Thrombosis Research (2008) 122 Suppl.

1, S55–S59

intl.elsevierhealth.com/journals/thre

Microparticles in vascular diseases

Françoise Dignat George*

INSERM U 608, Laboratoire d’Hématologie et d’Immunologie, Marseille, France

KEYWORDS
Cellular microparticles
Hypercoagulability
Tissue factor
Vascular disorders
Abstract
Cellular microparticles (MP) are small membrane vesicles that are released from cells
upon activation or apoptosis. They constitute a heterogeneous population of submi-
cron elements differing in cellular origin, number, size, antigenic composition and
functional properties. Circulating MP provide an additional procoagulant phospho-
lipid surface enabling the assembly of the clotting enzyme complexes and thrombin
generation. Their procoagulant properties rely on the exposure of phosphatidylserine
and on the possible presence of tissue factor, the main initiator of blood coagulation.
Microparticles constitute the main reservoir of blood-borne tissue factor.
Derived from various cells, most notably platelets, erythrocytes, leucocytes
and endothelial cells, circulating MP are detectable in the circulation of healthy
subjects. Elevated levels are encountered in diseases with vascular involvement
and hypercoagulability such as disseminated intravascular coagulation, diabetes,
immune-mediated thrombosis, kidney diseases, acute coronary syndromes or
systemic inflammatory disease, where they appear indicative of a poor clinical
outcome.
Converging evidence from experimental and clinical data underlines an
involvement of procoagulant MP in the initiation/dissemination of procoagulant and
inflammatory responses. In these clinical settings, the pharmacological modulation
of MP level or activity provides challenging issues.
© 2008 Elsevier Ltd. All rights reserved.

Introduction representative of their cellular origin. These

Cellular microparticles (MP) are submicrometric
fragments resulting from the remodeling of
plasma membrane in response to numerous con-
ditions, including activation and apoptosis [1]. MP
are generally referred to as 0.1 to 1 mm membrane
fragments exposing the anionic phospholipid
phosphatidylserine (PS) and membrane antigens
characteristics discriminate MP from exosomes,
which are smaller (<0.1 mm), originate from
intracellular multivesicular bodies, and differ in
antigenic composition [2].
Microparticle formation, composition and
functions

* Correspondence address: Françoise Dignat George. Profes-
seur d’Immunologie, Laboratoire d’hématologie et d’Immuno-
logie, UMR 608 UNIVERSITE INSERM, UFR de Pharmacie,
27 bld Jean Moulin, 13385 Marseille Cedex 05, France.
Tel.: +33 04 91 83 55 55 (secretariat: +33 04 91 83 56 00;
fax: +33 04 91 83 56 02.
E-mail address: dignat@pharmacie.univ-mrs.fr or
fdignatgeorge@ap-hm.fr (F. Dignat George)
All cell types shed MP according to an active
process controlling plasma membrane remodeling
and antigenic turnover. Among the mechanisms
proposed, disruption of the natural asymmet-
ric distribution of membrane phospholipids is
thought to be an important step explaining the
translocation of PS to the exoplasmic leaflet

0049-3848/ $ – see front matter © 2008 Elsevier Ltd. All rights reserved.
S56
Table 1
Microparticle cell origin and antigenic features
Cell origin Antigens
Erythrocytes CD235(a)
Monocytes CD14
Granulocytes CD66b
Lymphocytes CD4, CD8, CD3
Platelets CD41, CD42, CD61
Endothelium CD31, CD51, CD105, CD146, CD144
before membrane blebbing and MP shedding.
Although calcium entry, activation of calpain, and
scramblase activity have been reported to be
important processes [3], the exact mechanisms
governing MP formation are not completely
understood.
MP phospholipid composition, phenotypes, and
content vary according to the cellular origin and
to the inducer triggering their formation (Table 1).
Determination of the cellular origin of MP relies
on the expression of specific antigens. According
to the cell type, their formation occurs following
exposure to various agents, including calcium
ionophore, ATP depletion, lipopolysaccharides,
thrombin, complement, cytokines and autoan-
tibodies (Table 1). Therefore, vesiculation is a
well-regulated process generating MP that present
specific features, with significant implications on
their functional activity.
Indeed, MP behave as vectors of bioactive
molecules able to disseminate biological infor-
mation in the vascular compartment. Due to the
expression of both membrane PS, a procoagulant
phospholipid necessary for the assembly of the
blood clotting enzyme complex, and functional
tissue factor (TF), the major initiator of the
coagulation cascade, MP are catalytic procoag-
ulant surfaces involved in thrombogenesis [10,
11]. Recently, MP were identified as active
surfaces supporting plasmin generation in vitro,
a non-described facet of the multiple biological
processes involving endothelial MP (EMP) in the
control of vascular homeostasis [12]. Indeed,
MP also harbor inflammatory components (LpA,
cytokines), growth factors (TGF beta, VEGF), and
proteases (uPA, MMP) that are related to their
involvement in inflammation, immune response,
angiogenesis, and cancer [10,13].
Microparticle detection and measurement
A crucial step concerns the pre-analytical phase
involved in blood sampling and MP obtention,
according to standardized centrifugation steps.
F. Dignat George
Vesiculation agents Ref
Ionophore A23187, acid pH, ATP depletion [4]
LPS, phorbol esters [5]
LPS, phorbol esters [6]
LPS, phorbol esters [7]
ADP, collagen, thrombin, A23187 [8]
Complement, thrombin, autoantibodies, cytokines [9]
Different methodologies, which were reported
in a forum [14], are available for MP analysis.
Briefly, antibody-capture ELISA and flow cytometry
rely on antibodies detection of specific antigens
and/or annexin V binding, and allow MP to be
measured according to their cellular origin and/or
their expression of PS. Besides, assays measuring
MP-associated procoagulant activity are based on
the measurement of PS and TF function. Finally,
other assays combine ELISA capture on annexin V
or antibody and determination of procoagulant
activity of MP.
Microparticle in vascular diseases
Detectable in the peripheral blood in physiological
conditions, circulating MP originating from blood
and vascular cells are elevated in a variety of
diseases when comparing patients with matched
healthy volunteers. Based on information from
the main reviews published in the field [10,15 18],
alterations of MP levels and their principal clinical
significance in vascular disorders are summarized
in Table 2.
Sickle cell disease
MP derived from endothelial cells, platelets and
monocytes are detected in the circulation of
patients with sickle cell disease (SCD) during
chronic phases [19]. Procoagulant MP positive for
TF and originating from monocytes and endothe-
lial cells were dramatically increased during
vasoocclusive crisis. Moreover, these procoagulant
MP correlated with plasma markers of coagulation
activation, suggesting their possible involvement
in the thrombotic complications of patients with
SCD.
Paroxysmal nocturnal haemoglobinuria (PNH)
MP with a prothrombotic activity originating from
endothelial cells, platelets and monocytes, are
increased in the circulation of patients with PNH,
Microparticles in vascular diseases S57

Table 2
Alterations of MP levels and their principal clinical significance in vascular disorders a

Disease MP plasma MP cellular origin Main clinical significance Ref

Clinical setting alteration

Sickle cell disease ↑ All types TF+ MP increase during vaso-occlusive [16,19]
crisis
Paroxysmal nocturnal haemoglobinuria ↑ PMP, MMP, EMP MP support coagulation activation [4,16]
Antiphospholipid syndrome ↑ EMP EMP correlate with lupus anticoagulant [9,10,17]
Heparin-induced thrombocytopenia ↑ PMP, EMP PMP support coagulation activation [10]
Diabetes ↑ PMP, MMP, EMP,GMP MP Procoagulant activity varies with [10,17]
glycemic control
Chronic renal failure ↑ PMP, MMP, EMP GMP, ErMP EMP correlate with loss of endothelial [15]
function
Acute coronary syndrome ↑ EMP, PMP EMP correlate with angiography [15]
imaging and severity of stenosis
Stroke ↑ EMP, PMP EMP increase in small and large vessels [16]
and in multiinfarct dementia
Deep venous thrombosis and ↑ EMP, PMP PMP levels combined with D-dimer and [16]
pulmonary embolism P-selectin correlates with DVTdiagnosis
Sepsis ↑ PMP, MMP, EMP GMP MP support thrombin generation and [16]
correlate with prothrombin fragments
Cancer ↑ PMP, MMP TF+ MP correlate with metastasis and [17,18]
lower survival
a Abbreviations: EMP, endothelial microparticles; MMP, monocyte microparticles; PMP, platelet microparticles; GMP,
granulocyte microparticles; ErMP, erythrocyte microparticles.
b ↑ and ↓ indicate increase and decrease, respectively.

and could contribute to their prothrombotic sta-
tus [4]. The presence in the circulation of elevated
levels of EMP expressing constitutive markers such
as CD144 or CD105 is therefore indicative of
persistent endothelial damage associated with the
chronic haemolysis of PNH.
and P-selectin. PMP were found to be highly
thrombogenic with the potency to induce coag-
ulation activation. The ability of PMP to bind the
subendothelial matrix promotes the recruitment
of additional platelets and constitutes a PMP
reservoir [20].

Immune-mediated thrombosis Chronic vascular disorders

In “immune-mediated acquired thrombophilic dis-
orders” such as antiphospholipid syndrome (APS)
and heparin-induced thrombocytopenia (HIT),
auto-antibodies may have a specific contribution
to the thrombotic diathesis by contributing to the
release of MP.
Elevated levels of EMP were found in patients
with APS and correlated with lupus anticoag-
ulant [9], which is strongly associated with a
thrombotic propensity. In contrast, elevation of
platelet MP (PMP) also reported in systemic lupus
erythematosus patients is not related to disease
activity, nor with the presence of antiphospholipid
antibodies.
In patients with HIT [10], EMP express von
Willebrand factor and markers of endothelial
activation. Released PMP exposed GP1b, GPIIbIIIa,
In diabetes, a large panel of cells including
platelets, endothelial cells, granulocytes and
monocytes release MP. The patterns of MP and
their procoagulant activity vary according to
the type of diabetes. Indeed, EMP and PMP
appear particularly elevated in type-1 patients,
the highest EMP levels being detected in patients
suffering from microvascular complications. In
diabetic nephropathy, EMP were positively cor-
related with albuminuria. Procoagulant activity
borne by circulating MP was found informative
of the glycemic control, higher levels being
detected in patients with poorly controlled HbA1c
levels [10]. In type-2 diabetes, high levels of
monocyte- or platelet-derived MP were reported
indicative of nephropathy and retinopathy. During
acute myocardial infarction, the rise in platelet-,
S58
endothelial- and monocyte-derived MP may con-
tribute to enhanced thrombogenicity, regardless
of the diabetes type [10].
MP levels are increased during chronic renal
failure (CRF), suggesting that vesiculation may
participate in thrombogenesis and compensation
for the bleeding tendency in CRF. Elevated
PMP counts have been reported in CRF patients
(pre-dialyzed, under hemodialysis, and under
ambulatory peritoneal dialysis), with significantly
higher levels in patients with thrombotic events.
The high EMP levels detected in uremic patients
in the absence of a clinical history of vascular
disease may constitute an early indicator of
vascular injury [21]. In patients with end-stage
CRF, platelet, erythrocyte, and endothelial MP
are increased [22]. However, only EMP levels
correlate with loss of flow-mediated dilatation and
increased aortic pulse wave velocity, suggesting
that they are highly associated with arterial and
endothelial dysfunction in end-stage CRF.
Cardiovascular disorders
Detectable alterations of circulating MP reflecting
apoptosis/activation of blood and vascular cells
have been reported in patients with cardiovascu-
lar disorders such as acute coronary syndromes,
stroke, aortic aneurysm disease, peripheral vas-
cular disease or venous thromboembolism [15,16].
Elevated levels of circulating platelet-, monocyte-
or endothelial-derived MP are also associated with
most of the cardiovascular risk factors including
hypertension, obesity, cholesterol, or dyslipemia,
and appear indicative of a poor clinical outcome.
In these clinical situations, measurement and
characterization of MP cellular origin have turned
out to be useful for disease detection, diagnosis,
study of disease progression and pathophysiology,
and in assessing response to treatment. Several
therapies known to be beneficial in cardiovascular
disorders such as statins, anti-platelets agents,
anti-oxidants, beta-blockers, AT II receptors or
Ca-channel inhibitors were reported to reduce
both MP number and procoagulant activity [10].
These observations support the hypothesis that
part of the beneficial effect is linked to decreased
MP pathogenicity.
Sepsis
Elevated levels of MP derived from endothelial
cells, platelets, granulocytes and monocytes have
been reported in septic patients. The extent to
which TF-positive MP, alone or included in MP-
cell conjugates, participate in the disease process
F. Dignat George
remains to be elucidated [16]. The pathogenicity
of procoagulant MP has been demonstrated in
patients with meningococcal sepsis [23,24]. In-
deed, plasma from non-surviving patients presents
elevated numbers of TF-positive MP with a high
thrombin generation capacity. Such MP may play a
role in the initiation of disseminated intravascular
coagulation.
Cancer
Circulating levels of MP are augmented in
cancer, in particular in mucinous adenocarcioma,
known to be associated with venous thrombo-
embolism [17,18]. Interestingly, an increase in
TF-positive MP has been reported in patients
with disseminated breast cancer and pancreatic
cancer by reference to non-metastatic cancer
patients, suggesting a role of TF-positive MP in the
thrombotic complications of cancer patients.
Conclusion
MP behave not only as pathogenic effectors that
play a deleterious role in vascular diseases but
also as emerging biomarkers for the stratification
of the individual atherothrombotic risk and
the monitoring of therapeutics strategies. In
the future, the pharmacological modulation of
MP formation is a promising challenge in the
control of vascular complications.
Acknowledgments
I thank Laetitia Dou for careful reading of the text
and assistance in manuscript editing.
Conflict of interest statement
I certify that I have no conflict of interest.
References
[1] Freyssinet JM, Dignat-George F. More on: Measuring cir-
culating cell-derived microparticles. J Thromb Haemost
2005;3:613 14.
[2] Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ.
Activated platelets release two types of membrane
vesicles: microvesicles by surface shedding and exosomes
derived from exocytosis of multivesicular bodies and
alpha-granules. Blood 1999;94:3791 99.
[3] Simak J, Gelderman MP. Cell membrane microparticles
in blood and blood products: potentially pathogenic
agents and diagnostic markers. Transfus Med Rev
2006;20:1 26.
[4] Hugel B, Socié G, Vu T, Toti F, Gluckman E,
Freyssinet JM, et al. Elevated levels of circulating
procoagulant microparticles in patients with paroxysmal
nocturnal hemoglobinuria and aplastic anemia. Blood
1999;93:3451 6.
Microparticles in vascular diseases
[5] Satta N, Toti F, Feugeas O, Bohbot A, Dachary-
Prigent J, Eschwège V, et al. Monocyte vesiculation is
a possible mechanism for dissemination of membrane-
associated procoagulant activities and adhesion molecules
after stimulation by lipopolysaccharide. J Immunol
1994;153:3245 55.
[6] Watanabe J, Marathe GK, Neilsen PO, Weyrich AS, Harrison
KA, Murphy RC, et al. Endotoxins stimulate neutrophil
adhesion followed by synthesis and release of platelet-
activating factor in microparticles. J Biol Chem 2003;
278:33161 8.
[7] Martin S, Tesse A, Hugel B, Mart´ ınez MC, Morel O,
Freyssinet JM, et al. Shed membrane particles from T
lymphocytes impair endothelial function and regulate
endothelial protein expression. Circulation 2004;109:
1653 9.
[8] Combes V, Dignat-George F, Mutin M, Sampol J. A new
flow cytometry method of platelet-derived microvesicle
quantitation in plasma. Thromb Haemost 1997;77:220.
[9] Combes V, Simon AC, Grau GE, Arnoux D, Camoin L,
Sabatier F, et al. In vitro generation of endothelial
microparticles and possible prothrombotic activity in
patients with lupus anticoagulant. J Clin Invest 1999;104:
93 102.
[10] Morel O, Toti F, Hugel B, Bakouboula B, Camoin-Jau L,
Dignat-George F, et al. Procoagulant microparticles: dis-
rupting the vascular homeostasis equation? Arterioscler
Thromb Vasc Biol 2006;26:2594 2604.
[11] Furie B, Furie BC. Thrombus formation in vivo. J Clin
Invest 2005;115:3355 62.
[12] Lacroix R, Sabatier F, Mialhe A, Basire A, Pannell R, Borghi
H, et al. Activation of plasminogen into plasmin at the
surface of endothelial microparticles: a mechanism that
modulates angiogenic properties of endothelial progenitor
cells in vitro. Blood 2007;110:2432 9.
[13] Morel O, Toti F, Hugel B, Freyssinet JM. Cellular
microparticles: a disseminated storage pool of bioactive
vascular effectors. Curr Opin Hematol 2004;11:156 64.
[14] Jy W, Horstman LL, Jimenez JJ, Ahn YS, Biró E, Nieuwland
R, et al. Measuring circulating cell-derived microparticles.
J Thromb Haemost 2004;2:1842 51.
S59
[15] Boulanger CM, Amabile N, Tedgui A. Circulating
microparticles: a potential prognostic marker for
atherosclerotic vascular disease. Hypertension 2006;48:
180 6.
[16] Piccin A, Murphy WG, Smith OP. Circulating microparticles:
pathophysiology and clinical implications. Blood Rev
2007;21:157 71.
[17] Diamant M, Tushuizen ME, Sturk A, Nieuwland R. Cellular
microparticles: new players in the field of vascular
disease? Eur J Clin Invest 2004;34:392 401.
[18] Mackman N, Tilley RE, Key NS. Role of the extrinsic
pathway of blood coagulation in hemostasis and
thrombosis. Arterioscler Thromb Vasc Biol 2007;27:
1687 93.
[19] Shet AS, Aras O, Gupta K, Hass MJ, Rausch DJ,
Saba N, et al. Sickle blood contains tissue factor-
positive microparticles derived from endothelial cells and
monocytes. Blood 2003;102:2678 83.
[20] Hughes M, Hayward CP, Warkentin TE, Horsewood P,
Chorneyko KA, Kelton JG. Morphological analysis of
microparticle generation in heparin-induced thrombocy-
topenia. Blood 2000;96:188 94.
[21] Faure V, Dou L, Sabatier F, Cerini C, Sampol J, Berland Y,
et al. Elevation of circulating endothelial microparticles
in patients with chronic renal failure. J Thromb Haemost
2006;4:566 73.
[22] Amabile N, Guérin AP, Leroyer A, Mallat Z, Nguyen C,
Boddaert J, et al. Circulating endothelial microparticles
are associated with vascular dysfunction in patients
with end-stage renal failure. J Am Soc Nephrol
2005;16:3381 8.
[23] Soriano AO, Jy W, Chirinos JA, Valdivia MA, Velasquez HS,
Jimenez JJ, et al. Levels of endothelial and platelet
microparticles and their interactions with leukocytes
negatively correlate with organ dysfunction and predict
mortality in severe sepsis. Crit Care Med 2005;33:
2540 6.
[24] Nieuwland R, Berckmans RJ, McGregor S, Böing AN,
Romijn FP, Westendorp RG, et al. Cellular origin
and procoagulant properties of microparticles in
meningococcal sepsis. Blood 2000;95:930 5.