Summary
Background
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).
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 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).
Results
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 .
Conclusions
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.
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 maladie 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.
Introduction
According to Virchow’s triad, the pathophysiology of VTE relies on the presence of blood hypercoagulability, stasis of blood flow and vessel-wall damage . The factors involved in venous thrombogenesis could be defined as soluble coagulant factors, dysfunctional endothelium and circulating cells, especially platelets, lymphomonocytes and, potentially, CPMPs .
CPMPs are plasma membrane fragments that are released into the blood by stimulated cells during activation or apoptosis, and carry procoagulant phosphatidylserine and tissue factor on their surface . CPMPs circulate in healthy humans and support low-grade generation of thrombin . High levels of CPMPs have been reported in several scenarios, including, for example, in patients with an acute coronary syndrome or atrial fibrillation . Cardiovascular risk factors such as hypertension and diabetes have been associated with elevated CPMPs and their phenotypes. In interpreting the role of CPMPs in thrombogenesis, 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 levels of CPMPs – mainly PMPs and EMPs – by flow cytometry, in patients hospitalized for deep-vein thrombosis. However, they had different phenotypic representations, and the authors did not take into account potential confounders, such as cardiovascular risk factors . 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 correlation of leukocyte- and PMPs with thrombus weight and tissue factor activity . Recently, the crucial participation of circulating tissue factor-bearing microparticles released by tumour cells in cancer-associated hypercoagulability has been emphasized, depending on both the tumour cell origin and a critical threshold of microparticles . 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 .
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 thrombosis, and compared them with 45 healthy controls without (Controls noCVRFs ) and 45 patients with (Controls CVRFs ) cardiovascular risk factors, matched for age and sex. Controls CVRFs 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 antithrombotic therapy with low-molecular-weight or unfractionated heparin. Exclusion criteria were conditions known or suspected 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
Controls CVRFs comprised patients with no history of VTE or atrial fibrillation who were undergoing routine screening physical examinations for cardiac symptoms at our outpatient cardiology clinic, with an electrocardiogram documenting sinus rhythm.
Controls without cardiovascular risk factors
Controls noCVRF included patients undergoing screening examination before orthopaedic surgery, with no known cardiovascular risk factors, history of atrial fibrillation, prior VTE, clinical evidence of disease or current cardiovascular treatment, and who had an electrocardiogram documenting sinus rhythm. These subjects were assessed by careful examination 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 , with minor modifications.
Preparation of circulating procoagulant microparticle samples
All microparticle determinations were performed strictly according to Biro et al. . Briefly, citrated blood was taken soon after admission and centrifuged at 1500 g for 15 min at room temperature within the hour after sampling. The supernatant was centrifuged again at 13,000 g for 2 min to avoid platelet contamination. Thrombin and factor 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 μM each, and CaCl 2 at 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 microparticles at the calcium concentration used, four washing steps were performed with Tris buffer containing 1 mM CaCl 2 and 0.05% Tween 20, each for 5 min at 20 °C, and the last one without Tween. The phosphatidylserine content of microparticles, directly responsible for their procoagulant activity, was then measured in a prothrombinase assay. Microparticles were incubated with factor Xa (50 pmol/L), factor Va (360 pmol/L), prothrombin (1.3 μmol/L) and 2.3 mmol/L CaCl 2 for 15 min at 37 °C, and linear absorbance changes were recorded at 405 nm after the addition of chromozym TH (380 μmol/L).
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 status, transthoracic echocardiography was performed at the time of admission in all patients with APE. Systolic transtricuspid pressure gradient and left ventricular ejection fraction were also measured.
Statistical analysis
Based on previous studies , 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 factors. 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 possible confounders, we recruited in excess of this number of patients with APE and controls.
Categorical variables, expressed as percentages, were compared using the Chi 2 test or Fisher’s exact test. After a test for normality, continuous data are expressed as means and standard deviations or medians with IQRs as appropriate. Differences between patients and controls were evaluated using the two-sample t test or the Mann-Whitney U test. Correlations between annexin V-positive microparticles 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 analysis (after logarithmic transformation of the dependent variables) and presented using the estimated regression coefficient, expressed as percentage increase in microparticle 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 haemodynamic 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.
| Patient characteristics | APE ( n = 45) | Controls CVRFs ( n = 45) | Controls noCVRFs ( n = 45) | P value | ||
|---|---|---|---|---|---|---|
| APE vs Controls CVRFs | APE vs Controls noCVRFs | Controls CVRFs vs Controls noCVRFs | ||||
| 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 | – | – |
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