Thrombosis Research 127 Suppl.

3 (2011) S67S71

Contents lists available at ScienceDirect

Thrombosis Research
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s

Microparticles and pregnancy complications

Anat Aharona,b , Benjamin Brennera,b
a Thrombosis

and Hemostasis Unit, Rambam Health Care Campus; b Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel

article info
Keywords: Microparticles (MPs) Pregnancy complications Placenta Trophoblasts

abstract
Microparticles (MPs) are shed from cell membranes of a variety of cells, promote thrombus formation, mediate pro-inammatory effects and may cause endothelial dysfunction. Normal pregnancy is characterized by increased levels of MPs compared to non-pregnant healthy women but the prevalence, cell origin and the role of MPs in pregnancy-related complications remain controversial. Normal pregnancy is an acquired hyper-coagulable state due to an increase in procoagulants and decrease in natural anticoagulants. Pregnancy-related complications such as preeclampsia, intrauterine fetal growth restriction (IUGR) and fetal loss are associated with placental dysfunction and may cause signicant maternal and fetal morbidity and mortality. This article highlights the role of microparticles in maternal placental crosstalk and the interplay between microparticles, thrombosis and pregnancy complications. 2011 Elsevier Ltd. All rights reserved.

Introduction Pregnancy is an acquired hypercoagulable state, that may be accompanied by gestational vascular complications (GVC) including preeclampsia, intrauterine growth restriction (IUGR), pregnancy loss and placental abruption, the latter being a major cause of maternal morbidity and fetal mortality. Placental thrombotic ndings in women with GVC are associated with inherited and acquired thrombophilia; yet, the mechanisms leading toward thrombosis in women with GVC and the underlying placentalmaternal crosstalk promoting its development are not clearly understood [1]. The present review highlights the impact of microparticles (MPs) on maternal placental crosstalk and the interplay between MPs, thrombosis and pregnancy complications. Microparticles (MPs) are found in blood circulation under normal physiologic conditions, while their levels increase in a variety of disease states. MPs are shed from cell membranes upon activation or apoptosis. They vary in size (0.1 to 1 mm) as well as phospholipids and protein composition. MPs primarily express cell membrane proteins, reecting those of their cell origin. In blood, circulating MPs serve as a vehicle that bears and transfers proteins (receptors, growth and apoptotic factors, coagulation factors and others), RNA and DNA fragments from one cell to another. MPs can induce cell signaling that leads to such processes as invasion, angiogenesis or apoptosis [25]. In addition, MPs are involved in thrombosis, inammation and
* Corresponding author. Anat Aharon, PhD. Thrombosis and Hemostasis Unit, Rambam Health Care Campus, P.O. Box 9602, Haifa 31096, Israel. Tel.: +972 4 854 3520; fax: +972 4 854 3886. E-mail address: a_aharon@yahoo.com (A. Aharon).
0049-3848 /$ see front matter 2011 Elsevier Ltd. All rights reserved.

vascular dysfunction [6,7]. Therefore, MPs may have a crucial role in the maternal placental crosstalk. Microparticle levels and cell origin in normal pregnancy and in gestational complications The prevalence of MPs in gestational vascular complications still remains controversial. Levels of circulating MPs at 24 weeks of gestation had no predictive value for a subsequent development of pregnancy-related vascular complications such as induced hypertension, preeclampsia, intrauterine growth restriction, or small for gestational age infants [8]. However, Carp et al. demonstrated an increase in endothelial MPs in 12 out of 96 (12.5%) non-pregnant women with a history of three or more consecutive miscarriages compared with only two of 90 parous controls women (2.2%) (P < 0.008) [9] These ndings may indicate that combination of pregnancy with a basic vascular dysfunction (which is not related to pregnancy) may result in fetal loss. An overview of the studies measuring circulating numbers of MP and their cell origin in normal and complicated pregnancy is summarized in Tables 1 and 2. Data from different studies are inconsistent and demonstrate high variation in their results. Some of the trials showed an increase in MPs sub-populations in women with preeclampsia compared to normal healthy pregnant females, including increase in the total number of MPs [10] or total negatively charged phospholipids bearing MPs [14], elevation in platelet MPs [14] and activated platelet MPs [15,17], as well as increase in endothelial MPs [16,19] and white blood cell MPs [12,14,20]. Others studies revealed no differences in the total number of MPs or negatively charged phospholipids bearing MPs and platelet MPs between normal pregnant and preeclamptic

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A. Aharon, B. Brenner / Thrombosis Research 127 (2011) S67S71 Table 1 Overview of studies measuring circulating numbers of MP in non-pregnant women, healthy pregnant women and women with gestation vascular complications, mainly preeclampsia. Non-pregnant MPs, 106 /mL Healthy pregnant 68.7 (29.1160.5) n=9 1126 (9102455) n = 10 NS Preeclampsia 119.5 (39.0256.6) n = 10 *p < 0.05 1232 (8181653) n = 10 *NS Reference Orozco AF, et al. Placenta 2009 [10]

MPs, 106 /L

1207 (8762057) n = 10

Biro E, et al. Placenta 2007 [11]

Total PS-MPs (Annexin V) 106 /L plasma 2357 (10923468) n = 10 122 (18447) n = 19 1960 (4103714) n = 10 NS 429 (2601598) n = 15 p = 0.0005 7.51.35 n = 17 6.7 (2.216.6) n = 10 5.1 (1.512.8) n = 10 2256 (9943839) n = 10 NS 260 (10446) n = 24 *NS 11.681.09 n = 21 *P<0.05 2.6 (1.37.8) n=9 *P = 0.03 Van Wijk MJ, et al. Am J Obstet Gynecol 2002 [12]

(MPs/ml)

Bretelle F, et al. Thromb Haemost 2003 [13]

(nmol/L Eq PS)

Meziani E, et al. Am J Pathol 2006 [14]

(MP, 109 /L)

Lok CA, et al. Platelets 2007 [15]

p: p-value of the differences between normal pregnant women and non-pregnant women. *p: p-value of the differences between women with preeclampsia and normal pregnant women NS: non-signicant; n: number of patients.

women [11,12,16] or decrease in the total negatively charged phospholipids bearing MPs and platelet MPs [13,15] and activated platelet MPs [18], in preeclamptic women compared to those with normal healthy pregnancies. The broad variation in the origin of cell MPs obtained in healthy pregnancy and preeclampsia may be related to the differences in the characteristics of pregnancy vascular complications as well as to the diversity in study methods used for MPs characterization. Microparticle thrombogenicity Microparticles play a dominant role in coagulation initiation and thrombus formation [7]. MPs bearing tissue factor (TF), the central coagulation cascade initiator and other coagulation factors include TF pathway inhibitor (TFPI). In pregnancy, MP membrane coagulation antigens may reect the delicate thrombogenic balance between maternal and placental cells. Laude et al. found that microparticle samples obtained two months after an obstetrical event from women with a history of early or late pregnancy loss with no obvious medical explanation, demonstrated high prevalence of increased procoagulant microparticle levels when compared to controls [24]. These ndings may indicate that a combination of a basic hypercoagulabel state with other thrombogenic factors such as pregnancy may result in fetal loss. Furthermore, non- pregnant women with a history of preeclampsia produce more thrombin and platelet-derived MPs compared to healthy controls who delivered in the past [25]. We have recently reported that MPs obtained from healthy pregnant women demonstrate high procoagulant activity compared to non-pregnant females. The procoagulant activity further increases in MPs of women with gestational vascular complications (GVC) without a change in the TF expression, but with reduction in TFPI. The TF/TFPI ratio on MPs was higher in normal pregnancy compared to non-pregnant women with a further increase in women with GVC. The TF/TFPI MPs ratio appeared to be lower in women treated with low molecular weight heparin (LMWH) (due to a history of GVC or thrombotic events or because they harbor single or combined thrombophilia), compared to the GVC group without difference in the procoagulant activity. The TF/TFPI ratio on

circulating MPs reects the hypercoagulanle state that characterizes preeclampsia and other vascular complications and can serve as a potential biomarker for diagnostics of a prothrombogenic state in GVC as well as for monitoring the efciency of the anticoagulant treatment [21]. On the other hand, several studies in women with recurrent spontaneous abortions, reported that changes in the haemostatic balance were not associated with increased levels of circulating MPs. Furthermore, these trials demonstrated that microparticlebased thrombin generation was similar in normal and preeclamptic pregnancies [26]. In accordance with these ndings, the procoagulant activity generated by the total Annexin V MPs was unchanged in pathological pregnancies [13] and the numbers of negatively charged phospholipids-positive MPs, platelet- and endothelial-derived MPs or prothrombin fragments 1+2 and subsequent thrombin generation were nearly similar in women with recurrent spontaneous abortion and controls [27]. Likewise, levels of negatively charged phospholipid MPs in plasma collected during the third trimester did not differ between women with preeclampsia and controls, nor were they associated with heightened factor VII activity in women with preeclampsia [28]. As previously mentioned, the reported prevalence and thrombogenicity of MPs in gestational vascular complications remain controversial, which may be due to variations in microparticle isolation techniques and characterization methods as well as to differences in the denition of pregnancy pathology. Trophoblast-derived microparticles The placenta, a highly vascularized organ, is lined with trophoblast cells which are involved in local haemostatic mechanisms as well as in maternal and placental crosstalk. Syncytiotrophoblast microvilli are shed into the maternal circulation and their levels rise signicantly in preeclamptic women [29]. Unbound syncytiotrophoblast MPs have been detected in normal pregnancies by the second trimester, while their numbers considerably increased during the third trimester [23,29]. Excess shedding of syncytiotrophoblastderived MPs is a feature of early-onset preeclampsia, but not of normotensive intrauterine growth restriction [22]. While normal

A. Aharon, B. Brenner / Thrombosis Research 127 (2011) S67S71 Table 2 Overview of studies measuring MP cellular origin in non-pregnant women, healthy pregnant women and women with gestation vascular complications, mainly preeclampsia Non-pregnant Platelet MPs CD42b (nmol/L Eq PS) 2014 (4053261) n = 10 6.6 (1.915.6) n = 10 39 (17674) 4.20.94 N = 17 78714344 n = 20 5.830.65 n = 21 *P < 0.05 107516114 n = 20 *NS 107516114 n = 20 *NS 1818 (6383561), n = 10 *NS 37.5 (8451) *p < 0.05 Meziani E, et al. Am J Pathol. 2006 [14] Healthy pregnant Preeclampsia Reference

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CD31+/CD42+ (counts/lL)

Gonzalez VH, et al. Am J Obstet Gynecol 2003 [16]

Cd61 (106 /L plasma)

1618 (1173450) n = 10 NS 4.9 (1.414.9) n = 10 193 (14773) p = 0.0006

VanWijk MJ, et al. Am J Obstet Gynecol 2002 [12]

Cd61 (109 /L plasma)

Lok CA, et al. Platelets 2007 [15]

CD41,CD51 (/ml) Activated platelet MPs CD62p (%) 8.0 (4.913.1) n=9 0.7 (0.41.3) n = 42 431 n = 30

Bretelle F, et al. Thromb Haemost 2003 [13]

10.9 (7.017.4) n = 10 0.4 (0.21.0) n = 46 NS 491 n = 30 NS

15.4 (11.019.5) n = 10 *P = 0.02 1.0 (0.22.5) n = 46 *p = 0.029 33 n = 30 *P = 0.001

Lok CA, et al. Platelets 2007 [15]

CD62p (%) Quartile Range CD62p (109 /L)

Macey MG, et al. Thromb Res 2010 [17]

Harlow FH, et al. Am J Obstet Gynecol 2002 [18]

Endothelial MPs CD31+/CD41 (counts/ml) 7 (121) n = 19 11 (075) n = 10 13 (132) n = 15 p = 0.04 84062832 n = 20 61193592 n = 38 712160 n = 38 Nonidentied 0 (059) n = 10 NS 9 (232) n = 24 NS 147237724 n = 20 *P < 0.001 104975145 n = 52 *P < 0.001 1930966 n = 52 *P < 0.001 Nonidentied 23 (0122) n = 10 *NS Bretelle F, et al. Thromb Haemost 2003 [13]

CD31+/CD41b (counts/ml)

Gonzalez VH, et al. Am J Obstet Gynecol 2003 [16]

CD31+/CD41b (counts/ml)

Gonzalez VH, et al. Am J Obstet Gynecol 2004 [19]

CD62E+ (counts/ml)

Gonzalez VH, et al. Am J Obstet Gynecol 2004 [19]

CD31+ (nmol/L Eq PS) CD62e/CD144 (106 /L plasma)

Meziani E, et al. Am J Pathol 2006 [14] VanWijk MJ, et al. Am J Obstet Gynecol 2002 [12]

Lymphocyte MPs CD11a 2.90.65 n = 17 5.850.61 n = 21 *p < 0.01 Meziani E, et al. Am J Pathol 2006 [14]

Monocyte CD14 (106 /L plasma) 0 (048) n = 10 ~62 n = 10 0 (018) n = 10 NS ~60 n = 10 NS 0 (0253) n = 10 *NS ~90 n = 10 *P = 0.049 Van Wijk MJ, et al. Am J Obstet Gynecol 2002 [12] Lok et al. Am J Reprod Immunol 2009 [20]

CD14 (106 /L plasma)

Granulocyte CD66e (106 /L plasma) 7 (0267) n = 10 ~23 n = 10 8 (085) n = 10 NS ~37 n = 10 P = 0.022 79 (0287) n = 10 *P = 0.03 ~90 n = 10 *P = 0.086 Van Wijk MJ, et al. Am J Obstet Gynecol 2002 [12]

CD66e (106 /L plasma)

Lok CA, et al. Am J Reprod Immunol 2009 [20]

continued on next page

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Table 2 (continued) Non-pregnant Placenta MPs NDOG1 (FACS, %) NDOG2 (ELISA, ng/ml) 1.73 (0.37.2%) 0.49 N = 10 <5 6.03% (1.144.4%) P < 0.0001 20.4 N = 25 p < 0.001 ~30 p < 0.001 N = 24 5.79% (0.719.9%) *P = 0.012 45.4 N = 25 *P = 0.007 ~55 *p < 0.01 N = 24 Aharon A, et al. J Thromb Haemost 2009 [21] Goswami D, Placenta 2006 [22] Healthy pregnant Preeclampsia Reference

NDOG2 (ELISA, ng/ml)

Germain SJ, et al. Immunol. 2007 [23]

p: p-value of the differences between normal pregnant women and non-pregnant women *p: p-value of the differences between women with preeclampsia and normal pregnant women NS: non-signicant; ~: value extracted from gure; n: number of patients.

pregnancy, labor and placental separation have no effect on the shedding of syncytiotrophoblast-MPs, preeclampsia is associated with an increase in syncytiotrophoblast-MP shedding at full dilatation when compared to prelabor levels [30]. Trophoblast has a procoagulant nature, characterized by constitutively high TF levels [31]. Trophoblast differentiation process is accompanied with a membrane ip-op leading to exposure of negative phospholipids on their surface and the enzymes involved in regulation of cell membrane phospholipids asymmetry are implicated in microparticle generation [6]. Thus, trophoblast differentiation is often assumed to yield massive TF-bearing microparticle formation. We have recently reported that circulating MPs of pregnant women include TF-bearing MPs of placental syncytiotrophoblast origin. Whereas the relative contribution of syncytiotrophoblast MPs bearing TF was substantial in healthy pregnant women, a signicant decrease was noted in the GVC group. This represents an increase in the maternal source of TFbearing MPs that potentially reects the systemic nature of such pathologies [21]. Preeclampsia is characterized by an increase in inammation markers, endothelial activation and injury and shedding of trophoblast debris which may be related to placental ischemia and oxidative stress. The amount of syncytiotrophoblast-derived MPs in the preeclampsia plasma is in correlation with the degree of endothelial cell inhibitory activity and underlying the maternal syndrome of preeclampsia [29]. In addition, circulating syncytiotrophoblast-MPs bind to monocytes and stimulate the production of inammatory cytokines [23]. Placental MPs [labeled by fetal-derived human leukocyte antigen-G (HLA-G) and placental alkaline phosphatase (PLAP)] are elevated in maternal circulation at different times during gestation. DNA amounts per HLA-G MP increase in preeclamptic women which might indicate dysfunctional extravillous cytotrophoblasts [10]. Most mRNA of the human placental lactogen (hPL) and human chorion gonadotropin (hCG) in maternal plasma appear to be associated with subcellular particles, which may explain the stability of placental mRNA in maternal plasma [32]. An in-vitro study on cultured trophoblasts detected fetal DNA and RNA on placenta-derived syncytiotrophoblast MPs [33]. Microparticle effects The placental syncytiotrophoblast produces many proinammatory cytokines, which may be associated with their related MPs and the higher amounts of syncytiotrophoblast membrane MPs (STBM) circulating in maternal blood in preeclampsia might lead to the excessive maternal inammatory reaction [34]. The study examining the effects of in-vitro prepared STBM on

human peripheral blood monocytes of healthy non-pregnant women showed that STBMs stimulated production of inammatory cytokines such as TNF-a, interleukin (IL)12p70, and IL-18 but not IFN-g [23]; in addition, STBMs up-regulated the adhesion of molecule CD54, and induced the production of (IL)-8, IL-6, IL-1b [34]. The inammatory response is enhanced in preeclampsia compared with normal pregnancy and includes a further increase in secreted but not intracellular measures of TNF-a and IL-12p70 and inhibition of IFN-g production [23]. On the other hand, no evidence of increased complement activation on the microparticle surface was found in preeclamptic women [11]. MPs with bound C-reactive protein (CRP) were signicantly increased, but in contrast to healthy pregnant and nonpregnant women, this was not associated with elevated classical pathway activation on the surface of MPs. These results may indicate multiple effects of STBM in proinammatory response in normal pregnancy and preeclampsia. MPs from women with preeclampsia caused endothelial dysfunction in isolated myometrial arteries obtained from healthy pregnant women and induced vascular hyporeactivity to serotonin in human arteries and mouse aortas [35]; in addition, vascular hyporeactivity was observed in the arteries taken from mice treated in vivo with preeclamptic MPs, a phenomenon not observed with MPs isolated from healthy pregnant controls [14]. Conversely, Lok et al. demonstrated that expression of inammation-related genes in endothelial cells is not directly affected by MPs from preeclamptic patients [36] and Meziani showed that MPs from preeclamptics did not impair endothelial responses in either aorta or mesenteric arteries of mice [14] but the preeclamptic plateletderived MPs were associated with increased NO release [14]. Additionally, preeclamptic MPs have been shown to induce nuclear factor-B activation, nitric oxide synthase expression and enhance oxidative stress. MPs of this source also exhibit the capacity to induce vascular hyporeactivity of vessels from pregnant mice through overproduction of NO [37]. It can be summarized that MPs of preeclamptic women are associated with endothelial cells dysfunction. Summary Microvesicles, which play a key role in thrombosis, inammation, and vascular dysfunction, participate in the placental and maternal crosstalk in normal pregnancies as well as in gestational vascular complications. Preeclampsia is characterized by an increase in inammation markers, endothelial activation and injury and shedding of trophoblast debris. Due to the low shear stress in the intravillous space, some trophoblast-derived MPs bearing TF may accumulate in this area, initiating a local effect on placental

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haemostasis. Other trophoblast-derived MPs penetrate into the maternal circulation via the decidual veins and may lead to systemic maternal effects. Alternatively, increased blood pressure, inammation and other pathological conditions may result in increased levels of maternal TF-bearing MPs which then reach the placental intravillous space via the maternal spiral arteries, affecting placental haemostasis. Conict of Interest Statement There is no conict of interest to declare. References
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