TR-05976; No of Pages 5 Thrombosis Research xxx (2015) xxx–xxx
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Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia Elena Campello a, Luca Spiezia a, Claudia M. Radu a, Sonila Dhima b, Silvia Visentin b, Fabio Dalla Valle a, Daniela Tormene a, Barry Woodhams c, Erich Cosmi b, Paolo Simioni a,⁎ a b c
Department of Medicine (DIMED), 5th Chair of Internal Medicine, University of Padua, Italy Department for Health of Mothers and Children, University of Padua, Italy HaemaCon Ltd, Bromley, England
a r t i c l e
i n f o
Article history: Received 19 April 2015 Received in revised form 20 May 2015 Accepted 27 May 2015 Available online xxxx Keywords: Microparticles Pre-eclampsia Pregnancy Venous cord blood
s u m m a r y Introduction: Placenta microthrombi being one of the prevalent recurrent histological findings in women with preeclampsia (PE), it is reasonable to think that the study of coagulation alterations in cord blood could be more informative than that observed in maternal blood. The aim of the present study was to measure different subtypes of microparticles (MP) plasma levels in the maternal peripheral blood at labour and in the venous cord blood of pregnant women with PE compared to those in a group of women without PE. Materials and methods: Thirty-two pregnant women in labour, 16 with and 16 without PE, were enrolled. Blood samples were collected immediately after delivery from cord blood and from maternal peripheral blood. Total, cellular-derived and tissue factor- bearing MP were analyzed using flow-cytometry. Procoagulant activity of MP was assessed using the STA® Procoag PPL assay. Results: Total MP, platelet activated-derived (P-Selectin+), leukocyte-derived and TF + MP were higher in pregnancies complicated by PE as compared with normotensive women (p b 0.05). Platelet-derived MP (CD61+) levels were lower in PE than in healthy women and no difference was found in endothelial-derived MP levels between the two groups. The PPL clotting time was significantly shorter in PE compared with controls. When only venous cord blood was analysed, all MP detected were significantly higher in PE than in healthy normotensive women (p b 0.05). Conclusions: MP are very likely involved in the hypercoagulable and pro-inflammatory intravascular reactions during PE. Prospective studies in a larger population are needed to define the clinical meaning of MP measurement in the PE setting. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Preeclampsia (PE), a pathological condition characterized by hypertension and proteinuria, is one of the most serious causes of maternal and neonatal morbidity and mortality with a prevalence of 6-8% of pregnancies [1]. In pregnant women with PE, extensive activation of endothelial cells, leukocytes, and coagulation system have been reported [1–3]. Membrane microparticles (MP) consist of cell-derived vesicles formed from the outward blebbing of the plasma membrane and subsequent shedding into the extracellular space during apoptosis or cellular activation. MP are typically defined as 0.1–1.0 μm in size consisting of membrane proteins and cytosolic material derived from the cell from ⁎ Corresponding author at: 5th Chair of Internal Medicine, Thrombophilia and Hemophilia Center, Department of Medicine (DIMED), University of Padua Medical School, Via Ospedale Civile 105, 35100 Padua, Italy. E-mail address: [email protected] (P. Simioni).
which they originate [4–6]. There is increasing evidence in the literature that the levels of circulating MP may represent a possible marker or a causative agent of PE related vascular complications [7–9]. Some of the studies showed an increase in MP sub-populations in women with PE compared to normal healthy pregnant females, including an increase in the total number of MP [10,11], an elevation of both activated and non-activated platelet-MP [10,12,13], as well as an increase in endothelial MP [14] and white blood cell MP [10,15]. Other studies revealed no differences in the total platelet MP number between normal and pregnancy complicated by PE. Moreover, it was shown that MP are present in cord blood plasma in significantly higher concentration than in mother’s plasma [16]. Placenta microthrombi being one of the prevalent histological finding in women with PE [1], it is reasonable to think that the study of coagulation alterations in cord blood are more informative than that observed in maternal blood. The aim of the present study was to evaluate different subtypes of MP plasma levels in the maternal peripheral blood at labour and in
http://dx.doi.org/10.1016/j.thromres.2015.05.029 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: E. Campello, et al., Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.05.029
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E. Campello et al. / Thrombosis Research xxx (2015) xxx–xxx
the venous cord blood of pregnant women with PE as compared to those of a group of healthy women at delivery. 2. Materials and methods 2.1. Patients All consecutive pregnant women in labour with PE, referred for delivery to the Unit of Maternal Foetal Medicine at University Hospital of Padua, between September 2013 and January 2014, were considered. A group of healthy parturient with a single, uncomplicated pregnancy, age (± 3 yrs) matched with cases, who gave natural birth at term acted as control. PE was defined by high blood pressure (two separate readings taken at least 6 h apart of 140 mmHg or more in systolic blood pressure and/or 90 mmHg or more in diastolic blood pressure) and 300 mg of protein in a 24-h urine sample occurring after the 20th week of pregnancy. Exclusion criteria common to the overall study population were: i) age ≤18 years; ii) ongoing anticoagulant or antiplatelet treatment; iii) previous arterial or venous thrombosis; iv) comorbidities (i.e. cancer, diabetes, obesity, chronic hypertension, autoimmune, renal, or hepatic diseases, acute infections). Each woman received a peripheral blood sample before delivery and, subsequently, one sample of venous cord blood was obtained prior to clamping and cutting of the umbilical cord and before placental expulsion (III stage of labour). The study was performed according to the Declaration of Helsinki. Informed consent was obtained from all participants according to the University Hospital of Padua policy. 2.2. Blood sampling and conventional coagulation parameters Nine mL of venous blood was drawn from the antecubital vein with a light tourniquet, using a butterfly device with 21-gauge needle without venostasis. Blood was collected directly into syringes pre-filled with 1 mL of sodium citrate 109 mol/L. White blood cells and platelet count (r.v. 4.5-10.0 x109/L and 150–450 x109/L, respectively) were measured on the Sysmex Counter XE-2100 (Dasit Spa, Milan, Italy). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were automatically measured according to the standard methods. Platelet-poor plasma (PPP) was prepared within 1 hour of blood collection by double centrifugation (2 x 15 minutes at 2500 g) at room temperature. Prothrombin time (PT/INR), activated partial thromboplastin time (aPTT), D-Dimer, protein C (PC, chromogenic and coagulometric assays) and protein S (PS) were measured in all samples according to the methods described elsewhere [17,18]. Aliquots (1.5 ml) of PPP were immediately frozen and then stored at −80 °C until use. Samples, analyzed only after a single freeze-thaw cycle, were thawed by incubation for 5 minutes in a water bath at 37 °C immediately before assay. 2.3. MP assessment and characterization MP were identified by size and annexin V- fluorescein isothiocyanate (FITC) (Bender MedSystems GmbH, Vienna, Austria) labelling. The MP gate was established using a blend of mono-dispersed fluorescent beads of three diameters (0.5, 0.9 and 3 μm) (Megamix, BioCytex, Diagnostica Stago, France). MP were analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter, Miami Florida). To measure the different populations, the MP were co-labelled with antibodies against cell-type specific antigens and annexin V, as previously described [19,20]. Thirty microliters (μL) of freshly thawed PPP were incubated for 15 minutes at room temperature in the dark with 3 μL of monoclonal antibodies against cell-type specific antigens and 3 μL of annexin V-FITC. Platelet-derived MP were identified using CD61-PE (phycoerythrin) and activated platelet-derived using CD62P-PE (P-Selectin +) - both from Beckman Coulter, Miami, Florida; endothelial-derived MP using CD62E-PC5 (phycoerythrin-cyanin 5.1) (Beckman Coulter, Miami, Florida); leukocyte-derived MP using CD45-PC5 (BioLegend Europe,
The Netherlands) and Tissue factor-bearing (TF + MP) with CD142PE, (clone HTF-1, BD, Biosciences, Milan, Italy). The samples were diluted in 500 μL of annexin V kit binding buffer (Bender MedSystems GmbH, Vienna, Austria) before analysis. Thirty μL of counting beads with an established concentration (Flow Count TM Fluorospheres, Beckman Coulter, Miami, Florida) were added to each sample in order to calculate MP as absolute numbers per μL of PPP. 2.4. MP procoagulant activity Procoagulant activity of the MP was measured using the STA® Procoag PPL assay (Diagnostica Stago, Asnieres, France). The assay measures the clotting time in a system dependent on the total plasma phase procoagulant phospholipid content of the sample [21]. It differs from solid phase assays in that there is no pre-selection of annexin-V bound activity. The assay is performed using phospholipid depleted substrate plasma to eliminate the influence of any coagulation factors upstream. Factor Xa, in the presence of calcium, triggers the coagulation cascade and a shortening clotting time of the sample indicates an increased concentration of procoagulant phospholipids – a shorter clotting time indicating increased PPL activity. This activity linearly correlates with the functional activity of MP present in the sample [22]. 2.5. Statistical analysis Statistical analysis was performed using the PASW Statistics 17.0.2 (SPSS Inc.) for Windows. The demographic characteristics of patients were presented as means ± standard deviations. The study groups were compared with Student-t-test. Data of the flow cytometry and PPL were not normally distributed and therefore presented as median ad interquartile range (IQR) and analyzed with Mann–Whitney U-tests for differences between two groups. Frequencies were provided for all nominal values and differences were calculated using Chi-square test. Pearson’s correlation analysis was used to detect significant correlations between MP numbers and other parameters. A p-value b 0.05 was considered statistically significant. 3. Results 3.1. Patients Out of twenty-five pregnant women diagnosed with PE consecutively referred for delivery to the Unit of Maternal Foetal Medicine at University Hospital of Padua, 16 were enrolled in the study. Five were excluded because blood sample collection (n = 3) and/or informed consent (n = 2) were not available; 2 were under antiplatelet therapy treatment; 1 had an acute infection and 1 had experienced a venous thrombotic event. Sixteen healthy pregnant women during labor referred for delivery to the same Center, during the same period of time, acted as controls. Baseline characteristics of the study population are reported in Table 1. No differences in age, parity, and BMI between parturient women with and without PE were observed. As expected, both systolic and diastolic blood pressure was significantly higher in patients with PE than in normotensive pregnancies (p b 0.01). Birth weight and gestational age at delivery were significantly lower in the PE women compared with the normotensive parturient women (p b 0.05 for both comparisons). Most PE patients were delivered by cesarean section while most of the non-PE subjects had normal vaginal delivery (Table 1). 3.2. Coagulation parameters Preeclamptic women had slightly higher levels of PC activity (both chromogenic and coagulometric) and slightly lower platelet count than normotensive parturient subjects (p b 0.05 in all comparisons). No statistically significant differences were observed in INR, aPTT, PS
Please cite this article as: E. Campello, et al., Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.05.029
E. Campello et al. / Thrombosis Research xxx (2015) xxx–xxx Table 1 General characteristics of the overall study population.
Age, yrs Parity, n (%) Nulliparous Multiparous Gestational age, weeks BMI, Kg/m2 Weight increase, Kg Blood pressure, mmHg Systolic Diastolic Delivery, n (%) Vaginal birth Cesarean section Birth weight, g
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Table 3 MP levels in women with PE and in normotensive parturient women.
Pre-eclamptic parturients (N. 16)
Normotensive parturients (N.16)
MP (number/μL)
Pre-eclamptic parturients (N. 16)
Normotensive parturients (N. 16)
32 ± 6
31 ± 5
Total (annexin V+)
13 (81) 3 (19) 38 ± 6 days 23.2 ± 4.7 15.3 ± 4.7
10 (63) 6 (37) 37 ± 1 day 23.1 ± 4.3 11.2 ± 3.6
Platelet-derived (CD61+) P-Selectin +
158 ± 23 93 ± 10
123 ± 7⁎⁎ 76 ± 8⁎⁎
Leukocyte-derived
6 (37) 10 (63) 2879 ± 321
12 (75)⁎⁎ 4 (25) 3313 ± 344⁎
5728 [4782–6422] 997 [899–1129] 1298 [981–1401] 1295 [874–1445] 152 [102–202] 267 [190–301] 57 [47–63]
3435⁎⁎ [2957–4747] 1364⁎ [995–1583] 854⁎ [652–999] 1471 [1100–1645] 103⁎ [81–143] 176⁎⁎ [146–199] 65⁎
Endothelial-derived
TF+ PPL (sec.)
[55–76]
Data are expressed by mean ± SD where not specified otherwise. p are expressed vs controls: *p b 0.05, **p b 0.01. N, number; yrs, years; BMI, body mass index.
Data are expressed by median and range interquartile. p are expressed vs controls: *p b 0.05 **p b 0.01. MP, microparticles; TF+, tissue factor-bearing MP; PPL, phospholipid-dependent clotting time; sec, seconds.
activity, D-dimer, white blood cells count between the two groups (Table 2).
4. Discussion
3.3. MP levels in preeclamptic and normotensive parturient women Table 3 shows the median and IQR of the circulating MP levels in PE and normotensive parturient women. Significantly increased levels of total MP (annexin +), activated platelet-derived (P-Selectin +), leukocyte-derived MP, and TF + MP were seen in PE compared with normotensive parturient women (p b 0.05 in all comparisons). Platelet-derived MP (CD61+) were higher in controls women than in PE (p b 0.05). No significant difference was seen in endothelialderived MP levels between the two groups. The PPL clotting time was significantly shorter (p b 0.05) in PE compared with controls. 3.4. MP levels in venous cord blood We showed that the levels of all the MP studied were significantly higher in venous cord blood than in peripheral maternal venous blood both in PE and in healthy pregnancies (p b 0.05 for all comparisons). The levels of total MP (annexin V+), platelet-derived MP (CD61+), endothelial- and leukocyte-derived MP, and TF + MP in venous cord blood were significantly higher in patients with PE than in healthy parturient women (p b 0.05 in all comparisons). Concomitantly, the PPL clotting time was significantly shorter in venous cord blood from PE than in normal pregnancies (p b 0.01) (Table 4). Table 2 Coagulative and biochemical parameters in women with PE and in normotensive parturient women.
PT (INR) aPTT, sec PC- Chr, % PC- Coag, % PS- Act, % D-Dimer,μg/L White blood cells, x109/L Platelets, x109/L AST,UI/L ALT, UI/L
Pre-eclamptic Parturients (N. 16)
Normotensive parturients (N. 16)
1.01 ± 0.08 26.6 ± 1.9 133.3 ± 28.3 114.5 ± 37.5 57.4 ± 11.2 910 ± 180 12.1 ± 4.5 229 ± 66 21.5 ± 4.0 19.2 ± 6.9
0.99 ± 0.04 26.6 ± 1.9 112.27 ± 23.5⁎ 88.9 ± 24.8⁎ 50.6 ± 10.5 776 ± 254 11.9 ± 4.1 255 ± 46⁎ 19.0 ± 9.5 15.0 ± 10.5
Data are expressed by mean ± SD. p are expressed vscontrols: *p b 0.05. N, number; INR, International normalized ratio; aPTT activated partial thromboplastin time; PC, protein C; Chr, chromogenic activity; Coag, coagulometric activity; PS, protein S; AST, aspartate aminotransferase; ALT, alanine aminotransferase.
We found an increase in the number of total MP, activated plateletderived (P-Selectin+), leukocyte-derived and TF + MP in pregnancies complicated by PE as compared with normotensive parturient women. This was concomitant with increased PPL activity. On the contrary, lower numbers of platelet- (CD61+) and endothelial-derived MP were found in PE as compared with normotensive women. The actual prevalence of high MP levels in gestational vascular complications still remains controversial and, in particular, data from different studies regarding PE show wide variations [7,9]. Some studies have shown an increase in MP subpopulations in women with PE as compared with normal healthy pregnant women, including increases in total MP [11, 23], and leukocyte- or monocyte-derived MP numbers [10,15,23]. Elevated leukocyte-derived MP levels may reflect activation of leukocytes, which can occur in PE. In fact, monocytes and neutrophils possibly activate during placental crossing [24]. In agreement with previous studies [12,13], we showed a decrease in platelet-derived MP (CD61+), which mirrored the reduction in platelets count, and we found an increase in activated platelet-derived (P-Selectin +) MP. P-Selectin is a celladhesion molecule that translocates from alpha-granules to the platelet surface upon activation. P-Selectin is responsible for the adhesion of certain leukocytes and platelets to the endothelium. Probably MP derived from activated platelets (P-Selectin+) contribute to the development of early stage vascular dysfunction by facilitating the initial recruitment
Table 4 MP levels in venous cord blood of women with PE and of normotensive parturient women. MP (number/μL)
Pre-eclamptic parturients (N. 16)
Normotensive parturients (N.16)
Total (annexin V+)
5931 [5123–7199] 2456 [1400–2871] 1702 [1311–1942] 211 [149–234] 298 [254–357] 39 [31–47]
4631⁎ [3123–4999] 1566⁎⁎
Platelet-derived (CD61+) Endothelial-derived Leukocyte-derived TF+ PPL (sec.)
[1250–1875] 1542⁎ [1291–1677] 152⁎⁎ [112–184] 223⁎ [176–241] 42⁎⁎ [37–49]
Data are expressed by median and range interquartile. p are expressed vs controls: *p b 0.05 **p b 0.01. MP, microparticles; TF+, tissue factor-bearing MP; PPL, phospholipid-dependent clotting time; sec, seconds.
Please cite this article as: E. Campello, et al., Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.05.029
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E. Campello et al. / Thrombosis Research xxx (2015) xxx–xxx
of leukocytes, leukocyte-derived MP and platelets to the endothelium [25]. PE is known to be a two stage disease: the preclinical condition is impaired placentation (first stage) characterized by maternal endothelial dysfunction; in the second stage of PE, inflammatory leukocytes are also activated [26–28]. It is well known that the number of MP increases in conditions associated with enhanced systemic inflammatory responses (SIRs) in which intravascular systems are generally activated [29]. Several reports show that all components of the intravascular inflammatory network (inflammatory leukocytes, endothelium, coagulation system, acute phase response and complement) contribute to the responses in both normal pregnancy and, to a greater degree, in PE [30]. Hence, as in non-pregnant individuals with a SIR, it is to be expected that, in normal pregnancy and PE, there is enhanced MP release from intravascular cells, which has already been described [10,12,23]. Theoretically, endothelial cell activation should be detectable by elevated levels of endothelial-derived MP that mirror the endothelial cells turnover. In line with Lok et al. [24], we did not find a difference in the number of endothelial-derived MP between normotensive women and PE patients. We hypothesized a possibly consumption of endothelialderived MP during the inflammatory intravascular process or during the placental intravascular microthrombi. Hypercoagulability and inflammation are related and mutually reinforcing processes, involving inflammatory mediators (e.g., endotoxin, TNFα, and CD40 ligand), tissue factor expression on monocytes and the activated endothelium, and circulating TF–bearing MP. The trophoblast has a procoagulant nature, characterized by constitutively high TF levels [9]. Trophoblast differentiation process is accompanied with a membrane “flip-flop” leading to exposure of negative phospholipids on their surface and the enzymes involved in regulation of cell membrane phospholipids asymmetry are implicated in MP generation. Our study showed high levels of TF + MP in PE and also in physiological pregnancy, thus confirming that trophoblast differentiation yield massive TF-bearing MP formation [9]. In the present study we also evaluated the number of MP in venous cord blood of healthy foetuses and in the venous peripheral plasma of their both normotensive and PE mothers. We found that all MP were significantly higher in venous cord blood than in the maternal blood. We can confirm previous observations by Uszynski et al. [16] that MP are a constant component of the foetal blood and that MP may play a role of a powerful procoagulant, thus facilitating thrombin generation and compensating the “immaturity” of the foetal coagulation system. Moreover, when we analysed only venous cord blood, all MP detected were significantly higher in PE than in healthy normotensive parturient women. We hypothesized that changes in the balance of circulating microvesicles produced in the placental vessels might play a role in the alteration of maternal physiology and in the development of the maternal syndrome of pre-eclampsia [31]. Due to the low shear stress in the intravillous space, some foetus-placental-derived MP bearing TF may accumulate in this area, initiating a local effect on placental haemostasis. Other foetus-placental-derived MP penetrate into the maternal circulation via the decidual veins and may lead to systemic maternal effects [9]. The two major limitations of our study regard the relative small sample size enrolled and the lack of standardization in the MP analysis. As far as the small sample size was concerned we tried to overcome this limit using a longitudinal cohort of pregnant patients enrolled in a consecutive manner and as uniform as possible among them. As for methods, flow cytometric assays may not be sensitive enough to detect all sizes of MP, given that many of these fall below the detection threshold, though recommendations by the ISTH SSC Working Group on Vascular Biology [32–34] were followed. Moreover, an increased syncytiotrophoblast-derived MP number has also been described in PE patients, mainly in early-onset PE, compared with healthy pregnant women [9–35]. In our study, we could only explore the presence of MP derived from blood cells and TF-bearing with procoagulant properties and no cellular information is available on their placental origin.
In conclusion, our study shows that total MP, MP derived from activated platelet (P-Selectin+), leukocyte-derived MP and TF + MP are increased in pregnancies complicated by PE compared with normotensive pregnancies, confirming a pro-inflammatory intravascular reaction during this condition. On the contrary, platelet-derived MP (CD61+) levels are decreased in PE than in healthy women and no difference was seen in endothelial-derived MP levels between the two groups. Moreover, we showed that all subgroups of MP are higher in venous cord blood than in peripheral venous maternal blood both in healthy and in PE women. When only venous cord blood was analyzed, all MP detected were significantly higher in PE than in healthy normotensive parturient women. The transfer of foetus-placental-derived MP into the maternal circulation may have a role in the alteration of maternal physiology and may therefore be central in the development of the maternal syndrome of pre-eclampsia. Prospective studies on larger population are needed to evaluate the clinical utility of MP measuring in physiological and pathological pregnancies. Disclosure of conflict of interest None to declare except for BW who acted as a consultant to Stago via HaemaCon Ltd. References [1] J. Prochazkova, L. Slavik, J. Ulehlova, M. Prochazka, The role of tissue factor in normal pregnancy and in the development of preeclampsia. A review, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. (2014), http://dx.doi.org/10.5507/bp. 2014.061 (Epub ahead of print). [2] B.A. Lwaleed, L. Dusse, A.J. Cooper, Tissue factor dependent pathway and the diagnosis of pre-eclampsia, Semin. Thromb. Hemost. 37 (2011) 125–130. [3] L.M. Dusse, D.R. Rios, M.B. Pinheiro, A.J. Cooper, B.A. Lwaleed, Pre-eclampsia: relationship between coagulation, fibrinolysis and inflammation, Clin. Chim. Acta 412 (2011) 17–21. [4] N.S. Barteneva, E. Fasler-Kan, M. Bernimoulin, J.N. Stern, E.D. Ponomarev, L. Duckett, I.A. Vorobjev, Circulating microparitcles: square the circle, BMC Cell Biol. 14 (2013) 23. [5] Z.H. Wu, C.L. Ji, H. Li, G.X. Qiu, C.J. Gao, X.S. Weng, Membrane microparticles and diseases, Eur. Rev. Med. Pharmacol. Sci. 17 (2013) 2420–2427. [6] D. Burger, S. Schock, C.S. Thompson, A.C. Montezano, A.M. Hakim, R.M. Touyz, Microparticles: biomarkers and beyond, Clin. Sci. (Lond.) 124 (2013) 423–441. [7] J. Alijotas-Reig, C. Palacio-Garcia, E. Llurba, M. Vilardell-Tarres, Cell-derived microparticles and vascular pregnancy complications: a systematic and comprehensive review, Fertil. Steril. 99 (2013) 441–449. [8] F.K. Marques, F.M. Campos, L.P. Sousa, A. Teixeira-Carvalho, L.M. Dusse, K.B. Gomes, Association of microparticles and preeclampsia, Mol. Biol. Rep. 40 (2013) 4553–4559. [9] A. Aharon, B. Brenner, Microparticles and pregnancy complications, Thromb. Res. 127 (2011) S67–S71. [10] F. Meziani, A. Tesse, E. David, M.C. Martinez, R. Wangesteen, F. Schneider, R. Andriantsitohaina, Shed membrane particles from preeclamptic women generate vascular wall inflammation and blunt vascular contractility, Am. J. Pathol. 169 (2006) 1473–1483. [11] A.F. Orozco, C.J. Jorgez, W.D. Ramos-Perez, E.J. Popek, X. Yu, C.A. Kozinetz, F.Z. Bischoff, D.E. Lewis, Placental release of distinct DNA-associated micro-particles into maternal circulation: reflective of gestation time and preeclampsia, Placenta 30 (2009) 891–897. [12] C.A. Lock, R. Nieuwland, A. Sturk, C.M. Hau, K. Boer, E. Vanbavel, Microparticleassociated P-selectin reflects platelet activation in preeclampsia, Platelets 18 (2007) 68–72. [13] M.G. Macey, S. Bevan, S. Alam, L. Verghese, S. Agrawal, S. Beski, R. Thuraisingham, P.K. MacCallum, Platelet activation and endogenous thrombin potential in preeclampsia, Thromb. Res. 125 (2010) e76–e81. [14] V.H. Gonzalez-Quintero, J.J. Jime´nez, W. Jy, L.M. Mauro, L. Hortman, M.J. O’Sullivan, Y.S. Ahn, Elevated plasma endothelial microparticles in preeclampsia, Am. J. Obstet. Gynecol. 189 (2003) 589–593. [15] C.A. Lok, J. Jebbink, R. Nieuwland, M.M. Faas, K. Boer, A. Sturk, J.A. Van Der Post, Leukocyte activation and circulating leukocyte-derived microparticles in preeclampsia, Am. J. Reprod. Immunol. 61 (2009) 346–359. [16] M. Uszyński, E. Zekanowska, W. Uszyński, J. Kuczyński, A. Zyliński, Microparticles (MPs), tissue factor (TF) and tissue factor inhibitor (TFPI) in cord blood plasma. A preliminary study and literature survey of procoagulant properties of MPs, Eur. J. Obstet. Gynecol. Reprod. Biol. 158 (2011) 37–41. [17] P. Simioni, D. Tormene, L. Spiezia, G. Tognin, V. Rossetto, C. Radu, P. Prandoni, Inherited thrombophilia and venous thromboembolism, Semin. Thromb. Hemost. 32 (2006) 700–708. [18] V. Rossetto, L. Spiezia, F. Franz, L. Salmaso, L.V. Pozza, S. Gavasso, P. Simioni, The role of antiphospholipid antibodies toward the protein C/protein S system in venous thromboembolic disease, Am. J. Hematol. 84 (2009) 594–596.
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Please cite this article as: E. Campello, et al., Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.05.029
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Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia Elena Campello a, Luca Spiezia a, Claudia M. Radu a, Sonila Dhima b, Silvia Visentin b, Fabio Dalla Valle a, Daniela Tormene a, Barry Woodhams c, Erich Cosmi b, Paolo Simioni a,⁎ a b c
Department of Medicine (DIMED), 5th Chair of Internal Medicine, University of Padua, Italy Department for Health of Mothers and Children, University of Padua, Italy HaemaCon Ltd, Bromley, England
a r t i c l e
i n f o
Article history: Received 19 April 2015 Received in revised form 20 May 2015 Accepted 27 May 2015 Available online xxxx Keywords: Microparticles Pre-eclampsia Pregnancy Venous cord blood
s u m m a r y Introduction: Placenta microthrombi being one of the prevalent recurrent histological findings in women with preeclampsia (PE), it is reasonable to think that the study of coagulation alterations in cord blood could be more informative than that observed in maternal blood. The aim of the present study was to measure different subtypes of microparticles (MP) plasma levels in the maternal peripheral blood at labour and in the venous cord blood of pregnant women with PE compared to those in a group of women without PE. Materials and methods: Thirty-two pregnant women in labour, 16 with and 16 without PE, were enrolled. Blood samples were collected immediately after delivery from cord blood and from maternal peripheral blood. Total, cellular-derived and tissue factor- bearing MP were analyzed using flow-cytometry. Procoagulant activity of MP was assessed using the STA® Procoag PPL assay. Results: Total MP, platelet activated-derived (P-Selectin+), leukocyte-derived and TF + MP were higher in pregnancies complicated by PE as compared with normotensive women (p b 0.05). Platelet-derived MP (CD61+) levels were lower in PE than in healthy women and no difference was found in endothelial-derived MP levels between the two groups. The PPL clotting time was significantly shorter in PE compared with controls. When only venous cord blood was analysed, all MP detected were significantly higher in PE than in healthy normotensive women (p b 0.05). Conclusions: MP are very likely involved in the hypercoagulable and pro-inflammatory intravascular reactions during PE. Prospective studies in a larger population are needed to define the clinical meaning of MP measurement in the PE setting. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Preeclampsia (PE), a pathological condition characterized by hypertension and proteinuria, is one of the most serious causes of maternal and neonatal morbidity and mortality with a prevalence of 6-8% of pregnancies [1]. In pregnant women with PE, extensive activation of endothelial cells, leukocytes, and coagulation system have been reported [1–3]. Membrane microparticles (MP) consist of cell-derived vesicles formed from the outward blebbing of the plasma membrane and subsequent shedding into the extracellular space during apoptosis or cellular activation. MP are typically defined as 0.1–1.0 μm in size consisting of membrane proteins and cytosolic material derived from the cell from ⁎ Corresponding author at: 5th Chair of Internal Medicine, Thrombophilia and Hemophilia Center, Department of Medicine (DIMED), University of Padua Medical School, Via Ospedale Civile 105, 35100 Padua, Italy. E-mail address: [email protected] (P. Simioni).
which they originate [4–6]. There is increasing evidence in the literature that the levels of circulating MP may represent a possible marker or a causative agent of PE related vascular complications [7–9]. Some of the studies showed an increase in MP sub-populations in women with PE compared to normal healthy pregnant females, including an increase in the total number of MP [10,11], an elevation of both activated and non-activated platelet-MP [10,12,13], as well as an increase in endothelial MP [14] and white blood cell MP [10,15]. Other studies revealed no differences in the total platelet MP number between normal and pregnancy complicated by PE. Moreover, it was shown that MP are present in cord blood plasma in significantly higher concentration than in mother’s plasma [16]. Placenta microthrombi being one of the prevalent histological finding in women with PE [1], it is reasonable to think that the study of coagulation alterations in cord blood are more informative than that observed in maternal blood. The aim of the present study was to evaluate different subtypes of MP plasma levels in the maternal peripheral blood at labour and in
http://dx.doi.org/10.1016/j.thromres.2015.05.029 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: E. Campello, et al., Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.05.029
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E. Campello et al. / Thrombosis Research xxx (2015) xxx–xxx
the venous cord blood of pregnant women with PE as compared to those of a group of healthy women at delivery. 2. Materials and methods 2.1. Patients All consecutive pregnant women in labour with PE, referred for delivery to the Unit of Maternal Foetal Medicine at University Hospital of Padua, between September 2013 and January 2014, were considered. A group of healthy parturient with a single, uncomplicated pregnancy, age (± 3 yrs) matched with cases, who gave natural birth at term acted as control. PE was defined by high blood pressure (two separate readings taken at least 6 h apart of 140 mmHg or more in systolic blood pressure and/or 90 mmHg or more in diastolic blood pressure) and 300 mg of protein in a 24-h urine sample occurring after the 20th week of pregnancy. Exclusion criteria common to the overall study population were: i) age ≤18 years; ii) ongoing anticoagulant or antiplatelet treatment; iii) previous arterial or venous thrombosis; iv) comorbidities (i.e. cancer, diabetes, obesity, chronic hypertension, autoimmune, renal, or hepatic diseases, acute infections). Each woman received a peripheral blood sample before delivery and, subsequently, one sample of venous cord blood was obtained prior to clamping and cutting of the umbilical cord and before placental expulsion (III stage of labour). The study was performed according to the Declaration of Helsinki. Informed consent was obtained from all participants according to the University Hospital of Padua policy. 2.2. Blood sampling and conventional coagulation parameters Nine mL of venous blood was drawn from the antecubital vein with a light tourniquet, using a butterfly device with 21-gauge needle without venostasis. Blood was collected directly into syringes pre-filled with 1 mL of sodium citrate 109 mol/L. White blood cells and platelet count (r.v. 4.5-10.0 x109/L and 150–450 x109/L, respectively) were measured on the Sysmex Counter XE-2100 (Dasit Spa, Milan, Italy). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were automatically measured according to the standard methods. Platelet-poor plasma (PPP) was prepared within 1 hour of blood collection by double centrifugation (2 x 15 minutes at 2500 g) at room temperature. Prothrombin time (PT/INR), activated partial thromboplastin time (aPTT), D-Dimer, protein C (PC, chromogenic and coagulometric assays) and protein S (PS) were measured in all samples according to the methods described elsewhere [17,18]. Aliquots (1.5 ml) of PPP were immediately frozen and then stored at −80 °C until use. Samples, analyzed only after a single freeze-thaw cycle, were thawed by incubation for 5 minutes in a water bath at 37 °C immediately before assay. 2.3. MP assessment and characterization MP were identified by size and annexin V- fluorescein isothiocyanate (FITC) (Bender MedSystems GmbH, Vienna, Austria) labelling. The MP gate was established using a blend of mono-dispersed fluorescent beads of three diameters (0.5, 0.9 and 3 μm) (Megamix, BioCytex, Diagnostica Stago, France). MP were analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter, Miami Florida). To measure the different populations, the MP were co-labelled with antibodies against cell-type specific antigens and annexin V, as previously described [19,20]. Thirty microliters (μL) of freshly thawed PPP were incubated for 15 minutes at room temperature in the dark with 3 μL of monoclonal antibodies against cell-type specific antigens and 3 μL of annexin V-FITC. Platelet-derived MP were identified using CD61-PE (phycoerythrin) and activated platelet-derived using CD62P-PE (P-Selectin +) - both from Beckman Coulter, Miami, Florida; endothelial-derived MP using CD62E-PC5 (phycoerythrin-cyanin 5.1) (Beckman Coulter, Miami, Florida); leukocyte-derived MP using CD45-PC5 (BioLegend Europe,
The Netherlands) and Tissue factor-bearing (TF + MP) with CD142PE, (clone HTF-1, BD, Biosciences, Milan, Italy). The samples were diluted in 500 μL of annexin V kit binding buffer (Bender MedSystems GmbH, Vienna, Austria) before analysis. Thirty μL of counting beads with an established concentration (Flow Count TM Fluorospheres, Beckman Coulter, Miami, Florida) were added to each sample in order to calculate MP as absolute numbers per μL of PPP. 2.4. MP procoagulant activity Procoagulant activity of the MP was measured using the STA® Procoag PPL assay (Diagnostica Stago, Asnieres, France). The assay measures the clotting time in a system dependent on the total plasma phase procoagulant phospholipid content of the sample [21]. It differs from solid phase assays in that there is no pre-selection of annexin-V bound activity. The assay is performed using phospholipid depleted substrate plasma to eliminate the influence of any coagulation factors upstream. Factor Xa, in the presence of calcium, triggers the coagulation cascade and a shortening clotting time of the sample indicates an increased concentration of procoagulant phospholipids – a shorter clotting time indicating increased PPL activity. This activity linearly correlates with the functional activity of MP present in the sample [22]. 2.5. Statistical analysis Statistical analysis was performed using the PASW Statistics 17.0.2 (SPSS Inc.) for Windows. The demographic characteristics of patients were presented as means ± standard deviations. The study groups were compared with Student-t-test. Data of the flow cytometry and PPL were not normally distributed and therefore presented as median ad interquartile range (IQR) and analyzed with Mann–Whitney U-tests for differences between two groups. Frequencies were provided for all nominal values and differences were calculated using Chi-square test. Pearson’s correlation analysis was used to detect significant correlations between MP numbers and other parameters. A p-value b 0.05 was considered statistically significant. 3. Results 3.1. Patients Out of twenty-five pregnant women diagnosed with PE consecutively referred for delivery to the Unit of Maternal Foetal Medicine at University Hospital of Padua, 16 were enrolled in the study. Five were excluded because blood sample collection (n = 3) and/or informed consent (n = 2) were not available; 2 were under antiplatelet therapy treatment; 1 had an acute infection and 1 had experienced a venous thrombotic event. Sixteen healthy pregnant women during labor referred for delivery to the same Center, during the same period of time, acted as controls. Baseline characteristics of the study population are reported in Table 1. No differences in age, parity, and BMI between parturient women with and without PE were observed. As expected, both systolic and diastolic blood pressure was significantly higher in patients with PE than in normotensive pregnancies (p b 0.01). Birth weight and gestational age at delivery were significantly lower in the PE women compared with the normotensive parturient women (p b 0.05 for both comparisons). Most PE patients were delivered by cesarean section while most of the non-PE subjects had normal vaginal delivery (Table 1). 3.2. Coagulation parameters Preeclamptic women had slightly higher levels of PC activity (both chromogenic and coagulometric) and slightly lower platelet count than normotensive parturient subjects (p b 0.05 in all comparisons). No statistically significant differences were observed in INR, aPTT, PS
Please cite this article as: E. Campello, et al., Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.05.029
E. Campello et al. / Thrombosis Research xxx (2015) xxx–xxx Table 1 General characteristics of the overall study population.
Age, yrs Parity, n (%) Nulliparous Multiparous Gestational age, weeks BMI, Kg/m2 Weight increase, Kg Blood pressure, mmHg Systolic Diastolic Delivery, n (%) Vaginal birth Cesarean section Birth weight, g
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Table 3 MP levels in women with PE and in normotensive parturient women.
Pre-eclamptic parturients (N. 16)
Normotensive parturients (N.16)
MP (number/μL)
Pre-eclamptic parturients (N. 16)
Normotensive parturients (N. 16)
32 ± 6
31 ± 5
Total (annexin V+)
13 (81) 3 (19) 38 ± 6 days 23.2 ± 4.7 15.3 ± 4.7
10 (63) 6 (37) 37 ± 1 day 23.1 ± 4.3 11.2 ± 3.6
Platelet-derived (CD61+) P-Selectin +
158 ± 23 93 ± 10
123 ± 7⁎⁎ 76 ± 8⁎⁎
Leukocyte-derived
6 (37) 10 (63) 2879 ± 321
12 (75)⁎⁎ 4 (25) 3313 ± 344⁎
5728 [4782–6422] 997 [899–1129] 1298 [981–1401] 1295 [874–1445] 152 [102–202] 267 [190–301] 57 [47–63]
3435⁎⁎ [2957–4747] 1364⁎ [995–1583] 854⁎ [652–999] 1471 [1100–1645] 103⁎ [81–143] 176⁎⁎ [146–199] 65⁎
Endothelial-derived
TF+ PPL (sec.)
[55–76]
Data are expressed by mean ± SD where not specified otherwise. p are expressed vs controls: *p b 0.05, **p b 0.01. N, number; yrs, years; BMI, body mass index.
Data are expressed by median and range interquartile. p are expressed vs controls: *p b 0.05 **p b 0.01. MP, microparticles; TF+, tissue factor-bearing MP; PPL, phospholipid-dependent clotting time; sec, seconds.
activity, D-dimer, white blood cells count between the two groups (Table 2).
4. Discussion
3.3. MP levels in preeclamptic and normotensive parturient women Table 3 shows the median and IQR of the circulating MP levels in PE and normotensive parturient women. Significantly increased levels of total MP (annexin +), activated platelet-derived (P-Selectin +), leukocyte-derived MP, and TF + MP were seen in PE compared with normotensive parturient women (p b 0.05 in all comparisons). Platelet-derived MP (CD61+) were higher in controls women than in PE (p b 0.05). No significant difference was seen in endothelialderived MP levels between the two groups. The PPL clotting time was significantly shorter (p b 0.05) in PE compared with controls. 3.4. MP levels in venous cord blood We showed that the levels of all the MP studied were significantly higher in venous cord blood than in peripheral maternal venous blood both in PE and in healthy pregnancies (p b 0.05 for all comparisons). The levels of total MP (annexin V+), platelet-derived MP (CD61+), endothelial- and leukocyte-derived MP, and TF + MP in venous cord blood were significantly higher in patients with PE than in healthy parturient women (p b 0.05 in all comparisons). Concomitantly, the PPL clotting time was significantly shorter in venous cord blood from PE than in normal pregnancies (p b 0.01) (Table 4). Table 2 Coagulative and biochemical parameters in women with PE and in normotensive parturient women.
PT (INR) aPTT, sec PC- Chr, % PC- Coag, % PS- Act, % D-Dimer,μg/L White blood cells, x109/L Platelets, x109/L AST,UI/L ALT, UI/L
Pre-eclamptic Parturients (N. 16)
Normotensive parturients (N. 16)
1.01 ± 0.08 26.6 ± 1.9 133.3 ± 28.3 114.5 ± 37.5 57.4 ± 11.2 910 ± 180 12.1 ± 4.5 229 ± 66 21.5 ± 4.0 19.2 ± 6.9
0.99 ± 0.04 26.6 ± 1.9 112.27 ± 23.5⁎ 88.9 ± 24.8⁎ 50.6 ± 10.5 776 ± 254 11.9 ± 4.1 255 ± 46⁎ 19.0 ± 9.5 15.0 ± 10.5
Data are expressed by mean ± SD. p are expressed vscontrols: *p b 0.05. N, number; INR, International normalized ratio; aPTT activated partial thromboplastin time; PC, protein C; Chr, chromogenic activity; Coag, coagulometric activity; PS, protein S; AST, aspartate aminotransferase; ALT, alanine aminotransferase.
We found an increase in the number of total MP, activated plateletderived (P-Selectin+), leukocyte-derived and TF + MP in pregnancies complicated by PE as compared with normotensive parturient women. This was concomitant with increased PPL activity. On the contrary, lower numbers of platelet- (CD61+) and endothelial-derived MP were found in PE as compared with normotensive women. The actual prevalence of high MP levels in gestational vascular complications still remains controversial and, in particular, data from different studies regarding PE show wide variations [7,9]. Some studies have shown an increase in MP subpopulations in women with PE as compared with normal healthy pregnant women, including increases in total MP [11, 23], and leukocyte- or monocyte-derived MP numbers [10,15,23]. Elevated leukocyte-derived MP levels may reflect activation of leukocytes, which can occur in PE. In fact, monocytes and neutrophils possibly activate during placental crossing [24]. In agreement with previous studies [12,13], we showed a decrease in platelet-derived MP (CD61+), which mirrored the reduction in platelets count, and we found an increase in activated platelet-derived (P-Selectin +) MP. P-Selectin is a celladhesion molecule that translocates from alpha-granules to the platelet surface upon activation. P-Selectin is responsible for the adhesion of certain leukocytes and platelets to the endothelium. Probably MP derived from activated platelets (P-Selectin+) contribute to the development of early stage vascular dysfunction by facilitating the initial recruitment
Table 4 MP levels in venous cord blood of women with PE and of normotensive parturient women. MP (number/μL)
Pre-eclamptic parturients (N. 16)
Normotensive parturients (N.16)
Total (annexin V+)
5931 [5123–7199] 2456 [1400–2871] 1702 [1311–1942] 211 [149–234] 298 [254–357] 39 [31–47]
4631⁎ [3123–4999] 1566⁎⁎
Platelet-derived (CD61+) Endothelial-derived Leukocyte-derived TF+ PPL (sec.)
[1250–1875] 1542⁎ [1291–1677] 152⁎⁎ [112–184] 223⁎ [176–241] 42⁎⁎ [37–49]
Data are expressed by median and range interquartile. p are expressed vs controls: *p b 0.05 **p b 0.01. MP, microparticles; TF+, tissue factor-bearing MP; PPL, phospholipid-dependent clotting time; sec, seconds.
Please cite this article as: E. Campello, et al., Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.05.029
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of leukocytes, leukocyte-derived MP and platelets to the endothelium [25]. PE is known to be a two stage disease: the preclinical condition is impaired placentation (first stage) characterized by maternal endothelial dysfunction; in the second stage of PE, inflammatory leukocytes are also activated [26–28]. It is well known that the number of MP increases in conditions associated with enhanced systemic inflammatory responses (SIRs) in which intravascular systems are generally activated [29]. Several reports show that all components of the intravascular inflammatory network (inflammatory leukocytes, endothelium, coagulation system, acute phase response and complement) contribute to the responses in both normal pregnancy and, to a greater degree, in PE [30]. Hence, as in non-pregnant individuals with a SIR, it is to be expected that, in normal pregnancy and PE, there is enhanced MP release from intravascular cells, which has already been described [10,12,23]. Theoretically, endothelial cell activation should be detectable by elevated levels of endothelial-derived MP that mirror the endothelial cells turnover. In line with Lok et al. [24], we did not find a difference in the number of endothelial-derived MP between normotensive women and PE patients. We hypothesized a possibly consumption of endothelialderived MP during the inflammatory intravascular process or during the placental intravascular microthrombi. Hypercoagulability and inflammation are related and mutually reinforcing processes, involving inflammatory mediators (e.g., endotoxin, TNFα, and CD40 ligand), tissue factor expression on monocytes and the activated endothelium, and circulating TF–bearing MP. The trophoblast has a procoagulant nature, characterized by constitutively high TF levels [9]. Trophoblast differentiation process is accompanied with a membrane “flip-flop” leading to exposure of negative phospholipids on their surface and the enzymes involved in regulation of cell membrane phospholipids asymmetry are implicated in MP generation. Our study showed high levels of TF + MP in PE and also in physiological pregnancy, thus confirming that trophoblast differentiation yield massive TF-bearing MP formation [9]. In the present study we also evaluated the number of MP in venous cord blood of healthy foetuses and in the venous peripheral plasma of their both normotensive and PE mothers. We found that all MP were significantly higher in venous cord blood than in the maternal blood. We can confirm previous observations by Uszynski et al. [16] that MP are a constant component of the foetal blood and that MP may play a role of a powerful procoagulant, thus facilitating thrombin generation and compensating the “immaturity” of the foetal coagulation system. Moreover, when we analysed only venous cord blood, all MP detected were significantly higher in PE than in healthy normotensive parturient women. We hypothesized that changes in the balance of circulating microvesicles produced in the placental vessels might play a role in the alteration of maternal physiology and in the development of the maternal syndrome of pre-eclampsia [31]. Due to the low shear stress in the intravillous space, some foetus-placental-derived MP bearing TF may accumulate in this area, initiating a local effect on placental haemostasis. Other foetus-placental-derived MP penetrate into the maternal circulation via the decidual veins and may lead to systemic maternal effects [9]. The two major limitations of our study regard the relative small sample size enrolled and the lack of standardization in the MP analysis. As far as the small sample size was concerned we tried to overcome this limit using a longitudinal cohort of pregnant patients enrolled in a consecutive manner and as uniform as possible among them. As for methods, flow cytometric assays may not be sensitive enough to detect all sizes of MP, given that many of these fall below the detection threshold, though recommendations by the ISTH SSC Working Group on Vascular Biology [32–34] were followed. Moreover, an increased syncytiotrophoblast-derived MP number has also been described in PE patients, mainly in early-onset PE, compared with healthy pregnant women [9–35]. In our study, we could only explore the presence of MP derived from blood cells and TF-bearing with procoagulant properties and no cellular information is available on their placental origin.
In conclusion, our study shows that total MP, MP derived from activated platelet (P-Selectin+), leukocyte-derived MP and TF + MP are increased in pregnancies complicated by PE compared with normotensive pregnancies, confirming a pro-inflammatory intravascular reaction during this condition. On the contrary, platelet-derived MP (CD61+) levels are decreased in PE than in healthy women and no difference was seen in endothelial-derived MP levels between the two groups. Moreover, we showed that all subgroups of MP are higher in venous cord blood than in peripheral venous maternal blood both in healthy and in PE women. When only venous cord blood was analyzed, all MP detected were significantly higher in PE than in healthy normotensive parturient women. The transfer of foetus-placental-derived MP into the maternal circulation may have a role in the alteration of maternal physiology and may therefore be central in the development of the maternal syndrome of pre-eclampsia. Prospective studies on larger population are needed to evaluate the clinical utility of MP measuring in physiological and pathological pregnancies. Disclosure of conflict of interest None to declare except for BW who acted as a consultant to Stago via HaemaCon Ltd. References [1] J. Prochazkova, L. Slavik, J. Ulehlova, M. Prochazka, The role of tissue factor in normal pregnancy and in the development of preeclampsia. A review, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. (2014), http://dx.doi.org/10.5507/bp. 2014.061 (Epub ahead of print). [2] B.A. Lwaleed, L. Dusse, A.J. Cooper, Tissue factor dependent pathway and the diagnosis of pre-eclampsia, Semin. Thromb. Hemost. 37 (2011) 125–130. [3] L.M. Dusse, D.R. Rios, M.B. Pinheiro, A.J. Cooper, B.A. Lwaleed, Pre-eclampsia: relationship between coagulation, fibrinolysis and inflammation, Clin. Chim. Acta 412 (2011) 17–21. [4] N.S. Barteneva, E. Fasler-Kan, M. Bernimoulin, J.N. Stern, E.D. Ponomarev, L. Duckett, I.A. Vorobjev, Circulating microparitcles: square the circle, BMC Cell Biol. 14 (2013) 23. [5] Z.H. Wu, C.L. Ji, H. Li, G.X. Qiu, C.J. Gao, X.S. Weng, Membrane microparticles and diseases, Eur. Rev. Med. Pharmacol. Sci. 17 (2013) 2420–2427. [6] D. Burger, S. Schock, C.S. Thompson, A.C. Montezano, A.M. Hakim, R.M. Touyz, Microparticles: biomarkers and beyond, Clin. Sci. (Lond.) 124 (2013) 423–441. [7] J. Alijotas-Reig, C. Palacio-Garcia, E. Llurba, M. Vilardell-Tarres, Cell-derived microparticles and vascular pregnancy complications: a systematic and comprehensive review, Fertil. Steril. 99 (2013) 441–449. [8] F.K. Marques, F.M. Campos, L.P. Sousa, A. Teixeira-Carvalho, L.M. Dusse, K.B. Gomes, Association of microparticles and preeclampsia, Mol. Biol. Rep. 40 (2013) 4553–4559. [9] A. Aharon, B. Brenner, Microparticles and pregnancy complications, Thromb. Res. 127 (2011) S67–S71. [10] F. Meziani, A. Tesse, E. David, M.C. Martinez, R. Wangesteen, F. Schneider, R. Andriantsitohaina, Shed membrane particles from preeclamptic women generate vascular wall inflammation and blunt vascular contractility, Am. J. Pathol. 169 (2006) 1473–1483. [11] A.F. Orozco, C.J. Jorgez, W.D. Ramos-Perez, E.J. Popek, X. Yu, C.A. Kozinetz, F.Z. Bischoff, D.E. Lewis, Placental release of distinct DNA-associated micro-particles into maternal circulation: reflective of gestation time and preeclampsia, Placenta 30 (2009) 891–897. [12] C.A. Lock, R. Nieuwland, A. Sturk, C.M. Hau, K. Boer, E. Vanbavel, Microparticleassociated P-selectin reflects platelet activation in preeclampsia, Platelets 18 (2007) 68–72. [13] M.G. Macey, S. Bevan, S. Alam, L. Verghese, S. Agrawal, S. Beski, R. Thuraisingham, P.K. MacCallum, Platelet activation and endogenous thrombin potential in preeclampsia, Thromb. Res. 125 (2010) e76–e81. [14] V.H. Gonzalez-Quintero, J.J. Jime´nez, W. Jy, L.M. Mauro, L. Hortman, M.J. O’Sullivan, Y.S. Ahn, Elevated plasma endothelial microparticles in preeclampsia, Am. J. Obstet. Gynecol. 189 (2003) 589–593. [15] C.A. Lok, J. Jebbink, R. Nieuwland, M.M. Faas, K. Boer, A. Sturk, J.A. Van Der Post, Leukocyte activation and circulating leukocyte-derived microparticles in preeclampsia, Am. J. Reprod. Immunol. 61 (2009) 346–359. [16] M. Uszyński, E. Zekanowska, W. Uszyński, J. Kuczyński, A. Zyliński, Microparticles (MPs), tissue factor (TF) and tissue factor inhibitor (TFPI) in cord blood plasma. A preliminary study and literature survey of procoagulant properties of MPs, Eur. J. Obstet. Gynecol. Reprod. Biol. 158 (2011) 37–41. [17] P. Simioni, D. Tormene, L. Spiezia, G. Tognin, V. Rossetto, C. Radu, P. Prandoni, Inherited thrombophilia and venous thromboembolism, Semin. Thromb. Hemost. 32 (2006) 700–708. [18] V. Rossetto, L. Spiezia, F. Franz, L. Salmaso, L.V. Pozza, S. Gavasso, P. Simioni, The role of antiphospholipid antibodies toward the protein C/protein S system in venous thromboembolic disease, Am. J. Hematol. 84 (2009) 594–596.
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