Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new β-coronavirus strain identified for the first time in Wuhan, China, on late December 2019. On February 2020, the World Health Organization (WHO) named the infected disease “coronavirus disease 2019” (COVID-19) .
SARS-CoV-2 primarily spread from person to person via mucosae or conjunctiva after close contact with infected droplets, and respiratory and gastrointestinal systems epithelial cells are the main infection targets. First observations on disease onset resulted in progressive respiratory failure owing to alveolar damage, and classical symptoms included fever, cough, sore throat, malaise and myalgias. Nevertheless, later on, many reports also described gastrointestinal symptoms, including anorexia, nausea and diarrhea . Recently, evidences of cardiovascular and cerebrovascular systems involvement have been shown .
In a second time, in COVID-19 a high prevalence of coagulation abnormalities and thrombotic complications has been found, such as: Deep Vein Thrombosis (DVT), Pulmonary Embolism (PE), Venous Thromboembolism (VTE) and Disseminated Intravascular Coagulation (DIC) [4-5].
These clinical manifestations have been called “COVID-19 Associated Coagulopathy” (CAC), corresponding to a general worsening of patient conditions. Indeed, presence of small and mid-sized pulmonary arteries thrombosis and microangiopathy was found in COVID-19 patients, due to markedly low levels of oxygen. Subsequent respiratory failure causes Intensive Care Unit (ICU) admission and mechanical ventilation [6-8].
CAC is characterized by increased levels of routine clinical inflammation markers such as C Reactive Protein (CRP), Erythrocyte Sedimentation Rate (ESR), together with acute inflammatory response (cytokine storm): Interleukin-6 (IL-6), Interleukin-1 (IL-1) and Tumor Necrosis Factor α (TNFα). IL-6 levels are significantly higher in patients with severe conditions [9-10].
Regarding clinical laboratory data, coagulation parameters have been found altered in these patients [9,11], such as: activated partial thromboplastin time (aPTT), antithrombin (AT) and prothrombin time (PT). Many reports evidenced increased D-dimer and fibrinogen levels, involved in the development of thrombotic complications and in poor prognosis [12-13].
The real cause of COVID-19 patients’ coagulopathy has not yet been identified.
We can hypothesize three different pathological ways in CAC pathogenesis: I) SARS-CoV-2 infection in endothelial cells may cause loss of endothelial homeostasis and its physiological anticoagulant activity; II) the systemic inflammatory response and cytokine storm may increase atherosclerotic plaques rupture probability, in patients with previous cardiovascular events history; III) anti-phospholipids antibodies (aPLs) along with the development of circulating immune complexes (CICs) could be involved in thrombosis events.
We focused our study on the last hypothesis.
Anti-phospholipids antibodies positivity clinical manifestations include thrombosis, thrombocytopenia, coagulopathy and pregnancy complications with recurrent spontaneous abortions.
Furthermore, anti-phospholipids antibodies are essential clinical criteria in the Anti-phospholipid Syndrome (APS) diagnosis, a systemic autoimmune disease in which specific laboratory markers, such as: lupus anticoagulant (LAC), IgG and/or IgM anti-cardiolipin (aCL) and IgG and/or IgM anti-β2-glycoprotein-1 (aβ2GP1) antibodies are crucial to the diagnosis. These antibodies in fact represent the most frequent aPLs. IgG/IgM anti-prothrombin (aPT) and IgG/IgM anti-annexin-V are also detected in a minority of cases, especially in clinical APS patients with negative classical anti-phospholipids antibodies. In healthy population, positivity to anti-phospholipids antibodies has been found in about 5% of cases, with an uncertain relationship for increased risk to develop thrombotic events and APS. Complement activation is also required for the full APS clinical manifestation.
In addition, inflammatory responses and related immune dysregulation could trigger the development of circulating immune complexes (CICs), which leads to endothelial cells damage and organ inflammation through their tissue depositing and through complement system activation (C1q, C3), resulting in thrombotic complications. Moreover, macrophages could phagocytose CICs causing a hyperinflammatory response, typical in COVID-19 patients .
Preliminary evidences of a possible correlation between anti-phospholipids antibodies and coagulopathy in COVID-19 patients derives from Zhang et al., who reported 3 cases with thrombosis, aCL and aβ2GP1 positivity only for IgA class . It should be noticed that lack of IgG and/or IgM aCL and aβ2GP1 precludes the possible role of anti-phospholipids antibodies in CAC and furthermore, the analyzed patients had a history of cardiovascular disease episodes, which increased itself the risk of subsequent thrombosis events.
Based on these observations, Harzallah et al. tested LAC on 56 patients, IgG/IgM aCL and aβ2GP1 on 50 patients. LAC positivity was found in 25 patients (45%), whereas IgG and/or IgM aCL and aβ2GP1 were detected only in 5 patients (10%), three of them associated to LAC positivity . Some authors however highlight that high levels of CRP interfere with LAC detection methods .
In another study, Valle et al. reported a prospective observational study in 24 COVID-19 patients, who developed pneumonia and/or venous thromboembolism: 11 patients with PE (45.8%), 9 patients with DTV (37.5%) and 4 patients with VTE (16.6%); also in this cohort only 2 patients (8.3%) showed anti-phospholipids antibodies positivity (IgM aCL and IgM aβ2GP1), suggesting that anti-phospholipids antibodies might not be involved in COVID-19 coagulopathy and thrombosis mechanism .
The aPLs low prevalence was also reported by Borghi et al. on 122 COVID-19 patients . In this cohort IgG/IgA/IgM aβ2GP1 were the most frequent antibodies in 15.6%, 6.6% and 9.0% of patients, respectively; IgG/IgM aCL were detected in 5.7% and 6.6% of patients. Moreover, they detected IgG and IgM anti-phosphatidylserine/prothrombin (aPS/aPT) antibodies in 2.5% and 9.8% of patients, respectively. No association between thrombosis and aPLs was found. Nevertheless, combination of the various aPLs criteria and antibody profiles could be useful to better characterize the risk assessment of thrombotic events in COVID-19 patients .
To our knowledge, anti-prothrombin (aPT), anti-annexin-V antibodies and CICs in COVID-19 patients have not been reported in the scientific literature.
In this perspective, to better assess the anti-phospholipids antibodies role in CAC, we performed a more extended study panel including classical aPLs (IgG/IgM anti-cardiolipin and IgG/IgM anti-β2-glycoprotein-1), supported by other anti-phospholipids antibodies (IgG/IgM anti-prothrombin and IgG/IgM anti-annexin-V). We also detected IgG human circulating immune complexes (CICs) to evaluate the inflammatory status, through a possible complement system activation. These antibodies were assessed on a COVID-19 patients’ cohort compared to a control group (healthcare workers).