Coronavirus Disease 2019: COVID-19 Nov 7, 2020 0:55:56 GMT
Post by Admin on Nov 7, 2020 0:55:56 GMT
Fig. 3 IgG from COVID-19 patients potentiates thrombosis in mice.
(A) Schematic shows thrombus initiation in the inferior vena cava (IVC) of mice by local electrolysis leading to free radical generation and activation of the endothelium. (B, C) Mice were administered IgG from healthy individuals (control), from patients with COVID-19 who had high or low aPS/PT antibodies or from patients with catastrophic antiphospholipid syndrome (CAPS). Just prior to intravenous administration of IgG, mice were subjected to local electrolysis in the inferior vena cava. Thrombus length (B) and weight (C) were determined 24 hours after IgG injection. Scatter plots with individual data points (each point represents a single mouse) are presented. (D) Shown are photographs of representative thrombi from the experiments presented in panels B and C. (E) Serum samples from mice in the experiments presented in panels B and C were tested for NET remnants measured by an ELISA that detected myeloperoxidase (MPO)-DNA complexes. Scatter plots with individual data points (each point represents a single mouse) are presented. OD, optical density. (F) Schematic shows thrombus initiation in the inferior vena cava (IVC) of mice by stenosis that was induced via placement of a fixed suture over a spacer that was subsequently removed. (G, H) Mice were treated intravenously with IgG from a healthy individual (control) or from a patient with COVID-19 with high aPS/PT antibodies. Just prior to intravenous administration of IgG, stenosis was induced. 24 hours later thrombus length (G) and weight (H) were determined. Scatter plots with individual data points (each point represents a single mouse) are presented. (I) Shown are photographs of representative thrombi from the experiments presented in panels G and H. (J) Serum samples from mice in the experiments presented in panels G and H were tested for NET remnants measured by an ELISA that detected MPO-DNA complexes. Scatter plots with individual data points (each point represents a single mouse) are presented. OD, optical density. Horizontal black bars represent the mean. Comparisons were by either one-way ANOVA with correction for multiple comparisons by Dunnett’s method (B, C, E) or unpaired t test (G, H, J): *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Antiphospholipid autoantibodies (aPL antibodies) are a heterogeneous group of antibodies that underlie the pathogenesis of antiphospholipid syndrome via their interactions with phospholipid-binding plasma proteins such as β2GPI, prothrombin, thrombomodulin, plasminogen, antithrombin III, protein C, protein S, annexin II, annexin V, and likely others (22, 41–46). The association between various infections and the induction of aPL antibodies has long been recognized (47–52). For example, one study of 100 cases reported in Medline from 1983 to 2003 found the most commonly reported aPL antibody-associated infections to be skin infections (18%), pneumonia (14%), and urinary tract infections (10%); common pathogens included human immunodeficiency virus (17%), varicella-zoster virus (15%), and hepatitis C virus (13%) (50). Regarding specific aPL antibodies, aCL IgG and IgM (typically lacking anti-β2GPI antibody activity) have been most commonly reported (48, 52–57). The majority of these virus-associated aPL antibodies are thought to be transient (35, 54, 58). Although the clinical implications of transient virus-associated aPL antibodies remain to be fully defined, a recent review of 163 published cases of virus-associated aPL antibodies found thrombotic events in 116 cases (35). Even acknowledging the likelihood of sampling and publication bias, these data (along with the data presented here for individuals with severe COVID-19) suggest that some transient aPL antibodies may still have prothrombotic potential. Whether similar antibodies would be detected in patients with less symptomatic COVID-19 presentation—some of whom do experience thrombotic events—awaits further study.
The most severe presentation of antiphospholipid syndrome is its catastrophic variant, which fortunately impacts only a minority of patients with antiphospholipid syndrome, typically at times of stress such as infection, surgery, or withdrawal of anticoagulants (59). Catastrophic antiphospholipid syndrome involves derangements of both inflammatory and thrombotic pathways and impacts multiple organs in the body simultaneously (59). In the largest series of patients with catastrophic antiphospholipid syndrome assembled, the most commonly affected organs were kidneys (73%), lungs (60%), brain (56%), heart (50%), and skin (47%) (60). Whereas multi-organ failure certainly complicates severe cases of COVID-19, the lungs are typically the most severely affected organ. We speculate that local immune stimulation due to viral infection (including potentially the infection of endothelial cells) could synergize with circulating aPL antibodies and thereby lead to a particularly severe thrombo-inflammatory insult to the lungs of COVID-19 patients.
Many studies from the general thrombosis literature have revealed that activated neutrophils, and in particular NET formation, contribute to the propagation of thrombi affecting arterial, venous, and microscopic vascular beds (61, 62). NETs have also been recently implicated in the pathogenesis of antiphospholipid syndrome. Our group has reported that serum samples from patients with antiphospholipid syndrome, as well as purified aPL antibodies, trigger neutrophils to release NETs (23). The potential in vivo relevance of this observation has been confirmed in mouse models of aPL antibody-mediated large-vein thrombosis in which either depletion of neutrophils or digestion of NETs was protective (38). Neutrophils from patients with antiphospholipid syndrome also appear to have increased adhesive potential, which is dependent upon the activated form of integrin Mac-1. This pro-adhesive phenotype amplifies neutrophil-endothelium interactions, potentiates NET formation, and potentially lowers the threshold for thrombosis (63). Therapies that target NET formation have the potential to treat thrombotic diseases. For example, selective agonism of the adenosine A2A receptor suppresses aPL antibody-mediated NETosis in a protein kinase A-dependent fashion (39). A2A receptor agonism also reduces thrombosis in the inferior vena cava of both control mice and mice treated with aPL antibodies. Dipyridamole, which is known to potentiate adenosine receptor signaling by increasing extracellular concentrations of adenosine and interfering with the breakdown of cAMP, also suppresses aPL antibody-mediated NETosis and mitigates venous thrombosis in mice (64). Interestingly, a small study from China showed that dipyridamole suppressed D-dimer elevation and improved platelet counts in patients with COVID-19 (65). Whereas we have demonstrated here that dipyridamole mitigated NET release mediated by IgG from COVID-19 patients, prospective randomized clinical trials (NCT04391179) are needed to evaluate clinical outcomes among COVID-19 patients treated with dipyridamole (64).
aPL antibodies are defined based on their inclusion in the updated Sapporo classification criteria: namely, aCL IgG and IgM, aβ2GPI IgG and IgM, and lupus anticoagulant (25). Of these, lupus anticoagulant is generally accepted as the best indicator of a high-risk aPL antibody profile (66–71). There are certainly reports of patients with seronegative antiphospholipid syndrome, who have classic features of this disease but have tested negative for traditional aPL antibodies (72). Some non-criteria aPL antibodies discovered in the past 20 years have shown promising clinical utility in identifying antiphospholipid syndrome. Among those are aPS/PT IgG and IgM, as well as the IgA isotypes of aCL and aβ2GPI antibodies. Retrospective studies have suggested that aβ2GPI IgA is associated with thrombosis in lupus patients [odds ratio (OR) 2.8, 95% CI 1.3-6.2] (73). A recent review of 10 retrospective studies (1775 patients with lupus or primary antiphospholipid syndrome and 628 healthy controls) detected a strong association between aPS/PT antibodies and thrombotic events (OR 5.11; 95% CI 4.2-6.3) (74). Furthermore, serological agreement between aPS/PT IgG and IgM and high-risk aPL antibody profiles—especially the presence of lupus anticoagulant—has been demonstrated in a recent study of 95 well-characterized patients with primary antiphospholipid syndrome (75). Whereas the clinical implications of aPS/PT antibodies during viral infection remain to be comprehensively defined, we found here that IgG fractions containing high titers of these antibodies triggered NET release from neutrophils in vitro and accelerated thrombosis in vivo. Notably, IgG purified from COVID-19 patients with low aPS/PT serum titers demonstrated some activity in potentiating thrombosis (although high aPS/PT serum titer IgG fractions provided a more robust response). It is possible that aPL antibodies are but one species of a broader acute natural antibody response that is in fact prothrombotic in COVID-19 disease.
The orchestration of autoimmunity against phospholipids in COVID-19 is likely a complex interplay between genetic predisposition, historical antigen exposures, and a hyperactivated host immune response in the setting of a unique environmental trigger—infection with SARS-CoV-2 (76). It is not surprising that aPL antibodies of the IgM isotype (which are designed for rapid mobilization) predominate in our COVID-19 patient cohort where they correlate with markers of neutrophil activation and NET release. The relationship between aPL antibodies and NETs in COVID-19 is potentially bidirectional. NETs are a known source of autoantigens, and cytokines released in parallel with NETosis may also facilitate NET-associated autoantibody propagation (77–80). An example of a cytokine that could play such a role is B cell activating factor (BAFF), an important mediator of the maturation of B cells into antibody-producing cells (81). For example, neutrophil-derived BAFF likely participates in the production of anti-double-stranded DNA antibodies in lupus (78). In COVID-19, it is possible that production of aPL antibodies potentiates NET formation and BAFF release. This may further enhance the survival and differentiation of phospholipid-reactive B cells, and in some cases class-switching to the IgG isotype. The interplay between COVID-19 and humoral immunity is clearly an area that merits further study.
There are several potential clinical implications of these findings. Patients with catastrophic antiphospholipid syndrome are regularly treated with heparin, corticosteroids, and plasmapheresis (with the latter leading to a demonstrable improvement in outcomes) (82). Whereas both anticoagulation and corticosteroids have shown some promise to date in treatment of COVID-19, plasmapheresis has not been systematically explored. One wonders if this could provide benefit in the subgroup of COVID-19 patients with high titers of aPL antibodies. At the same time, convalescent plasma is receiving increasing attention as an approach to treating severe cases of COVID-19. Defining the extent to which convalescent plasma may contain aPL antibodies or other prothrombotic autoantibodies in addition to protective anti-SARS-CoV-2 antibodies, is another potential area for future investigation.