Phosphatidylserine Exposing Extracellular Vesicles in Pre-eclamptic Patients

Patients

The study population consisted of 66 women at 25–39 weeks of gestation, including 36 women with a normal pregnancy and 30 women with P-EC. All investigated females were part of the larger study previously published by our group (20). We enrolled patients referred to a tertiary care maternity hospital (The Obstetrics and Gynaecology Clinic “Narodni Front”) with a confirmed diagnosis of P-EC between April 2014 and November 2016, as previously described. According to the revised criteria of the International Society for the Study of Hypertension in Pregnancy, published in 2014, diagnostic criteria for P-EC include the development of hypertension in a woman with previously normal blood pressure accompanied with one or more of the following new onset conditions: proteinuria, other maternal organ dysfunction and uteroplacental dysfunction (intrauterine growth restriction—IUGR). If the woman with chronic hypertension also manifests one or more of the above features of P-EC, this is classified as chronic hypertension with superimposed P-EC (21). From each patient two blood samples were collected: (1) in the morning following admission to hospital and (2) 3–10 days after delivery (mean duration 5 days).

Healthy pregnant women of similar age and gestation with no previous history of thromboembolic events, cardiovascular diseases (CVD), and/or P-EC were included as the control group. Recruitment and blood sampling were carried out during their scheduled routine prenatal care visit, with no further follow-up of pregnancy outcome.

All patients and controls gave their written informed consent and underwent an interview on their smoking habits, ongoing medication and own or family history of pregnancy complications, venous thrombotic diseases, diabetes, and CVD. The study was approved by local Ethics Committee of Gynaecology and Obstetrics Clinic “Narodni Front” in accordance with the internationally accepted ethical standards.

Blood Sampling

Venous blood samples were collected into plastic tubes with 0.109 mol/L trisodium citrate (1 part trisodium citrate + 9 parts blood, pH 7.4). Platelet poor plasma (PPP) was obtained by double centrifugation at 2,600 g for 15 min at room temperature (with plasma supernatant harvesting in between). The final plasma supernatant was dispensed in aliquots of 500 μL and frozen at −70°C until analysis.

Analysis of Extracellular Vesicles

The 500 μL PPP samples were thawed at 37°C for 5 min and then prepared by sequential centrifugations at 2,000 × g for 20 min and at 13,000 × g for 2 min at room temperature (with plasma supernatant harvesting in between). All measurements were performed on a Beckman Gallios flow cytometer (Beckman Coulter, Brea, CA, USA), as previously described (22). After centrifugation, 20 μl of the supernatant was incubated in the dark with 5 μl lactadherin-FITC (Haematologic Technologies, Essex Junction, VT, USA), together with either 5 μL CD42a-PE (GPIX, Beckman Coulter, Brea, CA, USA), or 5 μL CD62E-APC (E-selectin, AH diagnostics, Stockholm, Sweden), or 5 μl CD142-PE (TF, BD, NJ, USA), or 5 μL CD106-PE (VCAM-1, AH diagnostics, Stockholm, Sweden). Further phenotyping included expression of PlGF (Anti-PlGF-FITC, Abcam, Cambridge, UK). Megamix-Plus FSC (Biocytex, Marseille, France), a mix of beads with diameters (0.1, 0.3, 0.5, and 0.9 μm), was used to determine the EV gate. EVs were defined as particles <1 μm in size and positive for the antibodies described above. Lactadherin was used to identify the initial population of phosphatidylserine exposing EVs, since it is more sensitive in detection of PS-rich EVs than annexin V. The platelet and endothelial components were confirmed by their expression of CD42a and CD62E, respectively. The results are presented as concentrations of detected EVs (EVs/μl plasma).

Global Haemostatic Assays

The EV concentrations were compared with the FVIII concentration and the results of global haemostatic assays, endogenous thrombin potential (ETP) and overall haemostatic potential (OHP), and turbidimetric parameters of fibrin clot formation, the polymerization rate (Vmax), and the number of protofibrils per fiber (Max Abs). All assays were carried out according to previously published methods (20).

Statistical Analysis

Statistical analyses were performed using SPSS 20.0 (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.) and R 3.4.2. (23). Depending on data distribution continuous variables are expressed as mean with standard deviation (SD) or median with inter quartile range (IQR), and compared using the parametric Student t-test and non-parametric Mann-Whitney U-test, as appropriate. Categorical variables are presented as count (%) and were compared by the Chi-square test. Pairwise comparisons were applied to compare the same index of one subject before and after delivery using the dependent samples t-test and the Wilcoxon Signed Rank test for variables with or without normal distribution, respectively. Correlations between independent variables were calculated using Spearman's rank correlation analysis. In order to control the analysis for confounding variables, logarithmic transformation of data was performed on several variables. Normally distributed variables were correlated using Pearson correlation analysis and partial correlation. Differences were considered significant for p < 0.05.

Patient and Control Characteristics

The clinical characteristics of the study subjects are presented in Table 1. Pregnant women with P-EC and controls with uncomplicated pregnancies were of similar age, parity, and gestational age at blood sampling. There were no significant differences in smoking status or family history of CVD. However, rates of previous pregnancy complications and reported positive family history of pregnancy complications were higher in the P-EC group. Women with P-EC had a significantly higher body mass index (BMI) than control subjects (p < 0.001).

Table 1

General characteristics of patients with P-EC (n = 30) and controls (n = 36).

	Controls	Patients with P-EC	p-value
Age (years)	30.6 ± 4.8	31.1 ± 6.2	0.753
BMI (kg/m⁾	25.6 ± 2.6	30.9 ± 6.2	<0.001
Smoking status (n)
- Non-smokers	30 (83%)	26 (87%)	0.745^a
- Smokers	6 (17%)	4 (13%)
Gestational age (weeks)	33.5 ± 3.1	33.4 ± 3.7	0.900
Parity (n)
- Primiparous	18 (50%)	14 (47%)	0.810^a
- Multiparous	18 (50%)	16 (53%)
Previous pregnancy complications (n)	5 (27%)	12 (75%)	0.005^a
Family history of pregnancy complications (n)
- Positive	2 (6%)	5 (17%)	0.041^a
- Negative	34 (94%)	25 (83%)
Family history of CVD (n)
- Positive	10 (28%)	10 (33%)	0.112^a
- Negative	26 (72%)	20 (67%)

Data reported as the mean ± standard deviation or frequency n (%).

^{Chi-square test}.

Extracellular Vesicles

PPP samples of 30 women with P-EC and 36 women with normal pregnancy were analyzed by flow cytometry and phenotyped according to protein expression. In total, 5 phenotypes of EVs were measured: PS+ CD42a^{, PS+ CD62E}^{, PS+ CD142}^{, PS+ CD106}^{, and PlGF}^.Figure 1 shows the gating strategy of EVs phenotyping by flow cytometry (Figure 1A) and representative dot-plots of PS+ platelet-derived EVs and PS+ VCAM-1^{EVs in a healthy pregnant woman (}Figures 1B,C) and a patient with pre-eclampsia before and after delivery (Figures 1D–G). The largest portion of PS-exposing EVs originated from platelets in all investigated groups (36.9% in normal pregnant women, 51.5 and 29.5% in women with pre-eclampsia before and after delivery, respectively).

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Figure 1

Representative dot-plots of platelet-derived (CD42a^{) and VCAM-1}^(CD106^{) EVs.}(A) EV gating strategy based on beads with diameter 0.3, 0.5, and 0.9 μm; (B,C) platelet-derived (CD42a^{) EVs and VCAM-1}^(CD106^{) EVs in a healthy pregnant woman;}(D,E) platelet-derived (CD42a^{) EVs in a patient with pre-eclampsia before and after delivery;}(F,G) VCAM-1^(CD106^{) EVs in a patient with pre-eclampsia before and after delivery.}

Phenotyping of EVs in Women With P-EC Compared to Healthy Pregnancy

Although the concentrations of PS+ EVs in women with P-EC and healthy pregnancy were revealed to be the same, comparing different phenotypes of PS-exposing EVs we demonstrated significantly higher concentrations of PS+ CD42a^{platelet-derived and PS+ VCAM-1}^{EVs in women with P-EC (}Table 2). However, no differences were observed in endothelial-derived (PS+ CD62E^{) EVs and TF-expressing PS+ EVs between P-EC patients and healthy pregnant women. Also, we found similar concentrations of PlGF-expressing EVs in the P-EC patients and healthy pregnant women (}Figures 2A,B).

Table 2

Concentration of circulating extracellular vesicles and levels of investigated haemostatic parameters in patients with P-EC before and after delivery (n = 30) and in controls (n = 36).

Parameter	Controls	Patients with P-EC
	Before delivery	Before delivery	After delivery
Extracellular vesicles
PS+ EVs/μL	957 (586.5–2022.5)	905 (700–2100)	2445.5 (1727–4235)^†††
PS+ CD42a^EVs/μL	353 (151.5–455)	466 (327–560)^*	720.5 (585–1266)^†††
PS+ CD62E^EVs/μL	108 (66–118)	91 (70–104)	136.5 (103–156)^†††
PS+ CD142^EVs/μL	34 (15.5–69.5)	29 (15–59)	87 (20–189)^†
PS+ CD106^EVs/μL	39.5 (27–47)	58 (40–80)^***	102 (75–153)^†††
PlGF^EVs/μL	295.5 (176.5–408.5)	198.5 (118–327)	263 (150–399)
Endogenous thrombin potential
ETP (AUC, %)	93.5 (90.5–105.0)	112.5 (106.0–119.0)^***	107.0 (101.5–118.0)
Peak height (%)	104.8 ± 10.3	115.2 ± 13.1^**	128.4 ± 17.9^††
Overall haemostatic potential
OCP (Abs-sum)	247.0 ± 32.9	229.4 ± 44.5	246.9 ± 50.8
OHP (Abs-sum)	177.1 ± 38.1	198.7 ± 40.7^*	219.3 ± 43.8
OFP (%)	27.3 (16.7–35.5)	12.2 (6.6–18.8)^***	8.8 (5.3–14.4)^†
Fibrin clot properties
Vmax (AU/min)	0.47 ± 0.14	0.55 ± 0.10^**	0.57 ± 0.16
Max Abs (AU)	1.36 ± 0.18	1.25 ± 0.21^*	1.30 ± 0.24
Factor VIII activity
FVIII (%)	189.1 ± 78.3	259.9 ± 115.4^**	343.2 ± 103.6^††

Data reported as the mean ± standard deviation or median with inter quartile range (IQR). EVs, extracellular vesicles; ETP, endogenous thrombin potential; OCP, overall coagulation potential; OHP, overall haemostatic potential; OFP, overall fibrinolysis potential; Vmax, polymerization rate; Max Abs, number of protofibrils per fiber. EV markers: CD42a, glycoprotein IX (platelet marker); CD62E, E-selectin (endothelial marker); CD142, Tissue Factor (TF); CD106, Vascular Cell Adhesion Molecule-1 (VCAM-1); PlGF, placental growth factor.

Controls vs. P-EC:

p < 0.05,

p < 0.01, and

p < 0.001.

P-EC before vs. after delivery:

p < 0.05,

p < 0.01, and

^{p < 0.001}.

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Object name is fmed-08-761453-g0002.jpg

Figure 2

Representative dot-plots of PlGF positive EVs in a healthy pregnant women (A) and a patient with pre-eclampsia before (B) and after (C) delivery.

Phenotyping of EVs in Women With P-EC After Delivery

The concentration of EVs and their phenotypes were also analyzed in women with P-EC before and 3–10 days after delivery. It is reasonable to expect that concentrations of EVs increase after delivery, which is associated with the delivery itself. Indeed, pairwise comparisons of results obtained in women with P-EC before and after delivery showed an ~2.5-fold increase in the concentration of PS+ EVs accompanied by the rise of all investigated EV phenotypes. As presented in Table 2, concentrations of PS+ CD42a^{, PS+ CD62E}^{, PS+ CD142}^{, and PS+ CD106}^{were significantly elevated in women with P-EC after delivery compared to the values before delivery. However, although slightly higher in the P-EC group after delivery, the concentration of EVs expressing PlGF did not differ significantly between P-EC patients before and after delivery (}Figures 2B,C).

Haemostatic Parameters and Their Correlation to EVs Concentrations

Results of global haemostatic assays (ETP and OHP), turbidimetric measurements of fibrin clot formation (Vmax and Max Abs), and factor VIII activity assay are presented in Table 2. Compared to gestational age-matched controls women with P-EC showed significantly elevated ETP, peak height and OHP values associated with depressed fibrinolysis [decreased overall fibrinolysis potential (OFP) values; 12.2 [6.6–18.8] % vs. 27.3 [16.7–35.5] %, p < 0.001]. Furthermore, P-EC patients exhibited significantly higher Vmax and lower Max Abs values, indicating the faster formation of the fibrin clot composed of thinner fibers. In the P-EC group after delivery, a significant increase of the peak height and an additionally decreased rate of fibrinolysis were observed, without significant change in fibrin clot properties. FVIII activity was above the normal range for non-pregnant individuals with a significant difference in all groups (p < 0.01).

Since this study focused on EVs presenting negatively charged phospholipids on their outer leaflet and their impact on coagulation disturbances, we performed Spearman correlations between the detected concentration of particles and measured haemostatic parameters (Table 3). Our results demonstrated that PS+ EVs correlated with overall coagulation potential (OCP; r = −0.47, p = 0.009), overall haemostatic potential (OHP; r = −0.49, p = 0.007) and fibrin formation parameters: maximum absorbance (Max Abs; r = −0.49, p = 0.011) and polymerization rate (Vmax; r = −0.41, p = 0.031). The same coagulation parameters correlated also with the EVs copresenting TF and PS on their outer leaflet (OCP r = −0.44, p = 0.016; OHP r = −0.49, p = 0.007; Max Abs r = −0.46, p = 0.019; Vmax r = −0.46, p = 0.013, respectively). While PlGF exposing EVs also showed significant correlation with Max Abs and Vmax (r = −0.40, p = 0.045 and r = −0.52, p = 0.004, respectively), PS+ VCAM-1^{EVs were correlated only with FVIII activity (}r = 0.39, p = 0.034). Interestingly, PS+ platelet-derived and endothelial-derived EVs showed no significant correlation with any of the investigated coagulation parameters. However, the Pearson correlation analysis showed the similar strength of association between EVs concentrations and measured haemostatic parameters, except no association was found between concentration of EVs exposing PlGF and Max Abs. After adjustment for maternal age and BMI we observed a moderate correlation between CD62E^{endothelial-derived EVs and ETP (}r = −0.42, p = 0.030), while the association between PS+ CD106^{EVs concentration and FVIII activity was no longer statistically significant. Regarding maternal complications (HELLP, renal complications, thrombocytopenia, placental abruption, and neurological disorders) and perinatal complications (IUGR and oligohydramnios) observed in our P-EC group we found no significant differences in the levels of investigated EVs between patients with and without complications. Correlation analysis revealed no association between the EVs concentrations and 1- or 5-min APGAR score.}

Table 3

Correlation between the results of haemostatic parameters and EV concentrations.

Variable	PS+ EVs/μL		PS+ CD42a^EVs/μL		PS+ CD62E^EVs/μL		PS+ CD142^EVs/μL		PS+ CD106EVs/μL		PlGFEVs/μL
	Rho	p	Rho	p	Rho	p	Rho	p	Rho	p	Rho	p
ETP	−0.18	0.36	−0.07	0.71	−0.20	0.29	−0.32	0.09	−0.05	0.78	−0.32	0.09
Peak height	−0.01	0.99	0.22	0.24	−0.12	0.53	−0.27	0.14	0.11	0.56	−0.16	0.39
OCP	−0.47	0.009	−0.14	0.47	−0.24	0.20	−0.44	0.016	0.01	0.99	−0.35	0.058
OHP	−0.49	0.007	−0.17	0.39	−0.24	0.22	−0.49	0.007	−0.07	0.72	−0.36	0.053
OFP	−0.10	0.58	0.00	0.99	−0.12	0.51	0.23	0.22	−0.07	0.71	0.10	0.59
Vmax	−0.41	0.031	−0.20	0.31	−0.28	0.14	−0.46	0.013	−0.23	0.24	−0.52	0.004
Max Abs	−0.49	0.011	−0.28	0.16	−0.28	0.17	−0.46	0.019	0.07	0.74	−0.40	0.045
FVIII	0.12	0.54	0.18	0.33	0.21	0.27	0.03	0.88	0.39	0.034	0.09	0.63

All the data were assessed using Spearman's rank correlation analysis and are reported as the correlation coefficients (Rho) with the corresponding p-values (p). ETP, endogenous thrombin potential; OHP, overall haemostatic potential; OCP, overall coagulation potential; OFP, overall fibrinolysis potential; Vmax, polymerization rate; Max Abs, number of protofibrils per fiber; FVIII, factor VIII activity; EVs, extracellular vesicles. EV markers: CD42a, glycoprotein IX (platelet marker); CD62E, E-selectin (endothelial marker); CD142, Tissue Factor (TF); CD106, Vascular Cell Adhesion Molecule-1 (VCAM-1); PlGF, placental growth factor. All significant associations are bolded.

^{Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia}

^{Haemostasis Department, Blood Transfusion Institute of Serbia and Faculty of Medicine, University of Belgrade, Belgrade, Serbia}

^{Gynaecology and Obstetrics Clinic “Narodni Front”, Faculty of Medicine, University of Belgrade, Belgrade, Serbia}

^{Department of Medical Sciences, Uppsala University, Uppsala, Sweden}

^{Department of Medicine, Division of Rheumatology, Karolinska Institutet and Rheumatology, Karolinska University Hospital Stockholm, Stockholm, Sweden}

Edited by: Eleni Gavriilaki, G. Papanikolaou General Hospital, Greece

Reviewed by: Panagiota Anyfanti, Aristotle University of Thessaloniki, Greece; Efthymia Vlachaki, Aristotle University of Thessaloniki, Greece

*Correspondence: Sanja Lalic-Cosic moc.oohay@ajnasl

This article was submitted to Hematology, a section of the journal Frontiers in Medicine

†These authors have contributed equally to this work and share last authorship

Edited by: Eleni Gavriilaki, G. Papanikolaou General Hospital, Greece

Reviewed by: Panagiota Anyfanti, Aristotle University of Thessaloniki, Greece; Efthymia Vlachaki, Aristotle University of Thessaloniki, Greece

Received 2021 Aug 19; Accepted 2021 Oct 4.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Abstract

Background: Pre-eclampsia (P-EC) is associated with systemic inflammation, endothelial dysfunction and hypercoagulability. The role of extracellular vesicles (EVs) in coagulation disturbances affecting the development and severity of P-EC remains elusive. We aimed to evaluate the concentration of EVs expressing phosphatidylserine (PS) and specific markers in relation to the thrombin and fibrin formation as well as fibrin clot properties, in pregnant women with P-EC in comparison to healthy pregnant women of similar gestational age.

Methods: Blood samples of 30 pregnant women diagnosed with P-EC were collected on the morning following admission to hospital and after delivery (mean duration 5 days). The concentration of the PS-exposing EVs (PS+ EVs) from platelets (CD42a^{, endothelial cells (CD62E}^{), and PS+ EVs expressing tissue factor (TF) and vascular cell adhesion molecule 1 (VCAM-1) were measured by flow cytometry. Further phenotyping of EVs also included expression of PlGF. Markers of maternal haemostasis were correlated with EVs concentration in plasma.}

Results: Preeclamptic pregnancy was associated with significantly higher plasma levels of PS+ CD42a^{EVs and PS+ VCAM-1}^{EVs in comparison with normotensive pregnancy. P-EC patients after delivery had markedly elevated concentration of PS+ CD42a}^{EVs, CD62E}^{EVs, TF}^{EVs, and VCAM-1}^{EVs compared to those before delivery. Inverse correlation was observed between EVs concentrations (PS+, PS+ TF}^{, and PlGF}^{) and parameters of overall haemostatic potential (OHP) and fibrin formation, while PS+ VCAM-1}^{EVs directly correlated with FVIII activity in plasma.}

Conclusion: Increased levels of PS+ EVs subpopulations in P-EC and their association with global haemostatic parameters, as well as with fibrin clot properties may suggest EVs involvement in intravascular fibrin deposition leading to subsequent microcirculation disorders.

Keywords: extracellular vesicles, endogenous thrombin potential, overall haemostatic potential, fibrin structure, endothelial dysfunction, pre-eclampsia

Abstract

Acknowledgments

The authors would like to thank Nida Mahmoud Hourani Soutari and Jelena Bozic for laboratory assistance.

Acknowledgments

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