Circulating Naturally-Occurring Anticoagulants before Treatment and after Recovery from SARS-CoV-2 Infection in Ghana

Background : Disturbance in naturally-occurring anticoagulants may contribute to the hypercoagulable state in COVID-19. This study determined the plasma antigen levels of protein C (PC), protein S (PS), antithrombin-III (AT-III), and thrombomodulin (TM) before treatment and after recovery from COVID-19. Materials and Methods : This cross-sectional study, conducted from February to August 2022 at Kumasi South Hospital, recruited sixty-five RT-PCR-confirmed COVID-19 participants. A venous blood sample was taken for full blood count (FBC) analysis using a 3-part fully automated haematology analyzer, and PC, PS, AT-III, and TM antigen levels measured using ELISA. The data were analyzed using SPSS version 26.0. P<0.05 was considered statistically significant. Results : Severe COVID-19 participants had relatively lower haemoglobin (p<0.001), RBC (p<0.001), HCT% (p<0.001) and platelets (p<0.001), but higher RDW-CV% (p=0.013), WBC (p<0.001), and absolute lymphocyte counts (p<0.001) compared to those with the non-severe form


Introduction
The upsurge in morbidities and mortalities from the novel coronavirus disease 2019 (COVID-19) may be attributed to the associated fatal acute respiratory distress syndrome (ARDS) experienced during disease progression [1]. The COVID-19-causing pathogen, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires angiotensinconverting enzyme-2 (ACE-2) for cell entry and adaptation, primarily in the respiratory tract. The widespread distribution of ACE-2 throughout the body enables SARS-CoV-2 entry into the general circulation and other parts of the body, inducing an inflammatory response and causing life-threatening complications [2,3].
Efficient regulation of the synthesis of the naturally-occurring anticoagulants, protein C (PC), protein S (PS), antithrombin-III (AT-III), and thrombomodulin (TM), mostly by hepatocytes and endothelial cells, is very essential to controlling haemostatic activities; dysregulation of the process triggers thrombosis or haemorrhage [17]. Physiologically, thrombin forms a complex with TM, and the resulting thrombin-TM complex activates the most potent anticoagulant, PC. Activated PC (APC), in the presence of its cofactor PS, through proteolytic digestion, inactivates essential cofactors in the coagulation cascade, activated factor VIII (FVIIIa) and activated factor V (FVa), as well as inactivating the potent anti-fibrinolytic agent, plasminogen activator inhibitor-1 (PAI-1). This eventually controls the rate of fibrin formation to avoid excess thrombi generation [18]. Also, AT-III, enhanced by heparin, inhibits the activities of thrombin to ensure complete regulation of the haemostatic processes [19]. and platelets were counted through the impedance principle. Flow cytometry enabled the counting of white blood cells (WBCs) and their differentials, while haemoglobin (Hb) estimation was achieved through the haemiglobin cyanide method at a wavelength of 540 nm. The procedure for the measurement of the blood cell indices was adopted from the study by Osei-Boakye et al. [33]. Diagnoses and stratification of anaemia among the study participants were based on the protocols described by the Chauhan et al. [34] study. Participants with Hb<11.5 g/dL were considered anaemic, and the severity of the anaemia was categorized into three based on the Hb concentration: mild (10.0-11.4 g/dL), moderate (7.0-9.9 g/dL), and severe (<7 g/dL) as recommended [34].

Plasma PC, PS, AT-III and TM Assay Using ELISA
The sandwich ELISA technique was used to measure the plasma levels of the anticoagulants PC, PS, AT-III, and TM. Commercially prepared ELISA reagents from Biobase, China, were used, and the tests were done according to the manufacturer's recommendations at Ankaase Methodist Hospital Laboratory, Ashanti Region, Ghana. The ELISA plates were splashed by manual technique, and the levels of the anticoagulants were measured with a microplate ELISA detector (Mindray MR-96A, China).

Statistical Analysis
IBM Statistical Package for the Social Sciences (SPSS) version 26.0 (IBM Corp., Armonk, NY, USA) was used to analyze the data. A one-sample Kolmogorov-Smirnov test and Shapiro-Wilk normality test were used to assess the distribution of the data. Descriptive data were presented as frequencies with corresponding percentages. Non-parametric data were presented in medians (25 th -75 th percentiles), while parametric data were in means ± standard deviation. Unpaired data were appropriately compared using the Student's T-test or Mann-Whitney U-test. Paired non-parametric data (on admission and after recovery) were compared with the Wilcoxon signed-rank test, and the paired parametric data were compared with the Paired Sample T-test. Statistical significance was set at p<0.05.

Prevalence and Severity of Anaemia among COVID-19 Patients
The overall prevalence of anaemia among the COVID-19 participants was 58.5% (38/65). Of the 38 anaemic COVID-19 participants, the majority (32/84.2%) had mild anaemia and 6 (15.8%) with moderate anaemia, but none of them had severe anaemia ( Figure 2).  Table 3 shows the plasma levels of the naturally-occurring anti-coagulants based on the COVID-19 severity of the study participants. The median circulating levels of the naturally-occurring anti-coagulants among the COVID-19 participants in the study were: PC (ng/mL): 21 Table 3.

Participants' Blood Cell Indices before Treatment and after Recovery from the SARS-Cov-2 Infection
Most blood cell indices significantly improved after a successful recovery from COVID-19. Hb (g/dL): 12.4 (11.6-13.6) vs 11.4 (8.  Table 4.  Table 5 shows the plasma antigen levels of the naturally-occurring anticoagulants before treatment and after recovery from COVID-19. Following successful recovery from SARS-CoV-2 infection, plasma levels of PC, PS, and AT-III significantly increased compared to the values before therapy commenced: PC (ng/mL): 32 Table 5.

Discussion
Deranged circulating levels of naturally-occurring anticoagulants may contribute to the noticeable thrombosis and deaths occurring during COVID-19 progression. This study determined plasma PC, PS, AT-III, and TM before treatment and after recovery from SARS-CoV-2 infection.
The COVID-19 participants in this study experienced cough, fever, and loss of taste and smell, and this is consistent with similar studies elsewhere [35][36][37]. The clinical manifestations exhibited by the SARS-CoV-2-infected participants could be related to the virus' vigorous interactions with ACE-2 in the host's epithelial cells, especially in the pulmonary region. The interaction consequently triggers a cytokine-mediated physiological response, activating both innate and adaptive immunity involving adrenergic stimulation pathways and enhancing the occurrence of the clinical features [38]. The damaged endothelial cells activate dendritic cells, which then trigger the release of cytokines and subsequent activation of T-cells, causing inflammation and its associated manifestations [39].
The present study agrees with the negative effects of severe COVID-19 on red cell parameters, as this has been described by earlier studies where RBC, HCT, and Hb were reduced in Ghana [40,41] and other parts of the world [4][5][6][7]. The 58.6% prevalence of anaemia among COVID-19 patients recorded in this study is higher than the 19.23% identified in Liaocheng, China [42], 35.6% [43], and 38.2% [44] of hospitalized COVID-19 patients in Wuhan, China, and 42.7% among COVID-19 survivors in a 30-day prospective cohort study during the initial wave of the outbreak in Italy [5]. The prevalence is, however, lower compared to the 62.8% found in a retrospective study in Riyadh, Kingdom of Saudi Arabia [6], 66.7% among COVID-19 non-survivors in Italy [5], and 74.3% from the CURE cohort (from Kaiser Permanente Georgia's (KPGA) electronic medical record (EMR), which used data generated from members of a southeastern integrated healthcare system testing positive for COVID-19 [7]. The anaemia may be due to the COVID-19-associated cytokine storm that influences iron absorption and re-uptake, and inhibits erythropoietin. IL-6 is a crucial controller of inflammation-induced iron dysregulation as it triggers the release of hepcidin, the chief regulator of iron homeostasis. The hormone hepcidin regulates iron efflux by interfering with the only cellular iron exporter, ferroportin 1 (FPN1), leading to cellular iron retention in iron-storage tissues such as macrophages and limiting iron absorption in the duodenum and jejunum [4]. Again, TNF-α and IL-1 inhibit EPO release and stimulation by the peritubular interstitial cells of the renals, and hepatocytes, leading to the downregulation of erythropoiesis [45][46][47].
The studies by Yan et al. [48] and Lippi & Plebani [13] observed an increase in total leucocytes with a corresponding elevation in lymphocytes in severe COVID-19 patients. This observation is linked to the probable overwhelming stimulation of the immune response by inflammatory cytokines during COVID-19 progression, triggering the release of immune cells to keep the virus in check [49]. Findings from the present study agree with the earlier observations [13,48]. Conversely, reduced lymphocyte counts have been identified among severe COVID-19 patients [6,50]. The disparity in the findings may be related to the variations in participants' selection in the studies.
The severe COVID-19 patients in this study had a reduced platelet count, which is similar to an earlier study in Northwest Ethiopia [51]. A similar study by Elderdery et al. [6] observed that 82.5% of individuals infected with SARS-CoV-2 developed thrombocytopaenia. This finding may be due to the suppression of medullary haemopoiesis, and direct viral interactions with megakaryocytes, affecting the primary synthesis of thrombocytes [52,53], SARS-CoV-2 disruption of the endothelium, stimulating platelet activation and aggregation [54], as well as the probable generation of anti-platelet proteins [55].
Plasma antigen levels of naturally-occurring anticoagulants: PC, PS, and AT-III were decreased in severe COVID-19 participants in this study, and this is comparable with similar studies elsewhere [9,10,22,24]. These findings could be due to the enhanced coagulopathy associated with severe COVID-19. To ensure effective haemostasis, activated PC in the presence of its cofactor PS deactivates FVa and FVIIIa to limit the rate of thrombi generation [10,24], and AT-III regulates the activities of the coagulation factor thrombin, which helps to control excessive coagulation [28,56]. Continuous excessive consumption of endogenous anticoagulants depletes their plasma levels, and minimizes the expected inhibition of the coagulation process to regulate the rate of clot formation. Again, the presence of ACE-2 on hepatocytes permits SARS-CoV-2 interaction with the liver cells and eventually suppresses the liver's ability to synthesize the anticoagulants, leading to their low concentrations in the plasma in severe COVID-19 patients [57,58]. Additionally, the Kim and Kim [10] study suggests that activated plasma anticoagulants, such as PC, have a short halflife and rapidly get depleted from circulation during the disease's progression. However, the present study recorded increased circulating levels of TM among the severe COVID-19 participants, and this agrees with the findings from an earlier study in the United States [25]. This could be due to the increased loss of TM from the endothelial cell surface resulting from the excessive disruption of the endothelium by the explosive cytokine release during severe COVID-19 progression [25].
Blood cell indices and the anticoagulants were significantly changed after recovery from COVID-19. The improved erythrocyte parameters after severe COVID-19 participants recovered from the infection identified in this study agree with findings from earlier studies [4,45,46,59]. Effective regulation of iron homeostasis due to the suppression of the inflammatory response and the sufficient release of EPO by the peritubular interstitial cells of the kidneys after recovery from COVID-19 could eventually promote erythropoiesis and increase peripheral numbers of the RBC parameters [4,46]. Conversely, another study in China noticed reduced haemoglobin concentrations among COVID-19 patients posttreatment and associated the anaemia with the probable effect of the drugs [60].
Following successful recovery from the SARS-CoV-2 infection, platelet counts in peripheral blood were restored to normal in the current study. This could be related to a probable drop in the SARS-CoV-2 viral load with a subsequent limited inflammatory response resulting in well-regulated thrombocyte consumption and sufficient hepatic thrombopoietin release [60].
Comparatively, the plasma levels of PC, PS, and AT-III were higher after recovery from COVID-19 than the values at admission. This could be due to the restoration of endogenous synthesis by the liver following the suppression of the inflammatory response. Also, the reduced consumption of anticoagulants after recovery from the SARS-CoV-2 infection, when the rate of coagulation may be physiologically regulated, could account for the findings [24,49]. Additionally, effective endothelial repairs following limited inflammation and suppressed endotheliopathy with a subsequent reduction in the release of TM from the endothelial cells could account for the lower TM levels found after recovery from COVID-19 [25].
This study could not assess the entire haemostatic system of the study participants.

Conclusion
Severely infected COVID-19 patients had higher PC, PS, and AT-III, but lower TM plasma levels. The significant changes in circulating anticoagulants may contribute to the hypercoagulable state of COVID-19. Blood cell indices were negatively affected during COVID-19 disease progression. Complete recovery from the SARS-CoV-2 infection normalised the haematological indices. Assessment of naturally-occurring anticoagulants and the provision of appropriate anticoagulants are recommended in the management of COVID-19 to prevent thrombotic complications. A future study to assess the entire haemostatic system of COVID-19 patients is recommended.

Data Availability Statement
Data presented in the study are available on request from the corresponding author.

Funding
The authors received no financial support for the research, authorship, and/or publication of this article.

Acknowledgements
Authors are grateful to the contributions of the staff of the Department of Biomedical Laboratory Sciences, School of Allied Health Sciences, University for Development Studies, Tamale, Ghana, and the staff of the COVID-19 Laboratory, Kumasi South Regional Hospital, Kumasi, Ghana. We salute all the participants who willingly availed themselves to be part of this study.

Institutional Review Board Statement and Ethical Approval
The Committee on Human Research, Publications and Ethics (CHRPE) of Kwame Nkrumah University of Science and Technology (KNUST), Kumasi (Ref: CHRPE/AP/012/22) provided approval for the study. Permission was obtained from the Management of Kumasi South Regional Hospital, and informed consent was given by the participants.

Informed Consent Statement
Not Applicable.