Background Animal models and few clinical reports suggest the involvement of the complement system in the onset of severe manifestations of coronavirus disease-2019 (COVID-19). However, complement contribution to endotheliopathy and hypercoagulability has not been elucidated yet. Objective To evaluate the association among complement activation, endothelial damage and disease severity or activity in COVID-19 patients. Methods In this single-centre cohort study, 148 patients with COVID-19 of different severity were evaluated upon hospital admission and 30 days later. Markers of complement activation (SC5b-9 and C5a) and endothelial perturbation (von Willebrand factor [vWF], tissue-type plasminogen activator [t-PA], plasminogen activator inhibitor-1 [PAI-1], soluble thrombomodulin [sTM], and soluble endothelial selectin [sE-selectin]) were measured in plasma. Results The patients had high plasma levels of SC5b-9 and C5a (p = 0.0001 for both) and vWF, t-PA and PAI-1 (p = 0.0001 for all). Their SC5b-9 levels correlated with those of vWF (r = 0.517, p = 0.0001) and paralleled disease severity (severe vs mild p = 0.0001, severe vs moderate p = 0.026 and moderate vs mild p = 0.001). The levels of sE-selectin were significantly increased only in the patients with severe disease. After 30 days, plasma SC5b-9, C5a and vWF levels had significantly decreased (p = 0.0001 for all), and 43% of the evaluated patients had normal levels. Conclusions Complement activation is boosted during the progression of COVID-19 and dampened during remission, thus indicating its role in the pathophysiology of the disease. The association between complement activation and the biomarkers of endothelial damage suggests that complement may contribute to tissue injury and could be the target of specific therapy.

Antiphospholipid antibodies (Abs) are associated with thrombosis and are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome. It is generally accepted that the major autoantigen for aPL Abs is beta (2) glycoprotein I, which mediates the binding of aPL Abs to target cells (i.e., endothelial cells, monocytes, platelets, trophoblasts, etc.) leading to thrombosis and fetal loss. This article addresses molecular events triggered by aPL Abs on endothelial cells, platelets, and monocytes and complement activation, as well as a review of the current knowledge with regard to the putative receptor(s) recognized by aPL Abs on target cells as well as novel mechanisms that involve fibrinolytic processes. A section is devoted to the description of thrombotic and inflammatory processes that lead to obstetric complications mediated by aPL Abs. Based on experimental evidence using in vitro and in vivo models, new targeted therapies for treatment and/or prevention of thrombosis and pregnancy loss in antiphospholipid syndrome are proposed.

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 anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-b 2 GPI), and anti-phosphatidylserine/ prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-b 2 GPI 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-b 2 GPI 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.

Objective. It has been suggested that only antibodies against domain 1 (D1) of b 2 -glycoprotein I (b 2 GPI) are pathogenic and diagnostic. The role of antibodies against other b 2 GPI domains is still debated. This study was undertaken to evaluate the clinical relevance of domain specificity profiling of anti-b 2 GPI IgG antibodies in antiphospholipid syndrome (APS) patients and in control groups of patients with systemic autoimmune rheumatic diseases and in asymptomatic antiphospholipid antibody (aPL) carriers.Methods. We evaluated 159 subjects with persistently positive, medium or high-titer anti-b 2 GPI IgG, including 56 patients with thrombotic (obstetric or nonobstetric) primary APS, 31 women with obstetric primary APS, 42 aPL-positive patients with systemic autoimmune rheumatic diseases, and 30 asymptomatic aPL carriers. One hundred healthy donors were included. Anti-b 2 GPI D1 and D4/5 IgG were tested on research enzyme-linked immunosorbent assays containing recombinant b 2 GPI domains.Results. As compared to other groups, aPL carriers displayed higher frequency/titer of anti-D4/5 IgG. Unlike anti-D4/5, anti-D1 IgG antibodies were more frequent and at higher titer in triple than in single or double aPL-positive subjects. An anti-D1 to anti-D4/5 ratio of ‡1.5 was predictive of systemic autoimmunity (odds ratio 3.25 [95% confidence interval 1.45-7.49], P 5 0.005). Neither anti-D1 nor anti-D4/5 antibodies were associated with APS clinical criteria.Conclusion. Anti-D1 IgG is the preferential specificity not only in vascular and obstetric primary APS, but also in patients with systemic autoimmune rheumatic disease with no clinical features of APS. Conversely, aPL carriers do not have a polarized profile toward D1. Combined testing for anti-b 2 GPI IgG with different domain specificity allows a more accurate aPL profiling, with polarization toward anti-D1 IgG as a possible fingerprint of systemic autoimmunity.

In vitro studies have documented ␤2 glycoprotein I (␤2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of ␤2GPI to these cells and the conditions that favor their interaction have not been investigated. We analyzed the in vivo distribution of cyanine 5.5-labeled ␤2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The ␤2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar ␤2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after ␤2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that ␤2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes. (Blood. 2011;118(15): 4231-4238) IntroductionAntiphospholipid syndrome (APS) is characterized by vascular thrombosis and adverse pregnancy outcome associated with circulating antiphospholipid antibodies (aPL) which are believed to play an important pathogenic role in the development of the clinical manifestations of the syndrome. 1,2 Human ␤2-glycoprotein I (␤2GPI) has been recognized as the major antigenic target for antiphospholipid antibodies and in vivo models have shown that antibodies directed against this molecule are able to mediate thrombus formation. 3,4 Beta2GPI is a heavily glycosylated glycoprotein that circulates in blood at a concentration of 150-300 g/mL 5 and is synthesized mainly in the liver, although expression of ␤2GPI mRNA has also been detected in endothelial cells (ECs), central nervous system, astrocytes, and placenta. The physiologic function of this protein is still unclear, but the apparently healthy life of humans and mice deficient in ␤2GPI suggests that its role is not all that critical. [6][7][8][9] Most of the information now available on ␤2GPI has been collected following the observation that this protein is the main target of antiphospholipid (aPL) antibodies. 10,11 Patients with circulating antibodies to ␤2GPI are at increased risk of venous and arterial thrombosis as well as of pregnancy complications including miscarriage, preeclampsia and retarded fetal growth. 1,12 For this reason antibodies with this specificity have been included among the criteria for the diagnosis of aPL syndrome (APS). 13,14 The induction of fetal loss and promotion of thrombosis in animal models as a result of immunization with ␤2GPI or passive transfer of antibodies further support the pathogenic role of these antibodies. [15][16][17...

Key Points• A recombinant antibody recognizing the D1 domain of b2 glycoprotein I induces fetal loss and clot formation in animal models.• The CH2-deleted antibody fails to activate complement and prevents the procoagulant and proabortive effects of patient antibodies.A single-chain fragment variable (scFv) recognizing b2-glycoprotein 1 (b2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against b2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-b2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-b2GPI antibodies from APS patients and displaced b2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy. (Blood. 2014;123(22):3478-3487)

Recurrent thrombosis and miscarriages are the main clinical manifestations of antiphospholipid syndrome (APS). Although most patients display both clinical signs, some patients can have isolated vascular or obstetric variants. Emerging data raise the question of whether obstetric and vascular APS are the same or different diseases. An important difference between the two conditions is that a thrombophilic state is a common feature in vascular APS, whereas clot occlusions of the decidual spiral arteries are seldom observed in obstetric APS, and infarctions are found in only one-third of APS placentae. Conversely, inflammation, which is undetectable in vascular APS, is frequently observed in the placentae of patients with obstetric APS and has been documented in the placentae of pregnant mice with fetal loss mediated by antiphospholipid antibodies. Attempts to identify different antibodies or epitopes responsible for the two clinical manifestations of APS have so far been unsuccessful. Possible mechanisms exist that might explain the development of the two clinical presentations, including the tissue distribution and expression level of the main target antigen of antiphospholipid antibodies, β2 glycoprotein I (β2GPI). The identification of the factors that promote the onset of either obstetric or vascular APS has important diagnostic and therapeutic implications.

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