A new study funded by the U.S. NIH that involved researchers from the University of Michigan-USA and the U.S. Lung and Blood Institute at Bethesda has found that SARS-CoV-2 antiphospholipid antibodies causes endothelial cell activation and dysfunction that often leads to severe blood clotting in COVID-19 patients.
The study findings were published in the peer reviewed journal: Arthritis & Rheumatology. https://onlinelibrary.wiley.com/doi/10.1002/art.42094
The earlier preprint version is available on MedRxiv.
There are several likely synergistic mechanisms by which SARS-CoV-2 infection may result in COVID-19-associated coagulopathy including cytokine release that activates leukocytes, endothelium, and platelets; direct activation of various cells by viral infection; and high levels of intravascular neutrophil extracellular traps (NETs) (1).
The latter are inflammatory cell remnants that amplify thrombosis (2). COVID-19-associated coagulopathy may manifest with thrombosis in venous, arterial, and microvascular circuits. The incidence of venous thromboembolism is particularly notable in severe COVID-19 (10% to 35%) with autopsy series suggesting that as many as 60% of those who succumb to COVID-19 are impacted (3).
Recently, there have been a number of descriptions of what appears to be de novo autoantibody formation in individuals with severe COVID-19. One example replicated by multiple groups is the detection of antibodies reminiscent of the antiphospholipid antibodies (aPL) that mediate antiphospholipid syndrome (APS) in the general population.
In APS, patients form durable autoantibodies to phospholipids and phospholipid-binding proteins such as prothrombin and beta-2-glycoprotein I (β2GPI). These autoantibodies then engage cell surfaces, where they activate endothelial cells, platelets, and neutrophils and thereby tip the blood:vessel wall interface toward thrombosis. While viral infections have long been known to trigger transient aPL (4), mechanisms by which these potentially short-lived antibodies may be pathogenic have not been deeply characterized.
Intriguingly, the circulating B cell compartment in COVID-19 appears similar to the autoimmune disease lupus, whereby naïve B cells rapidly take an extrafollicular route to becoming antibody-producing cells (6), and in doing so bypass the normal tolerance checkpoints against autoimmunity provided by the germinal center.
Here, we were initially interested in the extent to which circulating NET remnants might be an important activator of endothelial cells. We then also necessarily turned our attention to aPL as potential markers of COVID-19 serum and polyclonal IgG fractions with strong endothelial cell activating-potential.
Here, we report that serum from COVID-19 patients activate cultured endothelial cells to express surface adhesion molecules integral to inflammation and thrombosis, namely ICAM-1, E-selectin, and VCAM-1.
Furthermore, we found that for at least a subset of serum samples from patients with COVID-19, this activation could be mitigated by depleting total IgG. The role of aPL in activating endothelial cells has been demonstrated both in vitro and in vivo (9).
For example, IgG fractions from APS patients have long been known to activate HUVEC, as reflected by increased monocyte adherence and expression of adhesion molecules. It is intriguing that while most characterization of endothelium in the APS field has focused on activation of the endothelium by anti-β2GPI antibodies (10), these were only rarely detected in our cohort.
Interesting recent work demonstrates the ability of phospholipid-binding, “cofactor- independent” antibodies to also activate endothelium in APS (11).
Of course, it should be noted that all experiments performed here were with total IgG fractions and not affinity-purified aPL. Therefore, aPL may mark antibody profiles in severe illness, quite possibly polyclonal, that activate the endothelium and steer the normally quiescent blood:vessel interface toward inflammation and coagulation.
In addition to our findings (5), several other groups (12) have also provided evidence for the de novo formation of pathogenic autoantibodies in COVID-19. For example, an interesting study used a high-throughput autoantibody discovery technique to screen a cohort of COVID-19 patients for autoantibodies against 2,770 extracellular and secreted proteins (13).
The authors found a tendency for autoantibodies to be directed against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. This is further bolstered by our data suggesting correlations amongst aPL species in serum from patients with COVID-19.
Beyond COVID-19, we were intrigued to find that approximately one-quarter of sepsis patients had at least one positive aPL test, mostly anti-PS/PT IgM. Given that anti-PS/PT IgM levels tracked with the ability of plasma to activate endothelial cells, we posit that a similar autoreactive antibody-mediated, endothelial cell-activating mechanism may occur in some patients with sepsis, a state in which infection and the host immune response conspire to perturb the vasculature (14).
Of particular note, infections causing critical illness have long been known to be potential triggers of autoantibodies, and in particular aPL (4, 15). Although infection-associated aPL have typically been described as transient (16), a recent systematic review found that one-third of individuals found to have positive aPL in the setting of a virus- associated thrombotic event continue to have durably-positive aPL for at least several months
There are several potential clinical implications of our findings. A consideration that warrants further investigation is whether patients with severe COVID-19 should be screened for aPL to evaluate their risk of thrombosis and progression to respiratory failure, and whether patients with high aPL titers might benefit from treatments used in traditional cases of severe APS such as therapeutic anticoagulation, complement inhibition, and plasmapheresis.
At the same time, determining the extent to which aPL are direct mediators of the endothelial cell phenotypes observed here, or perhaps highlight polyclonal antibody fractions likely to activate endothelial cells, is an important question deserving of future research. This study has additional limitations including a lack of a direct readout of macrovascular thrombosis available in other well- conducted studies (17) given aggressive anticoagulation used at our center early in the pandemic, and an as yet incomplete understanding of mechanisms by which aPL-associated IgG fractions activate endothelial cells. However, given the urgency of COVID-19 research, we believe these issues are counterbalanced by the relatively large sample size and the heretofore unknown discovery of endothelial cell-activating antibody profiles in some COVID-19 serum.
Indeed, these data also put the diffuse organ involvement of COVID-19 into context, where a non-specific humoral response to the illness may disrupt the normally quiescent endothelium and potentiate vascular inflammation. As we await definitive solutions to the pandemic, these findings add important context to the complex interplay between SARS-CoV-2 infection, the human immune system, and vascular immunobiology.