Factors influencing the level of circulating procoagulant microparticles in acute pulmonary embolism

Article · June 2010with92 Reads
DOI: 10.1016/j.acvd.2010.06.005 · Source: PubMed
Abstract
Flow cytometry has shown levels of platelet-derived microparticles (PMPs) and endothelial-derived microparticles (EMPs) to be elevated in deep-vein thrombosis. Cardiovascular risk factors can also contribute to hypercoagulability due to circulating procoagulant microparticles (CPMPs). To investigate in a case-control study the respective contribution of pulmonary embolism and cardiovascular risk factors to the level of hypercoagulability due to CPMPs. CPMP, PMP and EMP levels were measured in 45 consecutive patients (age 67.9 +/- 11.6 years; 66.7% men) admitted to an intensive care unit for acute pulmonary embolism (APE), 45 healthy control subjects with no history of venous thromboembolism or vascular risk factors (Controls(noCVRFs)), and 45 patients with cardiovascular risk factors (Controls(CVRFs)). APE was diagnosed by spiral computed tomography or scintigraphy. CPMP levels were assessed using a prothrombinase assay on platelet-depleted plasma (results expressed as nmol/L equivalent). CPMP levels were higher in APE patients than in Controls(noCVRFs) (medians 4.7 vs 3.2 nmol/L, interquartile ranges [IQRs] 2.9-11.1 vs 2.3-4.6 nmol/L; p=0.02). Similar results were reported for PMPs (medians 2.2 vs 1.9 nmol/L, IQRs 1.7-5.8 vs 1.4-2.4 nmol/L; p=0.02), whereas EMP levels were not significantly different. However, CPMP procoagulant activity was not significantly different in APE patients and Controls(CVRFs). CPMPs and PMPs were significantly elevated in APE patients vs Controls(noCVRFs), but this correlation was not significant when APE patients were compared with Controls(CVRFs). Our observations highlight the importance of adjusting for the presence of cardiovascular risk factors in conditions in which microparticle levels are raised.
3 Figures
Archives of Cardiovascular Disease (2010) 103, 394—403
CLINICAL RESEARCH
Factors influencing the level of circulating
procoagulant microparticles in acute
pulmonary embolism
Facteurs infuenc¸ant le niveau des microparticules procaogulantes dans
l’embolie pulmonaire
Laurence Bala, Stéphane Ederhya,
Emanuele Di Angelantonioa, Florence Totib,
Fatiha Zobairib, Ghislaine Dufaitrea,
Catherine Meulemana, Ziad Mallatc, Franck Boccaraa,
Alain Tedgui c, Jean-Marie Freyssinetb, Ariel Cohena,
aCardiology Department, Saint-Antoine University and Medical School, Assistance
Publique—Hôpitaux de Paris, université Pierre-et-Marie-Curie, 184, rue du
Faubourg-St-Antoine, 75571 Paris cedex 12, France
bInserm U770, hôpital de Bicêtre, université Louis-Pasteur, faculté de médecine, institut
d’hématologie et immunologie, 67400 Strasbourg, France
cInserm U970, Paris Cardiovascular Research Centre, université Paris-Descartes and
Assistance Publique—Hôpitaux de Paris, 75015 Paris, France
Received 12 May 2010; received in revised form 15 June 2010; accepted 17 June 2010
Available online 13 August 2010
KEYWORDS
Microparticles;
Pulmonary embolism;
Cardiovascular risk
factors
Summary
Background. — Flow cytometry has shown levels of platelet-derived microparticles (PMPs) and
endothelial-derived microparticles (EMPs) to be elevated in deep-vein thrombosis. Cardiovas-
cular risk factors can also contribute to hypercoagulability due to circulating procoagulant
microparticles (CPMPs).
Aims. — To investigate in a case-control study the respective contribution of pulmonary
embolism and cardiovascular risk factors to the level of hypercoagulability due to CPMPs.
Methods. — CPMP, PMP and EMP levels were measured in 45 consecutive patients (age
67.9 ±11.6 years; 66.7% men) admitted to an intensive care unit for acute pulmonary embolism
(APE), 45 healthy control subjects with no history of venous thromboembolism or vascular risk
Abbreviations: APE, acute pulmonary embolism; ControlsCVRFs, controls with cardiovascular risk factors; ControlsnoCVRFs , controls without
cardiovascular risk factors; CPMP, circulating procoagulant microparticle; EMP, endothelial-derived microparticle; IQR, interquartile range;
PMP, platelet-derived microparticle; VTE, venous thromboembolism.
Corresponding author. Fax: +33 1 49 28 28 84.
E-mail address: ariel.cohen@sat.aphp.fr (A. Cohen).
1875-2136/$ — see front matter © 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.acvd.2010.06.005
Circulating procoagulant microparticles in pulmonary embolism 395
factors (ControlsnoCVRFs), and 45 patients with cardiovascular risk factors (ControlsCVRFs ). APE
was diagnosed by spiral computed tomography or scintigraphy. CPMP levels were assessed
using a prothrombinase assay on platelet-depleted plasma (results expressed as nmol/L
equivalent).
Results. — CPMP levels were higher in APE patients than in ControlsnoCVRFs (medians 4.7 vs
3.2 nmol/L, interquartile ranges [IQRs] 2.9—11.1 vs 2.3—4.6 nmol/L; p=0.02). Similar results
were reported for PMPs (medians 2.2 vs 1.9 nmol/L, IQRs 1.7—5.8 vs 1.4—2.4 nmol/L; p= 0.02),
whereas EMP levels were not significantly different. However, CPMP procoagulant activity was
not significantly different in APE patients and ControlsCVRFs.
Conclusions. — CPMPs and PMPs were significantly elevated in APE patients vs ControlsnoCVRFs,
but this correlation was not significant when APE patients were compared with ControlsCVRFs.
Our observations highlight the importance of adjusting for the presence of cardiovascular risk
factors in conditions in which microparticle levels are raised.
© 2010 Published by Elsevier Masson SAS.
MOTS CLÉS
Microparticules ;
Embolie pulmonaire ;
Facteurs de risque
cardiovasculaires
Résumé
Contexte. — La thrombose veineuse profonde est associée à une augmentation du niveau de
microparticules (MP) d’origine endothéliale et plaquettaire mesurée en cytométrie de flux. Les
facteurs de risque cardiovasculaires (FRCV) ont aussi une influence importante sur le niveau
des microparticules.
Objectifs. — Nous avons évalué le niveau des microparticles procoagulantes circulantes chez
des patients admis pour embolie pulmonaire (EP) aiguë et étudié le rôle respectif de la mal-
adie veineuse thrombo-embolique et des facteurs de risque cardiovasculaire sur le niveau
d’hypercoagulabilité lié aux microparticules.
Méthodes. — Les microparticules procoagulantes circulantes, les microparticules plaquettaires
et d’origine endothéliale ont été mesurées chez 45 patients consécutifs (âge 67,9 ±11,6,
66,7 % d’homme) admis en unité de soins intensifs pour une embolie pulmonaire aiguë, chez
45 patients sans facteur de risque cardiovasculaire et chez 45 patients avec des facteurs de
risque cardiovasculaire. L’embolie pulmonaire était documentée soit par angioscanner soit par
scintigraphie pulmonaire de ventilation et perfusion. L’activité procoagulante des MP circu-
lantes a été mesurée en utilisant un plasma pauvre en plaquettes et un test fonctionnel à la
prothrombinase.
Résultats. — Les microparticules procoagulantes circulantes étaient plus élevées chez les
patients admis pour une EP (médiane 4,7 nmol/L, interquartile range [IQR] 2,9—11,1) que chez
les patients sans FRCV (médiane 3,2 nmol/L, IQR 2.3—4.6 ; p=0,01). Le niveau des MP d’origine
plaquettaire était plus élevé chez les patients présentant une EP comparativement aux patients
sans FRCV (médiane 2,2 nmol/L, IQR 1,7—5,8 versus 1,9, 1,4—2,4 ; p=0,02). Le niveau des MP
d’origine endothéliale était, en revanche, comparable dans les deux populations. Cependant,
le niveau des MP procoagulantes n’était pas significativement différent des patients avec FRCV,
et ce, quel que soit le phénotype considéré.
Conclusion. — Les MP procoagulantes totales et plaquettaires sont significativement plus
élevées chez les patients admis pour embolie pulmonaire aiguë comparativement à des patients
sans facteur de risque cardiovasculaire. Cette relation n’est plus retrouvée lorsque ces patients
sont comparés à des sujets témoins avec facteurs de risque cardiovasculaire. Ces données
démontrent l’importance de prendre en compte les facteurs de risque cardiovasculaire dans
l’interprétation du niveau des MP.
© 2010 Publi´
e par Elsevier Masson SAS.
Introduction
According to Virchow’s triad, the pathophysiology of
VTE relies on the presence of blood hypercoagula-
bility, stasis of blood flow and vessel-wall damage
[1]. The factors involved in venous thrombogenesis
could be defined as soluble coagulant factors, dys-
functional endothelium and circulating cells, especially
platelets, lymphomonocytes and, potentially, CPMPs
[2,3].
CPMPs are plasma membrane fragments that are released
into the blood by stimulated cells during activation or apop-
tosis, and carry procoagulant phosphatidylserine and tissue
factor on their surface [4—7]. CPMPs circulate in healthy
humans and support low-grade generation of thrombin [8].
High levels of CPMPs have been reported in several sce-
narios, including, for example, in patients with an acute
coronary syndrome [9—11] or atrial fibrillation [12]. Car-
diovascular risk factors such as hypertension and diabetes
[13—15] have been associated with elevated CPMPs and their
396 L. Bal et al.
phenotypes. In interpreting the role of CPMPs in thrombo-
genesis, we need to take into account potential cofounders,
the most prevalent of which appear to be cardiovascular risk
factors.
Two previous clinical studies have reported elevated lev-
els of CPMPs — mainly PMPs and EMPs — by flow cytometry,
in patients hospitalized for deep-vein thrombosis. How-
ever, they had different phenotypic representations, and the
authors did not take into account potential confounders,
such as cardiovascular risk factors [16,17]. Experimental
studies in vivo are supportive of the involvement of human
cell-derived microparticles in venous thrombogenesis in a
tissue factor-dependent manner, and have described a cor-
relation of leukocyte- and PMPs with thrombus weight and
tissue factor activity [18,19]. Recently, the crucial partic-
ipation of circulating tissue factor-bearing microparticles
released by tumour cells in cancer-associated hypercoagula-
bility has been emphasized, depending on both the tumour
cell origin and a critical threshold of microparticles [20—24].
In addition, we have shown in a case-control study that CPMP
levels, defined by their procoagulant activity, were elevated
in patients without cancer hospitalized with APE, and we
analysed the influence of cardiovascular risk factors on this
correlation [25].
Methods
Study subjects
Between November 2004 and December 2005, we included
45 consecutive patients admitted to our intensive care unit
for APE associated with or without deep-vein thrombo-
sis, and compared them with 45 healthy controls without
(ControlsnoCVRFs) and 45 patients with (ControlsCVRFs ) cardio-
vascular risk factors, matched for age and sex. ControlsCVRFs
and patients with APE were also matched for the presence
of hypertension. Demographic and clinical characteristics
were recorded prospectively upon enrolment. The study
was approved by the institutional review board and was
performed in accordance with institutional guidelines. All
patients gave written informed consent before participating
in the study.
Acute pulmonary embolism cases
APE was confirmed by spiral computed tomography (n= 25),
ventilation-perfusion scintigraphy (n= 20) or both (n= 12).
Treatment on admission consisted of standard antithrom-
botic therapy with low-molecular-weight or unfractionated
heparin. Exclusion criteria were conditions known or sus-
pected to increase levels of CPMPs independently, such
as acute coronary syndromes, acute heart failure, stroke,
sepsis, chronic inflammatory disease, antiphospholipid
syndrome, heparin-induced thrombocytopenia, thrombotic
thrombocytopenic purpura and atrial fibrillation.
Transient VTE risk factors were defined as pregnancy,
oestrogen therapy, surgery (< 60 days), trauma, confined
to bed (> 5 days) and recent journey (> 10 hours). Cancer
and thrombophilia were defined as chronic VTE risk factors.
Haemodynamic status was considered over three levels:
submassive APE (stable haemodynamics with signs of right
heart failure on transthoracic echocardiography), massive
APE (unstable haemodynamics with right heart failure on
transthoracic echocardiography) and shock.
Controls with cardiovascular risk factors
ControlsCVRFs comprised patients with no history of VTE
or atrial fibrillation who were undergoing routine screen-
ing physical examinations for cardiac symptoms at our
outpatient cardiology clinic, with an electrocardiogram doc-
umenting sinus rhythm.
Controls without cardiovascular risk factors
ControlsnoCVRF included patients undergoing screening exam-
ination before orthopaedic surgery, with no known cardio-
vascular risk factors, history of atrial fibrillation, prior VTE,
clinical evidence of disease or current cardiovascular treat-
ment, and who had an electrocardiogram documenting sinus
rhythm. These subjects were assessed by careful examina-
tion of their medical histories and by blood tests.
Circulating procoagulant microparticles
Blood was collected in the acute phase when the diagnosis
of VTE was assessed and just before anticoagulation was
started. Measurement of CPMPs was performed as described
previously [26], with minor modifications.
Preparation of circulating procoagulant
microparticle samples
All microparticle determinations were performed strictly
according to Biro et al. [18]. Briefly, citrated blood was
taken soon after admission and centrifuged at 1500 g for
15 min at room temperature within the hour after sam-
pling. The supernatant was centrifuged again at 13,000 g
for 2 min to avoid platelet contamination. Thrombin and fac-
tor Xa inhibitors (d-phenylalanyl-prolyl-arginyl chloromethyl
ketone and 1,5-dansyl-glutamyl-glycyl-arginyl chloromethyl
ketone, respectively) were added to plasma samples at
a final concentration of 50 M each, and CaCl2at a final
concentration of 50 mM.
Quantitation of circulating procoagulant
microparticles
After capture of microparticles onto annexin V-coated wells
(for 30 min at 37 C), taking advantage of the strong affinity
of annexin V for aminophospholipids present in microparti-
cles at the calcium concentration used, four washing steps
were performed with Tris buffer containing 1 mM CaCl2and
0.05% Tween 20, each for 5 min at 20C, and the last one
without Tween. The phosphatidylserine content of micropar-
ticles, directly responsible for their procoagulant activity,
was then measured in a prothrombinase assay. Micropar-
ticles were incubated with factor Xa (50 pmol/L), factor
Va (360pmol/L), prothrombin (1.3 mol/L) and 2.3 mmol/L
CaCl2for 15 min at 37 C, and linear absorbance changes
were recorded at 405 nm after the addition of chromozym
TH (380 mol/L).
Circulating procoagulant microparticles in pulmonary embolism 397
Quantitation of platelet-derived microparticles and
endothelial-derived microparticles
After specific capture of PMPs onto anti-glycoprotein Ib
antibody-coated wells and of EMPs onto anti-CD31 antibody-
coated wells, quantitation was achieved after several
washing steps using a prothrombinase assay as described
above. Microparticle levels are expressed as nmol/L of phos-
phatidylserine equivalent.
Miscellaneous measurements
Quantification of C-reactive protein was determined by
immunonephelometric tests and circulating brain natri-
uretic peptide levels by enzyme immunoassays.
Transthoracic echocardiography
To evaluate right ventricular dysfunction and haemodynamic
status, transthoracic echocardiography was performed at
the time of admission in all patients with APE. Systolic
transtricuspid pressure gradient and left ventricular ejec-
tion fraction were also measured.
Statistical analysis
Based on previous studies [11,27,28], we hypothesized that
patients with APE would have microparticle levels increased
by approximately two standard deviations compared with
healthy controls and by one standard deviation compared
with subjects without APE but with cardiovascular risk fac-
tors. To achieve this with 90% power and p< 0.05 between
the three groups, 35 subjects per group were required. To
minimize the risk of a type II error and to account for pos-
sible confounders, we recruited in excess of this number of
patients with APE and controls.
Categorical variables, expressed as percentages, were
compared using the Chi2test or Fisher’s exact test. After a
test for normality, continuous data are expressed as means
and standard deviations or medians with IQRs as appro-
priate. Differences between patients and controls were
evaluated using the two-sample ttest or the Mann-Whitney
U test. Correlations between annexin V-positive microparti-
cles and endothelial and platelet microparticle levels were
evaluated using Spearman’s rank correlation coefficients.
The relationship between CPMP levels and patients’
characteristics was estimated using linear regression anal-
ysis (after logarithmic transformation of the dependent
variables) and presented using the estimated regression
coefficient, expressed as percentage increase in micropar-
ticle level for the presence of each risk factor or for a unit
increase in continuous variables, such as age. All analyses
were performed using STATA 9 statistical software (STATA,
College Station, TX, USA). A probability value of 0.05 was
considered statistically significant.
Results
The baseline characteristics of the three groups are given in
Table 1. The mean age of patients with APE was 67.9 ±11.6
years and 66.7% were men. Deep-vein thrombosis was
documented in 71.1% of patients, 26.7% (n= 12) had haemo-
Figure 1. Levels of circulating procoagulant microparticles in
controls with (ControlsCVRFs) and without (ControlsnoCVRFs ) cardio-
vascular risk factors and in patients with acute pulmonary embolism
(APE).
dynamic instability and 4.4% (n= 2) presented in cardiogenic
shock. APE was submassive in 17 (37.8%) patients. Sixteen
(35.5%) patients with APE had at least one transient VTE risk
factor and eight (17.8%) had a permanent risk factor (cancer,
n= 3; thrombophilia, n= 5). APE was idiopathic in 21 (46.7%)
patients.
There were no significant differences in terms of clini-
cal cardiovascular risk factors between patients with APE
and ControlsCVRFs, except for current smoking (Table 1). In
contrast, by design, ControlsnoCVRFs were significantly dif-
ferent from the other two groups with regard to clinical
cardiovascular risk factors. However, there was no signifi-
cant difference regarding age and sex between ControlsCVRFs
and ControlsnoCVRF, even if there was a slight predominance
of men in the former.
Annexin-positive microparticles in acute
pulmonary embolism patients and controls
Annexin V-positive microparticle levels were higher
in patients with APE (median 4.7 nmol/L, IQR
2.9—11.1 nmol/L) than in ControlsnoCVRFs (median
3.2 nmol/L, IQR 2.3—4.6 nmol/L; p=0.02), but there was no
significant difference compared with ControlsCVRFs(median
4.9 nmol/L, IQR 3.7—8.4 nmol/L; p= 0.99) (Fig. 1). Annexin
V-positive microparticle levels were significantly higher in
ControlsCVRFs than in ControlsnoCVRFs (p= 0.01; Fig. 1).
Moreover, after adjustment for age, sex and hyperten-
sion, CPMPs were significantly correlated with C-reactive
protein (p= 0.02) and brain natriuretic peptide levels
(p< 0.01), but not with the echographic haemodynamic sta-
tus evaluated by systolic transtricuspid pressure gradient
and left ventricular ejection fraction (Table 2).
Platelet-derived microparticles in acute
pulmonary embolism patients and controls
PMP levels were not significantly different between patients
with APE (median 2.2 nmol/L, IQR 1.7—5.8 nmol/L) and
ControlsCVRFs (median 5.5 nmol/L, IQR 2.6—11.3 nmol/L;
398 L. Bal et al.
Table 1 Baseline characteristics in patients with acute pulmonary embolism and controls with and without cardiovascular risk factors.
Patient characteristics APE (n= 45) ControlsCVRFs
(n= 45)
ControlsnoCVRFs
(n= 45)
Pvalue
APE vs
ControlsCVRFs
APE vs
ControlsnoCVRFs
ControlsCVRFs vs
ControlsnoCVRFs
Age (years) 67.9 ±11.6 67.1 ±9.9 67.0 ±9.5 — — —
Men 30 (66.7) 30 (66.7) 26 (57.8) 0.38 0.38
Hypertension 25 (55.6) 25 (55.6) 0
Diabetes mellitus 7 (15.6) 9 (20.0) 0 0.58 < 0.01 < 0.01
Hypercholesterolaemia 19 (42.2) 14 (31.1) 0 0.27
Current smoker 4 (9.1) 16 (35.6) 0 < 0.01 0.04 —
Coronary artery disease 3 (6.8) 9 (20.0) 0 0.07 0.08 < 0.01
History of heart failure 1 (2.2) 0 0
History of TIA or ischaemic stroke 2 (4.4) 1 (2.2) 0
History of atrial fibrillation 6 (13.3) 0 0
Concomitant treatment
Aspirin 8 (17.8) 1 (2.2) 0.01
Beta-blocker 9 (20.5) 10 (22.2) 0.84
Calcium channel blocker 6 (13.3) 4 (8.9) 0.50
Angiotensin-converting enzyme inhibitor 7 (15.9) 7 (15.6) 0.96
Diuretic 8 (17.8) 0 < 0.01 ——
Nitrate 1 (2.2) 1 (2.2) ————
Insulin 0 2 (4.4)
Oral antidiabetic therapy 2 (4.4) 6 (13.3) 0.14
Data are mean ±standard deviation or number (%).
APE: acute pulmonary embolism; ControlsCVRFs: controls with cardiovascular risk factors; ControlsnoCVRFs : controls without cardiovascular risk factors; TIA: transient ischaemic attack.
Circulating procoagulant microparticles in pulmonary embolism 399
Table 2 Baseline correlates of annexin V-positive microparticles and platelet and endothelial microparticle levels in 45
patients with an acute pulmonary embolism.
No. of patients (%) Pearson correlation
(95% CI)
% change in MPs
(95% CI)
Pvalue
Annexin V-positive MPs
Clinical presentation
Acute DVT 32 (71.1) 0.07 (0.02, 0.36) 1.18 (0.59, 2.36) 0.64
Baseline characteristics
Agea67.9 (11.6) 0.16 (0.43, 0.14) 0.99 (0.96, 1.01) 0.30
Hypertension 25 (55.6) 0.02 (0.27, 0.31) 1.05 (0.56, 1.97) 0.89
Diabetes mellitus 7 (15.6) 0.14 (0.42, 0.16) 0.66 (0.28, 1.56) 0.35
Hypercholesterolaemia 19 (42.2) 0.05 (0.24, 0.34) 1.12 (0.59, 2.12) 0.73
Current smoker 4 (9.1) 0.26 (0.04, 0.52) 2.57 (0.89, 7.42) 0.09
Coronary artery disease 3 (6.8) 0.02 (0.28, 0.32) 1.10 (0.31, 3.93)
Previous atrial fibrillation 6 (13.3) 0.23 (0.07, 0.49) 2.02 (0.82, 4.06) 0.13
Previous DVT 10 (22.2) 0.16 (0.15, 0.43) 1.48 (0.70, 3.13) 0.31
Aspirin 8 (17.8) 0.02 (0.27, 0.31) 1.06 (0.47, 2.4) 0.89
Biological criteria
C-reactive proteina59.2 (83.7) 0.36 (0.59, 0.07) 0.995 (0.99, 0.999) 0.02
BNPa,b279.6 (413.1) 0.45 (0.13, 0.69) 1.001 (1, 1.00) < 0.01
Echographic criteria
LVEF < 40% 3 (6.7) 0.17 (0.13, 0.44) 2.02 (0.58, 7.01)
STPGa135 (307.8) 0.08 (0.23, 0.37) 1.14 (0.69, 1.89) 0.62
Platelet-derived MPs (anti-GP1b)
Clinical presentation
Shock 0.37 (0.09, 0.60) 8.17 (1.70, 39.36)
Acute DVT 0.16 (0.14, 0.43) 1.52 (0.71, 3.24) 0.29
Baseline characteristics
Agea0.08 (0.36, 0.22) 0.99 (0.96, 1.02) 0.61
Hypertension 0.15 (0.15, 0.42) 1.42 (0.71, 2.84) 0.33
Diabetes mellitus 0.19 (0.46, 0.11) 0.54 (0.21, 1.39) 0.21
Hypercholesterolaemia 0.22 (0.08, 0.48) 1.67 (0.84, 3.33) 0.15
Current smoker 0.23 (0.07, 0.49) 2.55 (0.77, 8.43) 0.13
Coronary artery disease 0.10 (0.20, 0.39) 1.59 (0.39, 6.44)
Previous atrial fibrillation 0.28 (0.02, 0.53) 2.57 (0.96, 6.89) 0.07
Previous DVT 0.17 (0.13, 0.44) 1.61 (0.70, 3.68) 0.27
Aspirin 0.14 (0.16, 0.42) 1.55 (0.63, 3.82) 0.35
Biological criteria
C-reactive proteina0.16 (0.43, 0.14) 1.0 (0.99, 1.00) 0.30
BNPa,b 0.46 (0.15, 0.69) 1.00 (1, 1.00) <0.01
Ln MPs 1.62 (1.06) 0.88 (0.78, 0.93)
Echographic criteria
LVEF < 40% 0.09 (0.21, 0.38) 1.54 (0.38, 6.19)
STPGa 0.21 (0.09, 0.48) 1.47 (0.85, 2.54) 0.17
Endothelial-derived MPs (anti-CD31)
Clinical presentation
Shock 0.28 (0.02 , 0.53) 8.37 (0.91, 77.2)
Acute DVT 0.03 (0.32, 0.27) 0.91 (0.32, 2.61) 0.87
Baseline characteristics
Agea 0.02 (0.27, 0.32) 1.00 (0.96, 1.05) 0.88
Hypertension 0.08 (0.22, 0.36) 1.27 (0.49, 3.31) 0.62
Diabetes mellitus 0.06 (0.35, 0.24) 0.77 (0.21, 2.86) 0.70
Hypercholesterolaemia 0.08 (0.22, 0.37) 1.30 (0.50, 3.49) 0.60
Current smoker 0.22 (0.08, 0.49) 3.30 (0.66, 16.42) 0.15
Coronary artery disease 0.04 (0.33, 0.26) 0.79 (0.12, 5.31)
Previous atrial fibrillation 0.19 (0.11, 0.46) 2.44 (0.62, 9.65) 0.21
400 L. Bal et al.
Table 2 (Suite )
No. of patients (%) Pearson correlation
(95% CI)
% change in MPs
(95% CI)
Pvalue
Previous DVT 0.13 (0.17, 0.41) 1.62 (0.52, 5.05) 0.41
Aspirin 0.02 (0.28, 0.31) 1.08 (0.31, 3.75) 0.90
Biological criteria
C-reactive proteina0.12 (0.40, 0.18) 0.998 (0.99, 1.00) 0.44
BNPa,b 0.38 (0.05, 0.64) 1.00 (1, 1.00) 0.03
Ln MPs 1.62 (1.06) 0.53 (0.28, 0.72)
Echographic criteria
LVEF < 40% 0.09 (0.21, 0.37) 1.76 (0.26, 11.78)
STPGa 0.24 (0.07, 0.50) 1.84 (0.86, 3.91) 0.12
aMean.
bn= 34.
BNP: brain natriuretic peptide; CI: confidence interval; DVT: deep-vein thrombosis; GP: glycoprotein; Ln: natural logarithm; LVEF: left
ventricular ejection fraction; MP: microparticle; STPG: systolic transtricuspid pressure gradient.
p= 0.08) (Fig. 2). PMP levels were significantly higher in
patients with APE compared with ControlsnoCVRFs (median
1.9 nmol/L, IQR 1.4—2.4 nmol/L of phosphatidylserine
equivalent; p= 0.02) and in ControlsCVRFs compared with
ControlsnoCVRFs (p< 0.001).
After adjustment for age, sex and hypertension, PMPs
were associated with brain natriuretic peptide (p= 0.006)
but not with C-reactive protein levels (p= 0.302). Neither
pulmonary arterial pressure nor left ventricular systolic dys-
function were correlated with PMPs levels (Table 2).
Endothelial-derived microparticles in acute
pulmonary embolism patients and controls
EMP levels were not significantly different between patients
with APE (median 0.1 nmol/L, IQR 0.03—0.2 nmol/L) and
Figure 2. Levels of platelet-derived microparticles (PMP) in
controls with (ControlsCVRFs) and without (ControlsnoCVRFs ) cardio-
vascular risk factors and in patients with acute pulmonary embolism
(APE).
Figure 3. Levels of endothelial-derived microparticles (EMP) in
controls with (ControlsCVRFs) and without (ControlsnoCVRFs ) cardio-
vascular risk factors and in patients with acute pulmonary embolism
(APE).
ControlsCVRFs (median 0.2 nmol/L, IQR 0.1—0.2 nmol/L;
p= 0.44) (Fig. 3). EMP levels were not higher in patients with
APE compared with ControlsnoCVRFs (median 0.1 nmol/L, IQR
0.01—0.1 nmol/L; p=0.54). EMP levels in ControlsCVRFs were
not significantly higher than in ControlsnoCVRFs (p= 0.06).
After adjustment for age, sex and hypertension, EMPs
were only correlated with brain natriuretic peptide levels
(p= 0.026) (Table 2).
Baseline characteristics and circulating
procoagulant microparticles
Relations between other baseline characteristics and CPMPs
(annexin V-positive, PMPs and EMPs) were investigated
among patients with APE after adjustment for age, sex and
hypertension (Table 2). Current smoking, significantly rep-
resented in ControlsCVRFs, (Table 1;p= 0.003), appeared to
influence CPMP and PMP levels among patients with APE
Circulating procoagulant microparticles in pulmonary embolism 401
(change in microparticles 2.57%, p= 0.09 and 2.55%, p= 0.13,
respectively).
Discussion
To our knowledge, this is the first study to compare CPMP
levels and their phenotypes (using a functional assay based
on prothrombinase) in APE patients. Comparing APE patients
with two different control groups, our study design allowed
us to analyse the relative effect of cardiovascular risk fac-
tors and APE on microparticle levels. In APE patients, CPMP
procoagulant activity is significantly increased compared
with the physiological status represented by a population
with no prior thrombotic events or cardiovascular risk fac-
tors (p= 0.02). Nevertheless, procoagulant activity related
to CPMPs in APE patients was not significantly different to
that in ControlsCVRFs.
Microparticles and venous thromboembolism
VTE results from an imbalance between procoagulant,
anticoagulant and fibrinolytic activities. Platelet and
endothelial microparticles are considered markers of ongo-
ing or recent endothelial cell and platelet activation,
or apoptosis. Microparticle procoagulant activity, mainly
related to tissue factor activity in the presence of phos-
phatidylserine phospholipids, is dependent on the cellular
origin, the initial stimulus and the secondary microparticle-
induced cell activation [4,18,29]. Experimental animal
studies with high-resolution online videomicroscopy have
revealed that circulating microparticles mediate the accu-
mulation of tissue factor on platelet-rich thrombi, and also
in the venous thrombosis model [18,30]. Demonstrating the
increased procoagulant activity related to CPMPs in APE
patients vs ControlsnoCVRF, our results argue in favour of their
functional participation in venous thrombogenesis in the
interface of soluble coagulants factors, circulating cells and
dysfunctional endothelium. As far as microparticle subtypes
are concerned, only PMPs appeared to be significantly ele-
vated in APE patients vs ControlsnoCVRF. We have to take into
account the lower detection limits in the estimation of EMP
involvement in APE to interpret this result, in terms of the
variability in antibody affinity (CD31 vs CD62E or CD144), as
in the availability of the antigen or in the level of endothelial
markers borne by microparticles [31]. However, this feature
has been used in studies reported by other groups to mea-
sure EMPs (CD31) and PMPs (glycoprotein 1b) distinctly [16].
In the future, more specific endothelial phenotypes, such as
CD62E/CD144/CD146, should be targeted as a priority.
Data emerging from the literature are quite discordant
concerning microparticles and phenotypes involved in APE,
mainly because of the different techniques and method-
ological approaches used. To our knowledge, there are two
previous clinical reports on this subject that differ from our
study in terms of the design, the method of microparticle
quantitation, the population studied and the comparator
used [16,17]. In addition, increasing data correlated the
risk of deep-vein thrombosis in patients with cancer to
tissue factor-bearing tumour cell-derived microparticle lev-
els measured by functional test or flow cytometry, with a
pathophysiological role supported by P-selectin glycoprotein
ligand 1 (or other mucinous glycoproteins) and P-selectin
interaction [21,23].
Relation between cardiovascular risk factors
and microparticles in venous
thromboembolism
Our observations highlight the importance of adjusting for
the presence of cardiovascular risk factors in conditions in
which microparticle levels are raised.
Diabetes [13,15,32] and hypertension [14] are associated
with endothelial dysfunction and platelet activation, and
increase CPMP levels. In a previous report, our group [15]
found a higher level of CPMPs in patients with type 2 dia-
betes compared with healthy subjects. A strong positive
correlation was also found between endothelial and platelet
microparticle levels on the one hand, and the absolute
level of both systolic and diastolic blood pressures on the
other [14]. These findings and the present data emphasize
that cardiovascular risk factors, especially hypertension,
are confounders in the previously described relationship
between circulating microparticles and VTE disease [16,17].
We suggest several hypotheses to account for the intrigu-
ing lack of difference in CPMP procoagulant activity between
cases and ControlsCVRFs. First, we could consider the pre-
dominant role of cardiovascular risk factors as vascular cell
activators vs venous thrombosis risk factors such as hypox-
aemia induced by blood stasis. A second hypothesis relies on
an expected difference in microparticle consumption kinet-
ics between both populations, which would be faster in
cases of an acute thrombotic event, such as venous thrombo-
sis [22,33—35]. Indeed, the production and consumption of
microparticles are mainly local processes in the acute phase
of VTE. This is in line with the recent experimental study by
Ramacciotti et al., demonstrating not only variable kinetics
for each subtype of microparticle, but also their evolutive
thrombogenicity during the thrombotic process itself [19].
The sequestration of microparticles in the forming throm-
bus is also a diluting factor [23]. The last hypothesis is an
underestimation of the CPMP effective procoagulant activ-
ity due to a lack of sensitivity with our test using a specific
quantification of microparticle-linked phosphatidylserine in
a functional assay, compared with tissue factor activity eval-
uation [36].
Microparticles: cause or consequence in
venous thrombogenesis?
By evaluating microparticle-related procoagulant activity in
only the acute phase of venous thrombosis, our study is
unable to assess whether CPMPs have a causal role in venous
thrombogenesis. Nevertheless, we found some interesting
correlations.
The PMP subtype appeared to be an important source
of procoagulant microparticles, and was correlated with
CPMPs. This is in line with the hypothesis of platelet activa-
tion and participation in venous thrombogenesis as has been
underlined recently in the literature [2,3], linking venous
thrombosis to atherothrombosis. PMPs levels have been
associated positively with thrombus weight and proteomics
of microparticles after VTE revealed the upregulation of Gal
402 L. Bal et al.
3BP, a polypeptide from lectin family that plays a key role in
human platelet aggregation and function, promoting shed-
ding of microparticles and generation of leucocyte-platelet
aggregates [37].
In parallel, we observed a tendency towards an asso-
ciation of CPMPs and PMPs with severe clinical status, in
accordance with brain natriuretic peptide levels, but weakly
linked with C-reactive protein, an inflammatory marker with
a poor negative predictive value in pulmonary embolism
[38,39]. Recently, two studies correlated the severity of pul-
monary hypertension with CPMP levels in pulmonary artery
blood samples [31,40].
Finally, after adjustment for age, sex and hypertension,
CPMPs and PMPs appeared to be correlated in our cases
with current smoking, a major cardiovascular risk factor in
atherothrombosis. Moreover, Pomp et al. [41] showed that
current smoking remained a risk factor for venous thrombo-
sis among young people after adjustment for age, sex and
body mass index. In the literature, an increasing amount of
evidence suggests the likelihood of a link between arterial
and venous disease [42]. According to results from recent
studies, atherosclerosis and VTE share common risk fac-
tors, including age, obesity and current smoking [43,44].
In a large population-based study, Sorensen et al. [45]
provided strong evidence that patients with VTE are at
increased risk of subsequent arterial cardiovascular events
compared with population controls, which is most pro-
nounced during the first year of follow-up. This is consistent
with underlying common prothrombotic mechanisms such
as thrombogenesis, endothelial damage and inflammation
[46].
Study limitations
We did not evaluate the post-VTE prothrombotic state
related to CPMPs using a second measure of CPMP procoag-
ulant activity during the first year of follow-up. Moreover,
taking into account the limitation of our test related to
the choice of CD31 as single endothelial phenotypic marker
[47,48], we should also consider the involvement of other
microparticles subtypes, such as leukocyte microparticles.
Several studies demonstrated that platelets, leukocytes and
endothelial cells colocalize and interact in the milieu of a
forming thrombus [49—52]. By binding the PSGL-1 counter
receptor via P-selectin, platelets and PMPs could activate
monocytes, enhancing tissue factor expression and proco-
agulant leukocyte microparticle release [53]. As suggested
by experimental studies in vivo, leukocyte microparticle
release might play a key role in thrombus initiation to
vascular remodelling, and we are going to evaluate their
procoagulant activity in APE [19,49]. Finally, we might have
underestimated the procoagulant activity related to CPMP
by using a prothrombinase assay, whereas the estimation of
tissue factor procoagulant activity would be more relevant
[36].
Conclusions
Our study confirms the correlation between APE and CPMPs
through their own procoagulant functionality, relying mainly
on platelet activation. This relation no longer holds true
when patients with APE are compared with those with car-
diovascular risk factors, stressing the fact that potential
confounders should be taken into account when microparti-
cle levels are analysed in VTE.
Further research is needed to demonstrate in which con-
ditions (i.e., initial stimuli, cell origin, etc.) the circulating
pool of microparticles could be sufficient to precipitate a
venous thrombotic event and to evaluate the therapeutic
implications.
Conflict of interest statement
None.
Acknowledgment
Sophie Rushton-Smith, PhD, provided editorial assistance
in the final version of this manuscript, including editing,
checking content and language, formatting, referencing and
preparing tables and figures, and was funded by Saint-
Antoine University hospital and Medical School (association
Robert-Debré).
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    • Microparticles derived from platelets (PMP) are identified by the expression of platelet markers such as CD41 or CD61 [23]. High levels of PMPs are known to be strongly associated with thrombotic complications [24]. Horn et al. have recently shown that EMPs decrease 3 months after transcatheter aortic valve implantation (TAVI), reflecting improved endothelial function and wall shear stress [25].
    [Show abstract] [Hide abstract] ABSTRACT: Degenerative aortic stenosis (AS) is the most frequent form of acquired valvular heart disease. AS is known to entail endothelial dysfunction caused by increased mechanical shear stress leading to elevated circulatory levels of microparticles. Endothelial and platelet microparticles (EMP and PMP) are small vesicles that originate from activated cells and thrombocytes. We sought to evaluate whether transcatheter aortic valve implantation (TAVI) procedure would elicit effects on circulating EMP and PMP. 92 patients undergoing TAVI procedure for severe AS were included in this study. Samples were obtained at each visit before TAVI, 1 week post-procedure and at 1, 3 and after 6 months after TAVI and were evaluated using flow cytometry. A 12 month clinical follow-up was also performed. CD62E+ EMP concentration before TAVI was 21.11 % (±6.6 % SD) and declined to 20.99 % (±6.8 % SD) after 1 week, to 16.63 % (±5.4 % SD, p < 0.0001) after 1 month, to 17.08 % (±4.6 % SD, p < 0.0001) after 3 months and to 15.94 % (±5.4 % SD, p < 0.0001) after 6 months. CD31+/CD42b−, CD31+/Annexin+/− EMP remained unchanged. CD31+/CD41b+ PMP evidenced a slight, but statistically significant increase after TAVI and remained elevated during the entire follow-up. Apart from a procedure-related improvement in echocardiographic parameters, TAVI procedure led also to a decline in CD62E+ EMP. The reduction in pressure gradients with less hemodynamic shear stress seems also to have beneficially affected endothelial homeostasis. Electronic supplementary material The online version of this article (doi:10.1007/s00380-016-0885-z) contains supplementary material, which is available to authorized users.
    Full-text · Article · Aug 2016
    • All the sample collection in our study was done prior to PCI. In a study done by Bal et al., they found that PMPs did not show any correlation with EF [22].
    [Show abstract] [Hide abstract] ABSTRACT: Background: Microparticles (MP) are a nuclear fragments of membrane released by the damaged cell during stress. Elevated levels of MP have been found in patients with acute coronary syndrome (ACS) owing to the damage in the endothelium. Aim: To determine if the levels of endothelial and platelet microparticles (EMP & PMP) in patients with ACS influenced the severity of the disease. Materials and Methods: This was a prospective cohort study performed in 63 ACS patients (ST elevation myocardial infarction- STEMI-28, non ST elevation myocardial infarction -NSTEMI-35). After obtaining consent, blood samples were collected from the patients and processed by flow cytometry. Results: The NSTEMI group had higher levels of EMP {792.11(327.59-1661.49) vs 300.35 (176.3-550.46), p=0.001} and PMP {218.87(86.65-439.77) vs 114.45(50.34-196.75), p= 0.007} as compared to the STEMI group. However, it was found that the EMP (r=-0.438, p=0.001) and PMP (r= -0.316, p=0.024) negatively correlated with Global Registry of Acute Coronary Events score (GRACE in-hospital score) for the entire cohort. Conclusion: The levels of microparticles are elevated in ACS patients and may reflect a protective effect in patients with acute coronary syndrome. © 2015, Journal of Clinical and Diagnostic Research. All rights reserved.
    Article · Dec 2015
    • Pulmonary hypertension can be caused by chronic thromboembolic disease (CTEPH)[61]. Pulmonary embolism, as in many diseases in which apoptosis occurs[62], results in shedding of microparticles (MP)[63]. MP are shed from apoptotic sites, circulate via plasma, and act on distant sites to effect inflammation, hemostasis, and thrombosis[64].
    [Show abstract] [Hide abstract] ABSTRACT: Pulmonary hypertension (PH) is a fatal syndrome that arises from a multifactorial and complex background, is characterized by increased pulmonary vascular resistance and right heart afterload, and often leads to cor pulmonale. Over the past decades, remarkable progress has been made in reducing patient symptoms and delaying the progression of the disease. Unfortunately, PH remains a disease with no cure. The substantial heterogeneity of PH continues to be a major limitation to the development of newer and more efficacious therapies. New advances in our understanding of the biological pathways leading to such a complex pathogenesis will require the identification of the important proteins and protein networks that differ between a healthy lung (or right ventricle) and a remodeled lung in an individual with PH. In this article, we present the case for the increased use of proteomics-the study of proteins and protein networks- as a discovery tool for key proteins and protein networks operational in the PH lung. We review recent applications of proteomics in PH, and summarize the biological pathways identified. Finally, we attempt to presage what the future will bring with regard to proteomics in PH and offer our perspectives on the prospects of developing personalized proteomics and custom-tailored therapies.
    Full-text · Article · Feb 2015
    • There was a dramatic increase in platelet activation (35.2 vs 5.0 fluorescence intensity units for P-selectin; P<0.0001) in the setting of VTE, which was found in the recent study by Chirinos et al37. Likewise, elevated PMPs were detected in patients with VTE in two other studies34,35, and a strong correlation was found between total MPs and PMPs (ρ=0.99, P<0.0001) and between PMPs and TF-bearing MPs (ρ=0.94,
    [Show abstract] [Hide abstract] ABSTRACT: M Microparticles are small membrane fragments shed primarily from blood and endothelial cells during either activation or apoptosis. There is mounting evidence suggesting that microparticles perform a large array of biological functions and contribute to various diseases. Of these disease processes, a significant link has been established between microparticles and venous thromboembolism. Advances in research on the role of microparticles in thrombosis have yielded crucial insights into possible mechanisms, diagnoses and therapeutic targets of venous thromboembolism. In this review, we discuss the definition and properties of microparticles and venous thromboembolism, provide a synopsis of the evidence detailing the contributions of microparticles to venous thromboembolism, and propose potential mechanisms, by which venous thromboembolism occurs. Moreover, we illustrate a possible role of microparticles in cancer-related venous thromboembolism.
    Full-text · Article · Aug 2014
  • [Show abstract] [Hide abstract] ABSTRACT: Microparticles (MPs) derived from platelets, monocytes, endothelial cells, red blood cells, and granulocytes may be detected in low concentrations in normal plasma and at increased levels in atherothrombotic cardiovascular diseases. The elucidation of the cellular mechanisms underlying the generation of circulating MPs is crucial for improving our understanding of their pathophysiological role in health and disease. The flopping of phosphatidylserine (PS) to the outer leaflet of the plasma membrane is the key event that will ultimately lead to the shedding of procoagulant MPs from activated or apoptotic cells. Research over the last few years has revealed important roles for calcium-, mitochondrial-, and caspase-dependent mechanisms leading to PS exposure. The study of Scott cells has unraveled different molecular mechanisms that may contribute to fine-tuning of PS exposure and MP release in response to a variety of specific stimuli. The pharmacological modulation of MP release may have a substantial therapeutic impact in the management of atherothrombotic vascular disorders. Because PS exposure is a key feature in pathological processes different from hemostasis and thrombosis, the most important obstacle in the field of MP-modulating drugs seems to be carefully targeting MP release to relevant cell types at an optimal level, so as to achieve a beneficial action and limit possible adverse effects.
    Article · Jan 2011
  • [Show abstract] [Hide abstract] ABSTRACT: Microparticles are circulating fragments derived from blebbing and shedding of cell membranes through several mechanisms that include activation, apoptosis and cell damage. In the past they were largely considered as unimportant cell "dust", but more refined detection techniques have revealed large variations in their relative proportion and concentration in numerous disease states. Importantly, these conditions include the most prevalent causes of death and disability in our societies, namely cardiovascular, neoplastic, and inflammatory diseases. Microparticles carry procoagulant, proapoptotic and neoangiogenetic materials in the blood stream, and can also be viewed as a technique cells may adopt to rapidly modify their phenotype, independently from genetic signals. In this review, we focus on the role of these very small ( 1 micron) particles, not only as mere markers of an underlying pathologic state, but also as primordial intercellular messengers and defense mechanism that every viable cell can exploit in distress conditions. In this view, we suggest that this old communication system could be the target of future high-tech interventions, with potential broad consequences.
    Article · Mar 2011
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