Abstract
This study aimed to assess the von Willebrand factor (VWF) antigen levels and its proteolytic enzyme ADAMTS13 activity in preeclampsia. The study includes 10 non-pregnant women, 50 normal pregnancy, and 110 preeclamptic (PE) women at the same period of pregnancy. For all studied groups plasma ADAMTS13 activities were determined with the FRETs-VWF 73 assay, while VWF antigen levels with an immunoturbometric assay. The plasma ADAMTS13 activity was significantly reduced in PE as compared with normal pregnancy and non-pregnant women (P < 0.01 for both). In contrast, plasma VWF antigen and VWF RCO were significantly elevated in PE, as compared with normal pregnancy group as well as non-pregnant women (P < 0.01 for both). In conclusion, reduction of plasma ADAMTS13 and elevation in VWF might have a role in the pathogenesis of PE.
Keywords:
Introduction
Preeclampsia (PE) is a multisystem disorder of pregnancy, which complicates 3–5% of pregnancies in the western world. It is a major cause of maternal morbidity and mortality worldwide. The cardinal clinical features of the condition are hypertension and proteinuria occurring after 20 weeks gestation in women who were not previously known to be hypertensive. Other signs and symptoms include edema and headache, and in severe cases, the condition is associated with seizures (eclampsia), liver and kidney dysfunction as well as clotting abnormalities, Adult respiratory distress syndrome and fetal growth restriction (FGR).Citation1
Several studies have shown that in patients with pregnancies complicated by PE and HELLP syndrome, von Willebrand factor (VWF) antigen levels are markedly increased.Citation2,Citation3 VWF is synthesized by megakaryocytes and endothelial cells as a preproprotein. During maturation of VWF after multimerization, the preproprotein is cleaved off from the VWF.Citation4,Citation5
The main function of VWF is to mediate adhesion and aggregation of platelets, by forming a molecular bridge between exposed subendothelial matrix and the platelet surface GpIb-IX-V receptor complex.Citation6 Under physiologic conditions, VWF is unable to interact spontaneously with platelets. To induce an interaction, conversion of the VWF A1 domain into a GpIbα-binding conformation is required.Citation7 This conformation can be induced by binding of VWF to the exposed collagen. Moreover, newly secreted VWF multimers have been reported to interact spontaneously with platelets.Citation8 Under normal conditions, newly secreted VWF multimers are rapidly cleaved into smaller, less reactive multimers by the metalloproteinase ADAMTS13, which is synthesized mainly in hepatic satellite cells, but variable expression was observed in other cell type, including endothelial cells and platelets.Citation9
Although the previous studies reported that there is increase in VWF; the reports about the ADAMATS13 activities were controversial. So, the aim of this study is to assess the ADAMTS13 activity in parallel with the VWF antigen levels.
Patient inclusion
The study was conducted at the Departments of Laboratory Hematology and Obstetric Medicine, Mansoura University, Hospital, Egypt. A cross-sectional study was designed to include three women groups which are matched in age, gestational age, and include: (1) non-pregnant women (n = 10); (2) normal pregnancies (n = 50); (3) patients with PE (n = 110). PE was diagnosed in the presence of hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg on at least two occasions, 4 hours to 1 week apart, after the 20th week of gestation), and proteinuria (≥300 mg in a 24-hour urine collection, or one dipstick measurement ≥2+). Severe PE was defined as either severe hypertension (diastolic blood pressure ≥110 mmHg) and mild proteinuria or mild hypertension and severe proteinuria (a 24-hour urine sample containing 3.5 g protein or urine specimen ≥3+ protein by dipstick measurement). Patients were considered to have a normal pregnancy if they did not have any obstetrical, medical, or surgical complication of pregnancy, and delivered a term (≥37 weeks) neonate with a birth weight above the 10th percentile for gestational age.Citation10 Exclusion criteria were multifetal gestation, chronic hypertension, diabetes mellitus, autoimmune disease, angiopathy, renal disorder, maternal or fetal infection, and fetal congenital anomaly. This study was approved by committee of medical Ethics at Mansoura University Hospital, and written informed consent was obtained from each included woman.
Laboratory methods
Blood sample
Venous blood was collected from all subjects at the time of enrollment (time of PE diagnosis for cases and matched gestational age for controls), before any treatment, into a 1:10 final volume of 3.8% sodium citrate in vacutainer. Platelet-poor plasma was obtained by centrifugation at 3000 × g for 10 minutes. The aliquots of plasma samples were stored at −80°C until testing.
Determination of plasma ADAMTS13 activity
The fluorigenic substrate, FRETS-VWF73, was purchased from Peptides International (Louisville, KY, USA) and applied for the determination of ADAMTS13 enzyme activity according to the protocol provided by the supplier with minor modifications.Citation11 In brief, the test citrated plasma were diluted 1:20 in assay buffer (5 mM Bis-Tris, 25 mM CaCl2, 0.005% Tween 20, pH 6.0) and mixed with 5 µM FRETS-VWF73 substrate solution (20 µl each), in white 384-well plates. Fluorescence was measured at 37°C every 2 minutes for 1 hour in Chameleon microplate reader (Hidex, Turku, Finland) equipped with a 340-nm excitation and a 460-nm emission filter. The reaction rate was calculated by linear regression analysis of fluorescence over time. A two-fold dilution series of normal human plasma (mixed from citrated plasma samples of 10 healthy blood donors) was applied as standard curve, 100% ADAMTS13 activity was set at the reaction rate observed in the 1:20 diluted sample. The intra-assay coefficient of variation (CV) was <6%, the inter-assay CV was 7–10% (measured at 60 and 100% activity levels). Pooled citrated plasma of 10 healthy blood donors was used as reference.
Determination of plasma VWF levels
Plasma VWF antigen and VWF ristocetin cofactor (VWF:RCo) were measured by an immunoturbometric assay using the STA®-Liatest®VWF:Ag kit (Diagnostica Stago, Asnieres, France) and coagulation tests were performed on a Siemens Behring Coagulation Analyzer (BCS, Dade Behring, Marburg, Germany). Results are expressed as percentages of a standard composed of pooled human plasma of 10 healthy bloods. The CV was 13%.
Statistical analysis
The statistical analysis of data were done by using excel program and SPSS version 16 (statistical package for social science). The data were found to be not of normal distribution except for patients’ age and gestational age; therefore, non-parametric statistics was carried out. Qualitative data were described in the form of numbers and percentages. Quantitative data were described in the form of median and range. Statistical analysis were done by comparison between groups using χ2 test regarding qualitative data while quantitative nonparametric data comparison were performed using one-way analysis of variance and paired sample t-test. Multivariate analysis was performed according to forward stepwise model selection procedure. The odds ratios (OR) and their 95% confidence interval were calculated using the lowest tertile of the control group as the reference for VWF Ag and the highest tertile of this group as the reference for ADAMTS13 levels. Adjustments were done for VWF Ag or ADAMTS13 activity and further for PE risk factors (pregestational body mass index >30 kg/m2, nulliparity, multiple pregnancy, personal history of thrombosis, connective tissue disease, or antiphospholipid antibody syndrome). The relationship between the studied variables and PE or complications (occurrence of FGR <10th percentile (z score = −1.28) and preterm delivery before 37 weeks gestation (WG)) were determined using unconditional logistic regression analysis after dividing the levels into tertiles based on their distribution in the case group. The probability of being by chance (P value) was calculated for all parameters (P is significant if ≤0.05 at confidence interval 95%).
Results
Patients’ characteristics are shown in . All patients developed PE before 32 weeks gestational age. The mean maternal ages were not significantly different in the three studied groups (P > 0.05). Also there is no significant differences in the gestational age in the PE and normal pregnancy groups (P > 0.05).
Table 1. Demographic, obstetrical, and medical characteristics of the study population
There was significant difference in plasma ADAMTS13 activity between the PE and the healthy pregnant and non-pregnant groups (median [range] 80.8 [60.5–90.8] % then 110.0 [80.6–140.2]% and 130.1 [110.5–150.4]%, respectively (P < 0.01) (). Furthermore plasma VWF:Ag levels were significantly higher in PE patients than in healthy pregnant and nonpregnant women (median) [range] of 360.1 [240.6–570.3]% versus 190.2 [150.1–210.8]%, and 110.3 [100.1.2–120.3]%, respectively(P < 0.001).
The VWF RCO were significantly higher in PE patients than in healthy pregnant and non-pregnant women (median) [range] of 295.1 [166.4–398.5]% versus 169.3 [140.2–220.8]% and 145.0 [120.6–180.3]%, respectively (P < 0.001) ().
Table 2. VWF antigen levels, VWF RCO levels, and ADAMTS13 activity in different studied groups
ADAMTS13 activity was significantly lower in severe PE subgroup as compared with that in milder one. On the other hand, VWF antigen levels and VWF RCO were significantly elevated in severe PE subgroups as compared with that in milder PE subgroup (P < 0.01) ().
Table 3. VWF antigen level, VWF RCO levels, and ADAMTS13 activity in mild versus severe PE subgroups
The results in this study show that individuals with the lowest levels of ADAMTS13 (activity ≤70%) had a significantly increased risk of PE, independent of VWF:Ag levels. Further adjustments for PE risk factors did not modify these results ().
Table 4. Odds ratio for risk of PE for ADAMTS13 activity and VWF antigen
Discussion
VWF antigen levels were significantly higher in patients with PE as compared with normal pregnancy and non-pregnant women. Similar findings were reported by Molvarec et al.Citation9 Moreover, Analía et al.Citation12 found that the VWF increased markedly from non-pregnant values up to the end of early puerperium. In addition, Hulstein et al.Citation13 reported that Active VWF levels were increased 1.6-fold in patients with preeclampsia compared with healthy pregnant volunteers.
ADAMTS13 activity was significantly reduced in PE group as compared to healthy pregnant and non-pregnant women. This result was in parallel with the results reported by Stepanian et al.Citation14 who stated that the lowest levels of ADAMTS13 activity ≤70% or antigen ≤592 ng/ml, were significantly associated with preeclampsia. But it is not in accordance with the findings of Molvarec et al.Citation9 who mentioned that there were no significant differences in plasma ADAMTS13 activity between the PE, and healthy pregnant and non-pregnant groups.
The presence of increased amounts of active VWF in patients with PE can probably be ascribed to the decrease levels of ADAMTS13 activity. Owing to the decrease of ADAMTS13 activity, ultralarge VWF multimers, which are biologically more active, circulate in patients with PE. The activity of ADAMTS13 was found to be decreased in patients with PE when compared with healthy pregnant controls. These data indicate that reduction in the levels of ADAMTS13 may play a role in the onset of PE.Citation14
It has been suggested that preeclampsia is caused by extensive endothelial cell activation, which is part of an increased maternal inflammatory response when compared with normal pregnancy.Citation15 Endothelial cell activation has previously been reported to result in increased levels of soluble thrombomodulin, E-selectin, and VWF.Citation16 In this study we found increased VWF antigen levels in patients with pre-eclampsia, confirming the endothelial cell activation reported by other groups.Citation5,Citation17
In this cross-sectional study, we showed an independent association between ADAMTS13 plasma levels and PE. Decreased levels of ADAMTS13 activity (≤70%) or Ag (≤350 ng/ml) were significantly associated with PE after adjustment for VWF:Ag levels and for PE risk factors. This finding is in agreement with the study done by Stepanian et al.Citation14 On the other hand, our results are not in accordance with the study by Molvarec et al.,Citation9 who did not find any association between ADAMTS13 and PE. Several points may explain this discrepancy: first, the larger number of patients of our study (110 versus 67); second, the matching criteria between cases and controls in the current study; and third, an earlier gestational age at enrollment in our study (32 versus 38 weeks).Citation14
In conclusion, plasma ADAMTS13 activity is reduced in preeclampsia in parallel with increased VWF:Ag levels. However, further studies are needed to determine whether a decrease in plasma ADAMTS13 activity could predispose PE patients.
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