Clinical significance of assessing ADAMTS-13 and von Willebrand factor level in COVID-19 convalescent pregnant women

Introduction / Введение

The coronavirus disease 2019 (COVID-19) remains to be a global healthcare emergency. Although the disease was initially thought to be limited to the respiratory tract, soon it became clear that it was presented as a multisystem disease causing coagulopathy, kidney fai- lure, liver dysfunction, and heart failure [1]. The severe course of the disease is more common in the elderly and in comorbidity with chronic diseases such as diabetes mellitus, hypertension, chronic obstructive pulmonary disease, coronary heart disease, and chronic kidney disease [2]. SARS-CoV-2 infection is associated with abundant thrombotic complications, such as deep vein thrombosis and pulmonary embolism [3]. As a rule, a severe course of SARS-CoV-2 infection is observed upon strong inflammatory reaction due to neutrophil release and infiltration in various organs coupled to neutrophil extracellular traps (NETs), increased plasma levels of pro-inflammatory cytokines and chemokines, leading to cytokine storm, massive damage to the endothelium as well as activation of macrophages, platelets, and endothelial cells [4][5]. One of the leading causes of death in COVID-19-patients is thrombo-inflammatory concomitant diseases, such as hypercoagulability, thrombosis, and respiratory failure due to pulmonary microvascular thrombosis [6][7].

A spectrum of clinical manifestations widely varies from asymptomatic to severe in pregnant women with COVID-19 presented from mild, common cold-like to severe symptoms with thrombotic complications [8]. It is known that SARS-CoV-2 infection during pregnancy can be an additional trigger for severe thrombotic complications [9] because pregnancy serves as a preexisting physiological hypercoagulable state. Pathophysiological phenomena underlying the increased risk of obstetric complications are mainly presented by cytokine storm and activation of circulating cells such as macrophages, T-lymphocytes, and endothelial cells. SARS-CoV-2 promotes the development of endotheliitis in various organs and tissues, leading to endothelial damage [10] and, in turn, to thrombotic microangiopathy (TMA), which is related to direst consequences of infection [11]. An important etiological factor leading to TMA is the ADAMTS-13 metalloproteinase (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency. ADAMTS-13 belongs to the family of peptidase enzymes whose biological function is to cleave von Willebrand factor (vWF) multimers. Endothelial damage results in excessively released vWF multimers from Weibel–Palade bodies followed by ADAMTS-13 consumption. It is also important to remember that NETs can inhibit ADAMTS-13 activity followed by increased vWF concentration. In turn, excessive accumulation of ultrahigh molecular weight vWF multimers in complex with platelets can cause thrombosis of the microvasculature [12]. Many publications appear in global medical literature on potential role for a relationship between ADAMTS-13 and vWF in assessing acute conditions caused by COVID-19. Usually, the balance between ADAMTS-13 and vWF plays a crucial role in maintaining average circulation in vital organs. These data suggest that acute endothelial cell activation and dysregulation of the normal ADAMTS-13/vWF axis play an essential role in the pathogenesis of underlying COVID-19 immunothrombosis. Our previous study showed that increased vWF:Ag level, decreased concentration of ADAMTS-13 as well as an imbalance in the ADAMTS-13/vWF axis significantly correlate with low survival in severe coronavirus infection [13].

A recent study by H. Fogarty et al. demonstrated that the blood plasma levels of vWF:Ag and factor VIII (FVIII) remain markedly elevated 3 months later in convalescent patients after coronavirus disease compared with healthy control group [14]. Based on these data, a role for sustained endothelial cell activation in post-COVID-19 patients can be suggested. In this study, we set a goal to study clinically significant changes in the ADAMTS-13/vWF axis in pregnant women with previous coronavirus infection.

Aim: to study a clinical role of hemostasis activation particularly ADAMTS-13/vWF axis in pregnant women after COVID-19.

Materials and Мethods / Материалы и методы

Study design / Дизайн исследования

A prospective randomized case-control study involving 135 pregnant women was conducted at the following clinical sites: Maternity hospital No. 4 at Vorokhobov City Clinical Hospital No. 67, Perinatal Center at Vorokhobov City Clinical Hospital No. 67, Agafonov Republican Clini- cal Infectious Diseases Hospital (Republic of Tatarstan).

Patient groups / Группы обследованных

The patients were divided into 3 groups: group 1 – 45 patients with COVID-19 during pregnancy, group 2 included 45 pregnant women with acute COVID-19, control group – 45 healthy pregnant women.

Inclusion and exclusion criteria / Критерии включения и исключения

Inclusion criteria: age over 18; pregnant women diagnosed with COVID-19 during pregnancy (SARS-CoV-2 positive PCR test); singleton pregnancy; voluntary informed consent to participate in the study.

Inclusion criteria for the control group: age over 18; singleton pregnancy; physiological course of pregnancy; voluntary informed consent to participate in the study.

Exclusion criteria: age under 18 years; verified active infectious and inflammatory process; confirmed positive test for antibodies to HIV; markers of viral hepatitis, syphilis; previous ARVI, vaccinated; signs of thrombotic or hemorrhagic syndrome at first examination; refusal to participate in the study.

Study methods / Методы исследования

The patient's peripheral blood samples obtained on the day of hospitalization before treatment were placed in a 3.2 % buffered sodium citrate solution and centrifuged for 20 min at 3000 g, room temperature. Plasma samples were collected and stored at –80 °С. The level of von Willebrand factor antigen (vWF:Ag), ADAMTS-13 antigen (ADAMTS-13:Ag), ADAMTS-13 activity (ADAMTS-13:Aс), ADAMTS-13 inhibitor (ADAMTS-13:i) was assessed by using commercial TECHNOZYM^® test kits (Techno-clone Herstellung von Diagnostika und Arzneimitteln Gmb, Austria). According to the manufacturer, the normal reference ranges were: for ADAMTS-13:Ag – 0.41–1.41 U/mL, for ADAMTS-13:Ac – 0.4–1.3 IU/mL, for ADAMTS-13:i – less than 15 U/ml; for vWF:Ag – 0.5–1.5 IU/ml (50–150 %).

Ethical aspects / Этические аспекты

The study was approved by the Local Ethics Committee at the Sechenov University (Protocol No 04-22, dated of February 16, 2022). All patients participating in the study were informed about the scope of the study and the inclusion of examination data in current study. All patients received written informed consent.

The study was conducted following the ethical standards of the Declaration of Helsinki of the World Medical Association.

Statistical analysis / Статистический анализ

The data obtained were systematized in Microsoft Office Excel 2021 spreadsheets (Microsoft, USA). Statistical data processing was performed using the Jamovi program, version 1.2.5 (The jamovi project, Australia). Statistical analysis included the calculation of descriptive statistics: mean (M), median (Me), standard deviation (SD), and border of the 95 % confidence interval (95 % CI). While comparing quantitative data, the Mann–Whitney test was used. To test the statistical significance, a one-way analysis of variance was used by calculating Fisher's exact test at level less than 0.05 indicating significant differences. The Fisher's "p" value more than 0.05 indicated no differences.

Results and Discussion / Результаты и обсуждение

Clinical and anamnestic data as well as perinatal outcomes of the examined subjects are presented in Table 1.

The groups showed no significant difference in maternal characteristics. Two patients from group 1 and three from group 2 had arterial hypertension; in each group, 2 patients had mild preeclampsia during pregnancy. In case of COVID-19, none of the patients had signs of placental insufficiency and fetal growth retardation. In group 1, the majority of pregnant women had a mild COVID-19 (34/45; 75.5 %), 7/45 – (15.6 %) moderate form, and only 4/45 (8.9 %) – severe form, which did not significantly differ from group 2. Patients with moderate and severe COVID-19 were hospitalized for observation at infectious disease hospital. Severe cases of COVID-19 were not critical. In acute infection, 17 (37.7 %) patients from group 1 and 12 (26.6 %) from group 2 received low molecular weight heparin. No perinatal death was recorded in groups; 13.3 % (6/45) of patients in group 1, 46.6 % (21/45) of group 2, and 15.6 % (7/45) of group 3 had a history of pregnancy loss. It was found that preterm births accounted for 6.7 % (3/45) of observations in group 2. Vaginal delivery occurred in most women (91.1 %; 41/45) in either group (60.0 %; 27/45). Preterm births were observed only in group 2, accounting for 24.4 % (11/45).

Table 2 presents laboratory parameters of the pregnant women examined in the study.

The examined women showed significant differences in the level of vWF:Ag in acute COVID-19 (group 2) vs. control group (p < 0.001) as well as between pregnant women in the post-COVID (group 1) compared to control group (p < 0.001). There were noticeable interindividual differences in vWF:Ag level, which varied from 1.063 to 6.074 IU/ml in convalescents. Accordingly, plasma levels of vWF:Ag above the upper limit in our local reference range (0.5–1.5 IU/ml) were observed in 66.7 % (30/45) of patients with prior COVID-19 (group 1), whereas in this group it was 2.38 IU/ml being significantly higher compared to control group. In the latter, it was higher only in 40.0 % (18/45) of pregnant women, with an average concentrartion of 1.382 IU/ml (Fig. 1). Thus, a high blood plasma level of vWF:Ag in pregnant women after a coronavirus infection indicates ongoing endotheliopathy and continuing endothelial cell activation.

There were no significant differences in assessing ADAMTS-13:Ac among patients with a history of coronavirus infection (group 1) and control group as well as among patients with acute COVID-19 (group 2) vs. control group.

While assessing ADAMTS-13:i concentration, significant differences were observed between group 1 and group 2 vs. control group (Fig. 2): in healthy pregnant women (group 3) ADAMTS-13:i level was 3.144 ± 2.657 U/ml, which is profoundly lower than in group 2 – 7.3920 ± 5.817 U/ml (p < 0.001) and in group 1 – 5.619 ± 3.227 U/ml (p = 0.0002).

Significant differences were found in ADAMTS-13:Ag magnitude between pregnant women in acute COVID-19 (group 2) and healthy pregnant women (p < 0.001); no significant difference was found while comparing post-COVID (group 1) and healthy pregnant women (Fig. 3).

More than half (55.6 %; 25/45) of pregnant women during acute COVID-19 (group 2) experienced an increase in vWF:Ag concentration with simultaneously decreased ADAMTS-13 levels, which most likely resulted from the secondary consumption of ADAMTS-13 metal- loproteinase. Moreover, the vWF:Ag/ADAMTS-13:Ag ratio is significantly higher than in healthy patients (p < 0.001), demonstrating a disturbed vWF/ADAMTS-13 axis and regulation system of normal microcirculation.

In the post-COVID state (group 1), only in 6.7 % (3/45) cases was an increase in vWF:Ag magnitude with simultaneously decreased ADAMTS-13 level. Therefore, ADAMTS-13 is not consumed in such quantities as in acute COVID-19. Thus, the regulatory function of the vWF:Ag/ADAMTS-13:Ag axis remains intact in a significantly high proportion of patients (Fig. 4).

In the acute phase of COVID-19, the disease is hyper- inflammatory, prothrombotic and therefore negatively affect the level of specific thrombo-inflammatory markers, leading to endotheliopathy and increasing susceptibility to thrombotic and microvascular disorders (including microthrombosis and TMA) [15]. Therefore, it is not surprising that the ADAMTS-13/vWF axis may be involved in the TMA and cytokine storm observed during COVID-19 outside pregnancy. In non-pregnant adults, the more the ADAMTS-13/vWF axis is impaired, the more severe the disease course [13]. In this study, most pregnant women with SARS-CoV-2 infection had mild disease. However, regarding fetal and maternal outcomes, we found a significantly higher prevalence of preterm birth during acute COVID-19 (24.4 %). Noteworthy, as E. Grandone et al. pointed out that one of the most critical factors of preterm labor in women with COVID-19 is endotheliopathy, and an imbalance of vWF and ADAMTS-13 can contri- bute to multiorgan thrombosis with clinical TMA picture [16]. We consider extremely important to further elucidate these issues to confirm a role of endotheliopathy and other latent hemostasis disorders in emerging pregnancy complications.

Conclusion / Заключение

Our data provide new insight into the nature of persistent endotheliopathy and imbalance in the ADAMTS-13/vWF axis in post-COVID-19 pregnant women. Functioning of the latter is determined by assessing ADAMTS-13/vWF ratio that also accounts for risk of microcirculatory disorders as well as clinical complications. Thus, determining the ADAMTS-13/vWF ratio is crucial in clinical practice. Consistent with the critical role of immunothrombosis in acute COVID-19, our results support the hypothesis that persistent endotheliopathy and hemostatic dysfunction are sustained after COVID-19 during pregnancy, which is clinically relevant for the management of such patients. Further studies on larger patient cohort with longer follow-ups are required to confirm our data to open up new possibilities in treating post-COVID conditions.