Clinical significance of ADAMTS-13/vWF axis in pregnant women at different trimesters of gestation

Introduction / Введение

The hemostasis system being one of essential arms in hemostasis in health and disease of pregnancy has always been of great interest. Оwing to the advances in molecular biology occurred over the last decades, it was discovered that zinc-containing metalloproteinase ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) and von Willebrand factor (vWF) play an extremely important role in hemostasis [1–3].

Von Willebrand factor is a plasma glycoprotein produced by the endothelium [4] exerting multiple functions in the human body primarily resulting in platelet recruitment and binding [5]. Microscopic studies showed that in the absence of endothelial damage, vWF in a folded form functions to preventing platelet adhesion. When a blood vessel becomes damaged, vWF “spreads out” and attracts ADAMTS-13 metalloproteinase that cleaves ultra-large vWF multimers [6]. Upon a large area of endothelial damage, a massive vWF release from Weibel–Palade bodies occurs leading to the so-called "relative" vWF-protease deficiency. ADAMTS-13 deficiency results in accumulated ultra-large vWF molecules and platelet adhesion followed by platelet consumption and microvasculature thrombosis [1][5].

Normal pregnancy is characterized by developing hypercoagulant state. In recent years, due to the COVID-19 pandemic, it has been found that endothelial damage in hyperinflammation occurs along with the ADAMTS-13/vWF system dysregulation [7] resulting in the microcirculatory disorders and leading to multiple organ failure. Preeclampsia (PE) is one of the prominent cases of the endotheliopathy developing during pregnancy. Patients with PE were found to have substantially altered pro- and anticoagulant pathways, which result in developing the super-hypercoagulable state [8][9].

Aim: to assess the functioning of the ADAMTS-13/vWF axis during physiological pregnancy.

Materials and Methods / Материалы и методы

Study design / Дизайн исследования

At the clinical facilities of the Perinatal Center of Vorokhobov City Clinical Hospital № 67 and "Medical Women's Center" LLC, there was conducted a controlled non-randomized study enrolling 193 patients – 148 pregnant and 45 non-pregnant women.

Inclusion and exclusion criteria / Критерии включения и исключения

Inclusion criteria for main group: age over 18 years; healthy pregnant women with a physiological pregnancy at the I, II and III trimesters; written voluntary informed consent to participate in the study

Inclusion criteria for control group: age over 18 years; no pregnancy confirmed; written voluntary informed consent to participate in the study.

Exclusion criteria: convalescent women who had acute respiratory infections (ARI) and bacterial infection during pregnancy; with PE history; PE developed during pregnancy; unsatisfactory results of the first screening; positive glucose tolerance test; confirmed positive test for antibodies against human immunodeficiency virus, markers of viral hepatitis B and C and syphilis; history of malignant neoplasms, somatic pathology; preterm delivery; refusal to participate in the study.

Study methods / Методы исследования

Peripheral blood samples (volume: 9 ml) were collected from pregnant females (main group) at three time points: at first screening (gestational age: from 11 to 13 weeks), during oral glucose tolerance test (gestational age: from 24 weeks to 26 weeks 6 days), and at first period of labor. In healthy non-pregnant women (control group), peri- pheral blood samples (volume: 9 ml) were collected at a single time point. Peripheral blood sampling was performed with a dry sterile needle from the cubital vein into a plastic tube added with an anticoagulant (sodium citrate solution 3.8 %), at 9:1 ratio. Whole blood was centrifuged at 2,500 g for 20 min at 20–24 °C followed by storage at –80 °C. Plasma level of ADAMTS-13 inhibitor (ADAMTS-13:i), ADAMTS-13 antigen (ADAMTS-13:Ag), vWF antigen (vWF:Ag) and ADAMTS-13 activity (ADAMTS-13:Ac) was assessed using commercially available test kits Technoclon Herstellung von Diagnostika und Arzneimitteln Gmb (TECHNOZYM^®, Austria) followed by calculating the ADAMTS-13:Ac/vWF:Ag ratio. Normal refe- rence values were as follows: for ADAMTS-13:i < 15 U/ml, ADAMTS-13:Ag – 0.41–1.41 U/ml, vWF:Ag – 0.5–1.5 IU/ml (50–150 %) and activity ADAMTS-13:Ac – 0.4–1.3 IU/ml. Magnitude of the analyzed parameters in pregnant women at the I, II and III trimesters was compared with that of in non-pregnant women.

Ethical aspects / Этические аспекты

The study was approved by the Local Ethics Committee at the Sechenov University, Protocol No. 03-23 dated of February 16, 2023. All participants were informed about the study nature and the inclusion criteria as well as provided a signed voluntary informed consent and received relevant comprehensive information. The study was conducted in accordance with the ethical requirements of the Declaration of Helsinki of the World Medical Association

Statistical analysis / Статистический анализ

Statistical data processing retrieved from Microsoft Office Excel 2021 spreadsheets (Microsoft, USA) was performed using the Jamovi program, version 2.3.22 (The jamovi project, Australia). Statistical analysis included the calculation of descriptive statistics: mean (M), standard deviation (SD), median (Me), limit of the 95 % confidence interval (95 % CI). The Mann–Whitney U-test was used to compare inter-group parameters. Fisher's exact test was used to assess a significance for examined parameters: p ≤ 0.05 indicated the presence of significant differences, p > 0.05 – no differences.

Results and Discussion / Результаты и обсуждение

Patient groups / Группы обследованных

At the initial stage, there were enrolled 148 pregnant women. However, during the study, the following subjects were excluded: at trimester I – 7 pregnant women due to ARI and 11 due to unsatisfactory results of the first screening; at trimester II – 6 pregnant women due to ARI, 1 due to acute pyelonephritis and 4 due to a positive oral glucose tolerance test; at trimester III – 10 pregnant women due to ARI, 2 due to preterm delivery, 3 due to early PE, 19 due to late PE and 41 with physiological pregnancy due to delivery occurred in other medical facilities (Fig. 1).

The main group included 44 women with physiologically occurring pregnancies at the I, II and III trimesters, the control group – 45 healthy non-pregnant women planning pregnancy.

Clinical and anamnestic characteristics / Клинико-анамнестическая характеристика

Clinical and anamnestic examination data of study participants as well as perinatal outcomes are presented in Table 1.

The examined women did not differ in age, somatic status and body mass index, among them there were no cases of perinatal mortality. History of pregnancy loss before the week 10 in the main group was in 6 out of 44 (13.64 %), in the control group – in 4 out of 45 (8.89 %). A history of pregnancy loss before week 22 was observed only in the main group in 1 out of 44 (2.27%), its cause was premature rupture of the membranes. In the current pregnancy, vaginal delivery occurred in 33 out of 44 (75.0%) patients with physiological pregnancy at trimester III; delivery by caesarean section was performed in 11 of 44 (25.0 %), the delivery period ranged from 37 weeks to 41 weeks 2 days.

Laboratory results / Результаты лабораторных исследований

Table 2 presents the results of laboratory tests of the women examined.

The examined pregnant women showed statistical differences in ADAMTS-13:Ac magnitude at trimesters II and III compared with control group (p < 0.01) indicating that, normally, along with higher gestation period, ADAMTS-13 activity gradually declines. At the same time, at trimester I, it almost matched the level in non-pregnant women (Fig. 2).

While assessing ADAMTS-13:Ag (Fig. 3), significant differences were observed solely between main group at trimester III and the control group (p < 0.01).

ADAMTS-13:i values in main group were as follows: at trimester I – 1.268 ± 0.697 U/ml, trimester II – 1.417 ± 0.733 U/ml, and trimester III – 2.413 ± 1.659 U/ml. ADAMTS-13:i values in main group at trimester III vs. control group (1.486 ± 0.799 U/ml) differed significantly (p = 0.025), whereas ADAMTS-13:i magnitude was within the reference range (Fig. 4 ).

Significant differences in vWF:Ag level were noted between pregnant women in main group at trimesters II and III (p < 0.01), but not at trimester I vs. control group (Fig. 5).

As shown by many studies, vWF tends to increase in parallel with gestational age [10][11]. Other studies showing that vWF levels markedly increase in patients with PE compared with physiological pregnancy at trimester III [12][13]. However, some researchers reject such data and demonstrate no differences between these groups of patients [14]. Regarding ADAMTS-13, it is much more complicated; no global consensus about altered ADAMTS-13 activity during pregnancy was reached. Some studies demonstrate that its decline begins from trimester II of physiological pregnancy [15][16]; others state that it remains unaltered compared with that in physiological pregnancy and non-pregnant women [17]. Our study clearly demonstrates that along with increasing gestation age, ADAMTS-13 activity decreases, whereas vWF level steadily elevates. Most likely, it is due to the massive vWF release resulting in its consumption.

Assessing ADAMTS-13:Aс/vWF:Ag axis revealed significant differences (p < 0.01) between main group at trimesters II and III vs. control group (Fig. 6).

A decline in the ADAMTS-13:Ac/vWF:Ag ratio in COVID-19 demonstrated by numerous studies evidence about development of progressive endotheliopathy and several times precipitates a risk of thrombosis including thrombotic microangiopathy [18–20].

Conclusion / Заключение

Our study provides a novel insight into ADAMTS-13:Ac/vWF:Ag axis in women with normal pregnancies at trimesters I, II, III. The COVID-19 pandemic uncovered that decline in the ADAMTS-13:Ac/vWF:Ag axis was one of the most important severity predictors that also resulted in elevated risk of thrombotic complications, being the main marker of endotheliopathy. Moreover, it was also elucidated that higher gestation age is paralleled with lowered ADAMTS-13:Aс level along with rise in vWF:Ag that might evidence about developing endotheliopathy. However, due to the small single-center sample, further studies with a larger patient cohorts are required.