A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis.

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.

Antiphospholipid Antibodies and Infection: Non Nova Sed Nove.

The clinical significance of antiphospholipid antibodies (aPL) in the context of infections has attracted attention since their first discovery in patients with syphilis. In fact, the recognition of aPL in patients with infections has been described in parallel to the understating of the syndrome. Since the first description of aPL-positive tests in three patients with COVID-19 diagnosed in January 2020 in Wuhan, China, a large number of studies took part in the ongoing debate on SARS-2-Cov 2 induced coagulopathy, and many following reports speculated a potential role for aPL. In order to get further insights on the effective role of detectable aPL in the pro-thrombotic status observed in COVID-19 patients, we performed an observational age-sex controlled study to compare the aPL profile of hospitalized patients with COVID with those observed in a) patients with thrombotic APS and b) patients with cultural/serologically-proved infections. Our data showed positive aPL testing in about half of the patients (53%) with COVID-19 and patients with other viral/bacterial infections (49%). However, aPL profile was different when comparing patients with overt APS and patients with aPL detected in the contest of infections. Caution is therefore required in the interpretation and generalization of the role of aPL s in the management of patients with COVID-19. Before introducing aPL testing as a part of the routine testing in patients with COVID-19, larger well-designed clinical studies are required. While the pro-thrombotic status in patients with COVID-19 is now unquestionable, different mechanisms other than aPL should be further investigated.

Antiphospholipid antibodies and risk of post-COVID-19 vaccination thrombophilia: The straw that breaks the camel's back?

Antiphospholipid antibodies (aPLs), present in 1-5 % of healthy individuals, are associated with the risk of antiphospholipid syndrome (APS), which is the most common form of acquired thrombophilia. APLs may appear following infections or vaccinations and have been reported in patients with COronaVIrus Disease-2019 (COVID-19). However, their association with COVID-19 vaccination is unclear. Notably, a few cases of thrombocytopenia and thrombotic events resembling APS have been reported to develop in recipients of either adenoviral vector- or mRNA-based COVID-19 vaccines. The aim of this review is therefore to speculate on the plausible role of aPLs in the pathogenesis of these rare adverse events. Adenoviral vector-based vaccines can bind platelets and induce their destruction in the reticuloendothelial organs. Liposomal mRNA-based vaccines may instead favour activation of coagulation factors and confer a pro-thrombotic phenotype to endothelial cells and platelets. Furthermore, both formulations may trigger a type I interferon response associated with the generation of aPLs. In turn, aPLs may lead to aberrant activation of the immune response with participation of innate immune cells, cytokines and the complement cascade. NETosis, monocyte recruitment and cytokine release may further support endothelial dysfunction and promote platelet aggregation. These considerations suggest that aPLs may represent a risk factor for thrombotic events following COVID-19 vaccination, and deserve further investigations.

Anticardiolipin and other antiphospholipid antibodies in critically ill COVID-19 positive and negative patients.

BACKGROUND: Reports of severe COVID-19 being associated with thrombosis, antiphospholipid antibodies (APLA), and antiphospholipid syndrome have yielded disparate conclusions. Studies comparing patients with COVID-19 with contemporaneous controls of similar severity are lacking. METHODS: 22 COVID-19+ and 20 COVID-19- patients with respiratory failure admitted to intensive care were studied longitudinally. Demographic and clinical data were obtained from the day of admission. APLA testing included anticardiolipin (aCL), anti-ß2glycoprotien 1 (ß2GP1), antidomain 1 ß2GP1 and antiphosphatidyl serine/prothrombin complex. Antinuclear antibodies (ANAs) were detected by immunofluorescence and antibodies to cytokines by a commercially available multiplexed array. Analysis of variance was used for continuous variables and Fisher's exact test was used for categorical variables with α=0.05 and the false discovery rate at q=0.05. RESULTS: APLAs were predominantly IgG aCL (48%), followed by IgM (21%) in all patients, with a tendency towards higher frequency among the COVID-19+. aCL was not associated with surrogate markers of thrombosis but IgG aCL was strongly associated with worse disease severity and higher ANA titres regardless of COVID-19 status. An association between aCL and anticytokine autoantibodies tended to be higher among the COVID-19+. CONCLUSIONS: Positive APLA serology was associated with more severe disease regardless of COVID-19 status. TRIAL REGISTRATION NUMBER: NCT04747782.

Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome.

Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-ß2GPI), and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-ß2GPI antibodies was not reported. Objective: To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-ß2GPI antibodies. Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events. Results: Anti-ß2GPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM was detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of ß2GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-ß2GPI nor with thrombosis. Conclusions: aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against ß2GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.

Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19.

Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti-ß2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer's threshold and in 30% using a more stringent cutoff (≥40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.

Entangling COVID-19 associated thrombosis into a secondary antiphospholipid antibody syndrome: Diagnostic and therapeutic perspectives (Review).

The severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) is a novel ß coronavirus that is the etiological agent of the pandemic coronavirus disease 2019 (COVID­19) that at the time of writing (June 16, 2020) has infected almost 6 million people with some 450,000 deaths. These numbers are still rising daily. Most (some 80%) cases of COVID­19 infection are asymptomatic, a substantial number of cases (15%) require hospitalization and an additional fraction of patients (5%) need recovery in intensive care units. Mortality for COVID­19 infection appears to occur globally between 0.1 and 0.5% of infected patients although the frequency of lethality is significantly augmented in the elderly and in patients with other comorbidities. The development of acute respiratory distress syndrome and episodes of thromboembolism that may lead to disseminated intravascular coagulation (DIC) represent the primary causes of lethality during COVID­19 infection. Increasing evidence suggests that thrombotic diathesis is due to multiple derangements of the coagulation system including marked elevation of D­dimer that correlate negatively with survival. We propose here that the thromboembolic events and eventually the development of DIC provoked by SARS­CoV­2 infection may represent a secondary anti­phospholipid antibody syndrome (APS). We will apply both Baconian inductivism and Cartesian deductivism to prove that secondary APS is likely responsible for coagulopathy during the course of COVID­19 infection. Diagnostic and therapeutic implications of this are also discussed.

Concomitant new diagnosis of systemic lupus erythematosus and COVID-19 with possible antiphospholipid syndrome. Just a coincidence? A case report and review of intertwining pathophysiology.

In the midst of the COVID-19 pandemic, further understanding of its complications points towards dysregulated immune response as a major component. Systemic lupus erythematosus (SLE) is also a disease of immune dysregulation leading to multisystem compromise. We present a case of new-onset SLE concomitantly with COVID-19 and development of antiphospholipid antibodies. An 18-year-old female that presented with hemodynamic collapse and respiratory failure, progressed to cardiac arrest, and had a pericardial tamponade drained. She then progressed to severe acute respiratory distress syndrome, severe ventricular dysfunction, and worsening renal function with proteinuria and hematuria. Further studies showed bilateral pleural effusions, positive antinuclear and antidouble-stranded DNA antibodies, lupus anticoagulant, and anticardiolipin B. C3 and C4 levels were low. SARS-Cov-2 PCR was positive after 2 negative tests. She also developed multiple deep venous thrombosis, in the setting of positive antiphospholipid antibodies and lupus anticoagulant. In terms of pathophysiology, COVID-19 is believed to cause a dysregulated cytokine response which could potentially be exacerbated by the shift in Th1 to Th2 response seen in SLE. Also, it is well documented that viral infections are an environmental factor that contributes to the development of autoimmunity; however, COVID-19 is a new entity, and it is not known if it could trigger autoimmune conditions. Additionally, it is possible that SARS-CoV-2, as it happens with other viruses, might lead to the formation of antiphospholipid antibodies, potentially contributing to the increased rates of thrombosis seen in COVID-19.

Antiphospholipid antibodies in patients with COVID-19: A relevant observation?

BACKGROUND: High incidence of thrombosis in COVID-19 patients indicates a hypercoagulable state. Hence, exploring the involvement of antiphospholipid antibodies (aPL) in these patients is of interest. OBJECTIVES: To illustrate the incidence of criteria (lupus anticoagulant [LAC], anticardiolipin [aCL] immunoglobulin G [IgG]/IgM, antibeta2-glycoprotein I antibodies [aß2GPI] IgG/IgM) and noncriteria (anti-phosphatidyl serine/prothrombin [aPS/PT], aCL, and aß2GPI IgA) aPL in a consecutive cohort of critically ill SARS-CoV-2 patients, their association with thrombosis, antibody profile and titers of aPL. PATIENTS/METHODS: Thirty-one consecutive confirmed COVID-19 patients admitted to the intensive care unit were included. aPL were measured at one time point, with part of the aPL-positive patients retested after 1 month. RESULTS: Sixteen patients were single LAC-positive, two triple-positive, one double-positive, one single aCL, and three aCL IgG and LAC positive. Seven of nine thrombotic patients had at least one aPL. Sixteen of 22 patients without thrombosis were aPL positive, amongst them two triple positives. Nine of 10 retested LAC-positive patients were negative on a second occasion, as well as the double-positive patient. Seven patients were aPS/PT-positive associated to LAC. Three patients were aCL and aß2GPI IgA-positive. CONCLUSION: Our observations support the frequent single LAC positivity during (acute phase) observed in COVID-19 infection; however, not clearly related to thrombotic complications. Triple aPL positivity and high aCL/aß2GPI titers are rare. Repeat testing suggests aPL to be mostly transient. Further studies and international registration of aPL should improve understanding the role of aPL in thrombotic COVID-19 patients.

Revisiting hydroxychloroquine and chloroquine for patients with chronic immunity-mediated inflammatory rheumatic diseases.

Hydroxychloroquine and chloroquine, also known as antimalarial drugs, are widely used in the treatment of rheumatic diseases and have recently become the focus of attention because of the ongoing COVID-19 pandemic. Rheumatologists have been using antimalarials to manage patients with chronic immune-mediated inflammatory rheumatic diseases for decades. It is an appropriate time to review their immunomodulatory and anti-inflammatory mechanisms impact on disease activity and survival of systemic lupus erythematosus patient, including antiplatelet effect, metabolic and lipid benefits. We also discuss possible adverse effects, adding a practical and comprehensive approach to monitoring rheumatic patients during treatment with these drugs.

Clinically significant anticardiolipin antibodies associated with COVID-19.

The novel coronavirus strain known as SARS-CoV-2 has rapidly spread around the world creating distinct challenges to the healthcare workforce. Coagulopathy contributing to significant morbidity in critically ill patients with SARS-CoV-2 has now been well documented. We discuss two cases selected from patients requiring critical care in April 2020 in New York City with a unique clinical course. Both cases reveal significant thrombotic events noted on imaging during their hospital course. Obtaining serial inflammatory markers in conjunction with anti-phospholipid antibody testing revealed clinically significant Antiphospholipid syndrome (APS). This case series reviews the details preceding APS observed in SARS-CoV-2 and aims to report findings that could potentially further our understanding of the disease.