs were also found in patients with
acute ischemic stroke (77-79).
Furthermore, circulating EMPs are closely associated with vascular
dysfunction in patients with end-stage renal failure (80).
Preston et al (71) suggested that EMPs and PMPs increase in
severely hypertensive patients.
MPs bearing TF and endoglin as well as vascular cell adhesion
molecule-1 and chemo attractant protein-1 were elevated in patients
with pulmonary arterial hypertension compared with controls (81). It
was even suggested that MPs might prove to be valuable tools in deter-
mining the severity of pulmonary hypertension.
In patients with uncomplicated type 2 diabetes mellitus (DM),
Diamant et al (82) found elevated numbers of TF-exposing PMPs.
Although this MP-associated TF did not show any procoagulant activ-
ity, it might play a role in other processes such as angiogenesis, cell
growth and signal transduction. Patients with DM also showed ele-
vated levels of EMPs (83). In particular, CD144 EMP levels were sig-
nificantly higher in DM patients with coronary artery disease (CAD)
than in those without CAD, and allowed the identification of a sub-
population of DM patients who had CAD without typical chest
In patients with the metabolic syndrome, circulating MPs of vari-
ous origin are increased and impair endothelial function (84).
Ferreira et al (70) evaluated the possible relationship between
levels of EMPs and changes of postprandial hypertriglyceridemia in
healthy normolipemic subjects after a single high-fat meal. In these
subjects, the high-fat meal led to a significant elevation of plasma
EMPs, suggesting structural endothelial damage caused by triglycer-
ides, followed by the impairment of endothelial function (85-87).
Microparticle (MP) involvement in cardiovascular diseases
Coronary endothelial dysfunction
Acute coronary syndromes
Acute ischemic stroke
End-stage renal failure
Type 2 diabetes
The metabolic syndrome
EMPs Endothelium-derived MPs; PMPs Platelet-derived MPs; TF Tissue
TF exposing PMPs
Microparticles in cardiovascular diseases
Can J Cardiol Vol 26 No 4 April 2010e143
MPs AS A THERAPEUTIC TARGET IN
Due to their procoagulant and proinflammatory effects on vascular
walls and target organs (10,88), MPs might also be considered to be a
novel therapeutic target in cardiovascular diseases.
In 1998, Nomura et al (89) observed that cilostazol, a selective
cyclic AMP phosphodiesterase inhibitor and antiplatelet agent,
decreased the levels of PMPs in patients with noninsulin- dependent
DM (NIDDM). Subsequently, in hypertensive patients with or
without NIDDM, the same group found that treatment with losar-
tan (alone or in combination with simvastatin) significantly
decreased the levels of monocyte-derived MPs (90), and PMPs and
EMPs (91). Similar results on PMPs and monocyte-derived MPs
were obtained in patients with NIDDM treated with ticlopidine
In hypertensive patients, Labiós et al (93) observed that eprosartan
significantly reduced blood pressure and normalized the number of
MPs after blood shear exposure.
A powerful antiplatelet drug, the GP IIb/IIIa receptor antagonist
abciximab also reduces excessive PMP formation and shear stress-
induced platelet activation (94). Interestingly, the short-term high-
dose administration of vitamin C reduces the number of circulating
apoptotic MPs in patients with congestive heart failure and suppresses
EC apoptosis in vivo, which might contribute to the beneficial effect
of vitamin C supplementation on endothelial function (95).
More recently, Nomura et al (96) found that eicosapentaenoic acid
significantly reduced the number of circulating PMPs in hyperlipi-
demic diabetic patients, contributing to the prevention of vascular
complications. This effect was enhanced by the addition of pitavasta-
tin, and is in agreement with the results of a study investigating the
favourable effects of n-3 fatty acids on the levels of PMPs and
monocyte- derived MPs after myocardial infarction (97).
Because activated peroxisome proliferator-activated receptors
(PPARs) can inhibit inflammation and endothelial dysfunction, and
may also be effective in the primary prevention of cardiovascular
events (98), Esposito et al (99) evaluated the short-term effects of the
PPAR- gamma ligand pioglitazone on circulating EMPs in patients
with the metabolic syndrome. Pioglitazone reduced circulating EMPs
independently of the improvement of insulin sensitivity. Moreover,
experimental data on the PPAR- gamma agonist rosiglitazone showed
an inhibition of MP-induced vascular hyporeactivity through the reg-
ulation of proinflammatory proteins (100).
Overall, these results suggest that plasma MPs could be a promising
target in the treatment of cardiovascular diseases.
MPs of various origin may be considered to be ‘partners in crime’ in all
crucial steps of atherosclerosis (101). In fact, MPs play a role in inflam-
mation, coagulation, endothelial and vascular function, and apoptosis.
MPs could modulate the cross-talk between the cellular elements of
the coagulative and inflammatory system, through the transfer of sig-
nalling molecules and receptors of their cell of origin to other cell
Several studies have shown that MPs are increased in patients
with acute coronary syndromes, stroke, diabetes, pulmonary and sys-
temic hypertension, and hypertriglyceridemia. Thus, circulating MPs
could be considered to be important markers of cardiovascular risk
(102), with prognostic implications. However, the clinical impor-
tance of MPs in vascular disease states remains to be fully elucidated
because it is unclear whether MPs are a cause or a consequence of
these conditions. Conversely, available data from small studies sug-
gest that MPs could also be considered a novel therapeutic target in
cardiovascular diseases (10,88).
Recent progress in proteomics has shown that the protein content
of lymphocyte MPs is highly influenced by the cell culture medium
and type of stimulus used for MP generation (103). Thus, Chironi et al
(104) suggested special caution in extrapolating laboratory experimen-
tal results to the clinical setting because a given MP type may have
different compositions and biological behavioural patterns in vitro and
in vivo. Moreover, the different methods of measurement, the lack of
standardization and the various types of MPs to be measured may lead
to insufficiently reliable results (32). In addition, such methods of
measurement are not yet widely available.
Future research is needed before circulating MPs are considered to
be of significant clinical interest. Only a complete understanding of
the formation, composition, release and mechanisms of action of the
various MPs will allow the development of novel approaches in the
treatment of atherothrombosis-related diseases (105,106).
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