Factors Influencing the Level of Circulating Procoagulant Microparticles in Acute Pulmonary Embolism

Archives of Cardiovascular Disease (2010) 103, 394—403

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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 Bal a, Stéphane Ederhy a, Emanuele Di Angelantonio a, Florence Toti b, Fatiha Zobairi b, Ghislaine Dufaitre a, Catherine Meuleman a, Ziad Mallat c, Franck Boccara a, Alain Tedgui c, Jean-Marie Freyssinet b, Ariel Cohen a,∗

a Cardiology 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 b Inserm U770, hôpital de Bicêtre, université Louis-Pasteur, faculté de médecine, institut d’hématologie et immunologie, 67400 Strasbourg, France c Inserm 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 Summary Microparticles; Background. — Flow cytometry has shown levels of platelet-derived microparticles (PMPs) and Pulmonary embolism; endothelial-derived microparticles (EMPs) to be elevated in deep-vein thrombosis. Cardiovas- Cardiovascular risk cular risk factors can also contribute to hypercoagulability due to circulating procoagulant factors 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: [email protected] (A. Cohen).

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 Résumé Microparticules ; Contexte. — La thrombose veineuse profonde est associée à une augmentation du niveau de Embolie pulmonaire ; microparticules (MP) d’origine endothéliale et plaquettaire mesurée en cytométrie de flux. Les Facteurs de risque facteurs de risque cardiovasculaires (FRCV) ont aussi une influence importante sur le niveau cardiovasculaires 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 circulantes 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 Publie´ par Elsevier Masson SAS.

Introduction CPMPs are plasma membrane fragments that are released into the blood by stimulated cells during activation or apop- According to Virchow’s triad, the pathophysiology of tosis, and carry procoagulant phosphatidylserine and tissue VTE relies on the presence of blood hypercoagula- factor on their surface [4—7]. CPMPs circulate in healthy bility, stasis of blood flow and vessel-wall damage humans and support low-grade generation of thrombin [8]. [1]. The factors involved in venous thrombogenesis High levels of CPMPs have been reported in several sce- could be defined as soluble coagulant factors, dys- narios, including, for example, in patients with an acute functional endothelium and circulating cells, especially coronary syndrome [9—11] or atrial fibrillation [12]. Car- platelets, lymphomonocytes and, potentially, CPMPs diovascular risk factors such as hypertension and diabetes [2,3]. [13—15] have been associated with elevated CPMPs and their 396 L. Bal et al. phenotypes. In interpreting the role of CPMPs in thrombo- heart failure on transthoracic echocardiography), massive genesis, we need to take into account potential cofounders, APE (unstable haemodynamics with right heart failure on the most prevalent of which appear to be cardiovascular risk transthoracic echocardiography) and shock. factors. Two previous clinical studies have reported elevated levels of CPMPs — mainly PMPs and EMPs — by flow cytometry, Controls with cardiovascular risk factors in patients hospitalized for deep-vein thrombosis. How- ControlsCVRFs comprised patients with no history of VTE ever,they had different phenotypic representations, and the or atrial fibrillation who were undergoing routine screen- authors did not take into account potential confounders, ing physical examinations for cardiac symptoms at our such as cardiovascular risk factors [16,17]. Experimental outpatient cardiology clinic, with an electrocardiogram doc- studies in vivo are supportive of the involvement of human umenting sinus rhythm. cell-derived microparticles in venous thrombogenesis in a tissue factor-dependent manner, and have described a correlation of leukocyte- and PMPs with thrombus weight and Controls without cardiovascular risk factors tissue factor activity [18,19]. Recently, the crucial partic- ControlsnoCVRF included patients undergoing screening exam- ipation of circulating tissue factor-bearing microparticles ination before orthopaedic surgery, with no known cardio- released by tumour cells in cancer-associated hypercoagula- vascular risk factors, history of atrial fibrillation, prior VTE, bility has been emphasized, depending on both the tumour clinical evidence of disease or current cardiovascular treat- cell origin and a critical threshold of microparticles [20—24]. ment, and who had an electrocardiogram documenting sinus In addition, we have shown in a case-control study that CPMP rhythm. These subjects were assessed by careful examina- levels, defined by their procoagulant activity, were elevated tion of their medical histories and by blood tests. in patients without cancer hospitalized with APE, and we analysed the influence of cardiovascular risk factors on this correlation [25]. Circulating procoagulant microparticles Blood was collected in the acute phase when the diagnosis of VTE was assessed and just before anticoagulation was Methods started. Measurement of CPMPs was performed as described previously [26], with minor modifications. Study subjects

Between November 2004 and December 2005, we included Preparation of circulating procoagulant 45 consecutive patients admitted to our intensive care unit microparticle samples for APE associated with or without deep-vein thrombo- All microparticle determinations were performed strictly sis, and compared them with 45 healthy controls without according to Biro et al. [18]. Briefly, citrated blood was (Controls ) and 45 patients with (Controls ) cardio- noCVRFs CVRFs taken soon after admission and centrifuged at 1500 g for vascular risk factors, matched for age and sex. Controls CVRFs 15 min at room temperature within the hour after sam- and patients with APE were also matched for the presence pling. The supernatant was centrifuged again at 13,000 g of hypertension. Demographic and clinical characteristics for 2 min to avoid platelet contamination. Thrombin and fac- were recorded prospectively upon enrolment. The study tor Xa inhibitors (d-phenylalanyl-prolyl-arginyl chloromethyl was approved by the institutional review board and was ketone and 1,5-dansyl-glutamyl-glycyl-arginyl chloromethyl performed in accordance with institutional guidelines. All ketone, respectively) were added to plasma samples at patients gave written informed consent before participating a final concentration of 50 ␮M each, and CaCl at a final in the study. 2 concentration of 50 mM.

Acute pulmonary embolism cases Quantitation of circulating procoagulant APE was confirmed by spiral computed tomography (n = 25), microparticles ventilation-perfusion scintigraphy (n = 20) or both (n = 12). Treatment on admission consisted of standard antithrom- After capture of microparticles onto annexin V-coated wells ◦ botic therapy with low-molecular-weight or unfractionated (for 30 min at 37 C), taking advantage of the strong affinity heparin. Exclusion criteria were conditions known or sus- of annexin V for aminophospholipids present in microparti- pected to increase levels of CPMPs independently, such cles at the calcium concentration used, four washing steps as acute coronary syndromes, acute heart failure, stroke, were performed with Tris buffer containing 1 mM CaCl2 and ◦ sepsis, chronic inflammatory disease, antiphospholipid 0.05% Tween 20, each for 5 min at 20 C, and the last one syndrome, heparin-induced thrombocytopenia, thrombotic without Tween.The phosphatidylserine content of micropar- thrombocytopenic purpura and atrial fibrillation. ticles, directly responsible for their procoagulant activity, Transient VTE risk factors were defined as pregnancy, was then measured in a prothrombinase assay. Micropar- oestrogen therapy, surgery (< 60 days), trauma, confined ticles were incubated with factor Xa (50 pmol/L), factor to bed (> 5 days) and recent journey (> 10 hours). Cancer Va (360 pmol/L), prothrombin (1.3 ␮mol/L) and 2.3 mmol/L ◦ and thrombophilia were defined as chronic VTE risk factors. CaCl2 for 15 min at 37 C, and linear absorbance changes Haemodynamic status was considered over three levels: were recorded at 405 nm after the addition of chromozym submassive APE (stable haemodynamics with signs of right 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 phosphatidylserine equivalent.

Miscellaneous measurements

Quantiﬁcation of C-reactive protein was determined by immunonephelometric tests and circulating brain natriuretic peptide levels by enzyme immunoassays.

Transthoracic echocardiography

To evaluate right ventricular dysfunction and haemodynamic Figure 1. Levels of circulating procoagulant microparticles in status, transthoracic echocardiography was performed at controls with (ControlsCVRFs) and without (ControlsnoCVRFs) cardio- the time of admission in all patients with APE. Systolic vascular risk factors and in patients with acute pulmonary embolism transtricuspid pressure gradient and left ventricular ejec- (APE). tion fraction were also measured. dynamic instability and 4.4% (n = 2) presented in cardiogenic Statistical analysis shock. APE was submassive in 17 (37.8%) patients. Sixteen (35.5%) patients with APE had at least one transient VTE risk Based on previous studies [11,27,28], we hypothesized that factor and eight (17.8%) had a permanent risk factor (cancer, patients with APE would have microparticle levels increased n = 3; thrombophilia, n = 5). APE was idiopathic in 21 (46.7%) by approximately two standard deviations compared with patients. healthy controls and by one standard deviation compared There were no significant differences in terms of clini- with subjects without APE but with cardiovascular risk fac- cal cardiovascular risk factors between patients with APE tors. To achieve this with 90% power and p < 0.05 between and ControlsCVRFs, except for current smoking (Table 1). In the three groups, 35 subjects per group were required. To contrast, by design, ControlsnoCVRFs were significantly dif- minimize the risk of a type II error and to account for pos- ferent from the other two groups with regard to clinical sible confounders, we recruited in excess of this number of cardiovascular risk factors. However, there was no signifi- patients with APE and controls. cant difference regarding age and sex between ControlsCVRFs Categorical variables, expressed as percentages, were and ControlsnoCVRF, even if there was a slight predominance compared using the Chi2 test or Fisher’s exact test. After a of men in the former. test for normality, continuous data are expressed as means and standard deviations or medians with IQRs as appro- Annexin-positive microparticles in acute priate. Differences between patients and controls were pulmonary embolism patients and controls evaluated using the two-sample t test or the Mann-Whitney U test. Correlations between annexin V-positive microparti- Annexin V-positive microparticle levels were higher cles and endothelial and platelet microparticle levels were in patients with APE (median 4.7 nmol/L, IQR evaluated using Spearman’s rank correlation coefficients. 2.9—11.1 nmol/L) than in ControlsnoCVRFs (median The relationship between CPMP levels and patients’ 3.2 nmol/L, IQR 2.3—4.6 nmol/L; p = 0.02), but there was no characteristics was estimated using linear regression anal- significant difference compared with ControlsCVRFs(median ysis (after logarithmic transformation of the dependent 4.9 nmol/L, IQR 3.7—8.4 nmol/L; p = 0.99) (Fig. 1). Annexin variables) and presented using the estimated regression V-positive microparticle levels were significantly higher in coefficient, expressed as percentage increase in micropar- ControlsCVRFs than in ControlsnoCVRFs (p = 0.01; Fig. 1). ticle level for the presence of each risk factor or for a unit Moreover, after adjustment for age, sex and hyperten- increase in continuous variables, such as age. All analyses sion, CPMPs were significantly correlated with C-reactive were performed using STATA 9 statistical software (STATA, protein (p = 0.02) and brain natriuretic peptide levels College Station, TX, USA). A probability value of 0.05 was (p < 0.01), but not with the echographic haemodynamic sta- considered statistically significant. tus evaluated by systolic transtricuspid pressure gradient and left ventricular ejection fraction (Table 2). Results Platelet-derived microparticles in acute pulmonary embolism patients and controls The baseline characteristics of the three groups are given in Table 1. The mean age of patients with APE was 67.9 ± 11.6 PMP levels were not significantly different between patients years and 66.7% were men. Deep-vein thrombosis was with APE (median 2.2 nmol/L, IQR 1.7—5.8 nmol/L) and documented in 71.1% of patients, 26.7% (n = 12) had haemo- ControlsCVRFs (median 5.5 nmol/L, IQR 2.6—11.3 nmol/L; 398 L. Bal et al. vs CVRFs noCVRFs < 0.01 Controls Controls noCVRFs 0.04 — —— < 0.01 < 0.01 APE vs Controls CVRFs value < 0.01 < 0.01 P APE vs Controls noCVRFs 9.5 — — — : controls without cardiovascular risk factors; TIA: transient ischaemic attack. ± = 45) n noCVRFs ———— Controls ( CVRFs 9.9 67.0 ± = 45) n ( = 45) Controls 11.6 67.1 n ± : controls with cardiovascular risk factors; Controls CVRFs standard deviation or number (%). ± Baseline characteristics in patients with acute pulmonary embolism and controls with and without cardiovascular risk factors. AspirinBeta-blockerCalcium channel blockerAngiotensin-converting enzyme inhibitorDiuretic 7 (15.9) 7 (15.6) 6 (13.3) 9 (20.5) 4 — (8.9) 8 (17.8) 10 (22.2) 8 1 (17.8) (2.2) — 0.96 — 0 — 0.50 — 0.84 — 0.01 — — — — — — — NitrateInsulinOral antidiabetic therapy 2 (4.4) 6 1 (13.3) (2.2) 0 1 (2.2) — 2 (4.4) 0.14 — — — — — Coronary artery diseaseHistory of heart failureHistory of TIA or ischaemicHistory stroke of atrial fibrillationConcomitant treatment 2 3 (4.4) (6.8) 1 (2.2) 6 (13.3) 1 9 (2.2) (20.0) 0 0 0 0 0 0 0.07 — — — 0.08 — — — — — — HypercholesterolaemiaCurrent smoker 19 (42.2) 14 (31.1) 4 (9.1) 0 16 (35.6) 0 0.27 — — Table 1 Patient characteristicsAge (years) APE ( 67.9 Data are mean MenHypertensionDiabetes mellitus 7 (15.6) 25 (55.6) 30 (66.7) 25 9 (55.6) (20.0) 30 (66.7) 0 0 26 (57.8) — — 0.58 0.38 — 0.38 — APE: acute pulmonary embolism; Controls 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 % change in MPs P value (95% CI) (95% CI) 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 Agea 67.9 (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 proteina 59.2 (83.7) −0.36 (−0.59, −0.07) 0.995 (0.99, 0.999) 0.02 BNPa,b 279.6 (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) — STPGa 135 (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 Agea — −0.08 (−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 proteina — −0.16 (−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 % change in MPs P value (95% CI) (95% CI) 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 proteina — −0.12 (−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

a Mean. b n = 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 dysfunction 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 3. Levels of endothelial-derived microparticles (EMP) in controls with (ControlsCVRFs) and without (ControlsnoCVRFs) cardiovascular 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 Figure 2. Levels of platelet-derived microparticles (PMP) in among patients with APE after adjustment for age, sex and controls with (ControlsCVRFs) and without (ControlsnoCVRFs) cardio- hypertension (Table 2). Current smoking, signiﬁcantly rep- vascular risk factors and in patients with acute pulmonary embolism resented in ControlsCVRFs, (Table 1; p = 0.003), appeared to (APE). 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, ligand 1 (or other mucinous glycoproteins) and P-selectin respectively). interaction [21,23].

Relation between cardiovascular risk factors Discussion and microparticles in venous To our knowledge, this is the first study to compare CPMP thromboembolism levels and their phenotypes (using a functional assay based on prothrombinase) in APE patients. Comparing APE patients Our observations highlight the importance of adjusting for with two different control groups, our study design allowed the presence of cardiovascular risk factors in conditions in us to analyse the relative effect of cardiovascular risk fac- which microparticle levels are raised. tors and APE on microparticle levels. In APE patients, CPMP Diabetes [13,15,32] and hypertension [14] are associated procoagulant activity is significantly increased compared with endothelial dysfunction and platelet activation, and with the physiological status represented by a population increase CPMP levels. In a previous report, our group [15] with no prior thrombotic events or cardiovascular risk fac- found a higher level of CPMPs in patients with type 2 dia- tors (p = 0.02). Nevertheless, procoagulant activity related betes compared with healthy subjects. A strong positive to CPMPs in APE patients was not significantly different to correlation was also found between endothelial and platelet that in Controls . microparticle levels on the one hand, and the absolute CVRFs level of both systolic and diastolic blood pressures on the Microparticles and venous thromboembolism other [14]. These findings and the present data emphasize that cardiovascular risk factors, especially hypertension, are confounders in the previously described relationship VTE results from an imbalance between procoagulant, between circulating microparticles and VTE disease [16,17]. anticoagulant and fibrinolytic activities. Platelet and We suggest several hypotheses to account for the intrigu- endothelial microparticles are considered markers of ongo- ing lack of difference in CPMP procoagulant activity between ing or recent endothelial cell and platelet activation, cases and Controls . First, we could consider the pre- or apoptosis. Microparticle procoagulant activity, mainly CVRFs dominant role of cardiovascular risk factors as vascular cell related to tissue factor activity in the presence of phos- activators vs venous thrombosis risk factors such as hypox- phatidylserine phospholipids, is dependent on the cellular aemia induced by blood stasis. A second hypothesis relies on origin, the initial stimulus and the secondary microparticle- an expected difference in microparticle consumption kinet- induced cell activation [4,18,29]. Experimental animal ics between both populations, which would be faster in studies with high-resolution online videomicroscopy have cases of an acute thrombotic event, such as venous thrombo- revealed that circulating microparticles mediate the accu- sis [22,33—35]. Indeed, the production and consumption of mulation of tissue factor on platelet-rich thrombi, and also microparticles are mainly local processes in the acute phase in the venous thrombosis model [18,30]. Demonstrating the of VTE. This is in line with the recent experimental study by increased procoagulant activity related to CPMPs in APE Ramacciotti et al., demonstrating not only variable kinetics patients vs Controls , our results argue in favour of their noCVRF for each subtype of microparticle, but also their evolutive functional participation in venous thrombogenesis in the thrombogenicity during the thrombotic process itself [19]. interface of soluble coagulants factors, circulating cells and The sequestration of microparticles in the forming throm- dysfunctional endothelium. As far as microparticle subtypes bus is also a diluting factor [23]. The last hypothesis is an are concerned, only PMPs appeared to be significantly ele- underestimation of the CPMP effective procoagulant activ- vated in APE patients vs Controls . We have to take into noCVRF ity due to a lack of sensitivity with our test using a specific account the lower detection limits in the estimation of EMP quantification of microparticle-linked phosphatidylserine in involvement in APE to interpret this result, in terms of the a functional assay, compared with tissue factor activity eval- variability in antibody affinity (CD31 vs CD62E or CD144), as uation [36]. 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- Microparticles: cause or consequence in sure EMPs (CD31) and PMPs (glycoprotein 1b) distinctly [16]. venous thrombogenesis? In the future, more specific endothelial phenotypes, such as CD62E/CD144/CD146, should be targeted as a priority. By evaluating microparticle-related procoagulant activity in Data emerging from the literature are quite discordant only the acute phase of venous thrombosis, our study is concerning microparticles and phenotypes involved in APE, unable to assess whether CPMPs have a causal role in venous mainly because of the different techniques and method- thrombogenesis. Nevertheless, we found some interesting ological approaches used. To our knowledge, there are two correlations. previous clinical reports on this subject that differ from our The PMP subtype appeared to be an important source study in terms of the design, the method of microparticle of procoagulant microparticles, and was correlated with quantitation, the population studied and the comparator CPMPs. This is in line with the hypothesis of platelet activa- used [16,17]. In addition, increasing data correlated the tion and participation in venous thrombogenesis as has been risk of deep-vein thrombosis in patients with cancer to underlined recently in the literature [2,3], linking venous tissue factor-bearing tumour cell-derived microparticle lev- thrombosis to atherothrombosis. PMPs levels have been els measured by functional test or flow cytometry, with a associated positively with thrombus weight and proteomics pathophysiological role supported by P-selectin glycoprotein 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 when patients with APE are compared with those with car- human platelet aggregation and function, promoting shed- diovascular risk factors, stressing the fact that potential ding of microparticles and generation of leucocyte-platelet confounders should be taken into account when microparti- aggregates [37]. cle levels are analysed in VTE. In parallel, we observed a tendency towards an asso- Further research is needed to demonstrate in which con- ciation of CPMPs and PMPs with severe clinical status, in ditions (i.e., initial stimuli, cell origin, etc.) the circulating accordance with brain natriuretic peptide levels, but weakly pool of microparticles could be sufficient to precipitate a linked with C-reactive protein, an inflammatory marker with venous thrombotic event and to evaluate the therapeutic a poor negative predictive value in pulmonary embolism implications. [38,39]. Recently, two studies correlated the severity of pulmonary hypertension with CPMP levels in pulmonary artery blood samples [31,40]. Conflict of interest statement Finally, after adjustment for age, sex and hypertension, CPMPs and PMPs appeared to be correlated in our cases None. 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- Acknowledgment sis among young people after adjustment for age, sex and body mass index. In the literature, an increasing amount of Sophie Rushton-Smith, PhD, provided editorial assistance evidence suggests the likelihood of a link between arterial in the final version of this manuscript, including editing, and venous disease [42]. 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