International Consensus Statement on an Update of the Classification Criteria for Definite Antiphospholipid Syndrome(APS)

The antiphospholipid syndrome is a prothrombotic disorder associated with autoantibodies. It is associated with obstetrical complications (mainly spontaneous abortion) as well as venous and arterial thrombotic risks. Insights into disease mechanisms have led to new therapies.

https://www.nejm.org/doi/pdf/10.1056/NEJMra1112830

The Pathogenesis of the Antiphospholipid Syndrome

Oxidative stress causes profound alterations of various biological structures, including cellular membranes, lipids, proteins and nucleic acids, and it is involved in numerous malignancies. Reduced glutathione (GSH) is considered to be one of the most important scavengers of reactive oxygen species (ROS), and its ratio with oxidised glutathione (GSSG) may be used as a marker of oxidative stress. The main aim of this study was to determine GSH:GSSG ratio in the blood serum of paediatric cancer patients to use this ratio as a potential marker of oxidative stress. The whole procedure was optimised and the recoveries for both substances were greater than 80% under the optimised conditions. We analysed a group of paediatric patients (n=116) with various types of cancer, including neuroblastoma, anaplastic ependymoma, germ cell tumour, genital tract tumour, lymphadenopathy, rhabdomyosarcoma, nephroblastoma, Ewing's sarcoma, osteosarcoma, Hodgkin's lymphoma, medulloblastoma and retinoblastoma. We simultaneously determined the levels of reduced and oxidised glutathione, and thus, its ratio in the blood serum of the patients. The highest ratio was observed in retinoblastoma patients and the lowest in anaplastic ependymoma. We were able to distinguish between the diagnoses based on the results of the obtained GSH:GSSG ratio.

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Redox status expressed as GSH:GSSG ratio as a marker for oxidative stress in paediatric tumour patients.

https://chem.as.uky.edu/sites/default/files/Sultana%20et%20al%202013%20Free%20Radic.%20Biol.%20Med.%2062%20157-169.pdf

Lipid peroxidation triggers neurodegeneration: a redox proteomics view into the Alzheimer disease brain.

Tissue factor (TF) is the high-affinity receptor and cofactor for factor (F)VII/VIIa The TF-FVIIa complex is the primary initiator of blood coagulation and plays an essential role in hemostasis TF is expressed on perivascular cells and epithelial cells at organ and body surfaces where it forms a hemostatic barrier TF also provides additional hemostatic protection to vital organs, such as the brain, lung, and heart Under pathological conditions, TF can trigger both arterial and venous thrombosis For instance, atherosclerotic plaques contain high levels of TF on macrophage foam cells and microvesicles that drives thrombus formation after plaque rupture In sepsis, inducible TF expression on monocytes leads to disseminated intravascular coagulation In cancer patients, tumors release TF-positive microvesicles into the circulation that may contribute to venous thrombosis TF also has nonhemostatic roles For instance, TF-dependent activation of the coagulation cascade generates coagulation proteases, such as FVIIa, FXa, and thrombin, which induce signaling in a variety of cells by cleavage of protease-activated receptors This review will focus on the roles of TF in protective hemostasis and pathological thrombosis

Tissue Factor: An Essential Mediator of Hemostasis and Trigger of Thrombosis.

The pathological features of antiphospholipid syndrome (APS) are related to the activity of circulating antiphospholipid antibodies (aPLs) associated with vascular thrombosis and obstetric complications. Indeed, aPLs are not only disease markers, but also play a determining pathogenetic role in APS and exert their effects through the activation of cells and coagulation factors and inflammatory mediators for the materialization of the thromboinflammatory pathogenetic mechanism. Cellular activation in APS necessarily involves the interaction of aPLs with target receptors on the cell membrane, capable of triggering the signal transduction pathway(s). This interaction occurs at specific microdomains of the cell plasma membrane called lipid rafts. In this review, we focus on the key role of lipid rafts as signaling platforms in the pathogenesis of APS, and propose this pathogenetic step as a strategic target of new therapies in order to improve classical anti-thrombotic approaches with “new” immunomodulatory drugs.

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Advances in the Pathophysiology of Thrombosis in Antiphospholipid Syndrome: Molecular Mechanisms and Signaling through Lipid Rafts

Platelet factor 4 (PF4) is a protein with a pro-clotting function expressed by activated platelets with a high affinity for anionic glycosaminoglycans present on the platelet surface. It has been shown that the positively charged surface of PF4 tetramer interacts with the negatively charged regions of β2glycoprotein I (β2GPI) domains, stabilising the link between the antigen and the phospholipid surface, thus increasing the possibility of binding with the respective antibodies. In particular, a tetramer of PF4 selectively binds two molecules of β2GPI, favouring the dimerization of the same, which is crucial in platelet activation and therefore in thrombotic manifestations of antiphospholipid syndrome (aPS). PF4 may be a common denominator in syndromes such as aPS and heparin-induced thrombocytopenia (HIT), which share similar clinical manifestations as thrombocytopenia and thrombosis. Other syndromes, which share the same clinical and laboratory features of HIT despite not having previously received heparin, appear to be associated with the presence of anti-PF4 antibodies. Such pathologies could only be explained by HIT antibodies with heparin-independent platelet-activating properties. One of these could be vaccine-induced Immune thrombotic thrombocytopenia (VITT) post-somministration of ChAdOx1 nCoV-19 vaccine. Recent studies have shown structural similarities between heparin and β2GPI, which may be responsible for thrombotic events in those infected with SARS-Cov-2 and VITT, who never had heparin. In particular, oxidised-β2GPI (β2GPIox) may mimic heparin by structural analogy and link to PF4. Considering the structural similarities between heparin and β2GPIox, and demonstrating the immunogenicity of the hypothesised complex in APS, the alternative molecule could be represented by β2GPIox itself, thus explaining the thrombotic events following vaccination in subjects who have never received heparin.The aim of the study is to test the potential immunogenicity of the β2GPIox-PF4 complex and the presence of antibodies against this complex in patients with aPS, thrombosis during infection with SARS-CoV-2 or VITT.34 patients with proven diagnosis of APS, 17 patients with thrombosis related to infection SARS CoV-2 and 3 patients with VITT were enrolled. Only one aPS patient received heparin prior to testing. Antibodies to the β2GPIox-PF4 complex were evaluated by home made ELISA immunoenzyme testing. Competitive inhibition (homologous) experiments of the bond of PF4 with β2GPIox in the fluid phase were conducted in order to verify the binding specificity.Anti-β2GPIox-PF4 antibodies were detected in 11 of 34 aPS patients (32%) and all VITT patients (100%), while none of covid-19 patients tested positive. In addition, Anti-β2GPIox-PF4 antibodies positivity significantly correlated with the presence of anti-β2GPI antibodies (p=0.0259) and with a triple positive antibody profile (p=0.0017) (Figure 1). There was no significant association between anti-β2GPIox-PF4 antibodies’ title and clinical features of aPS patients.The results of this study show a new antibody positivity in aPS and VITT. In particular we have identified a high prevalence of anti-β2GPIox-PF4 antibodies which may be involved in the pathophysiology of both aPS and VITT. In addition the absence of anti-β2GPIox-PF4 antibodies in covid-19 patients suggest that the thrombotic mechanism that underlies VITT is different from covid-19 and, on the contrary, could resemble aPS.Figure 1.[1]Alessandri C et al. New autoantigens in the antiphospholipidsyndrome. Autoimmun Rev. 2011[2]Sikara MP et al. {beta}2 Glycoprotein I ({beta}2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome. Blood. 2010 Jan 21[3] Favaloro EJ et al. Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT/HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT/TTS. Antibodies (Basel). 2022 Jan 21NIL.None Declared.

/pdf/ab0127-antigenic-assessment-for-the-b2gpiox-pf4-complex-in-a-1j0c9w99.pdf

Ab0127 antigenic assessment for the β2gpiox-pf4 complex in a monocentric cohort of patients with aps, thrombosis during sars-cov-2 infection and vitt

the maturation and function of DCs glycoprotein I modulate 2 β Advanced glycation end products of human

Background Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterised by the presence of at least one clinical event among vascular thrombosis and/or pregnancy morbidity, in the presence of circulating antiphospholipid antibodies (aPL). High-mobility group box-1 (HMGB1) is a non-histonic protein belonging to the family of alarmins. It is associated with chromatin and has a dual function depending on the cell state: in basal conditions it is located in the nucleus and promotes the interaction of some transcription factors with DNA, in inflammatory conditions it is secreted in the extracellular space and exerts the functions of a pro-inflammatory cytokine. One of the main receptor system responsible for the HMGB1 activity is the ”receptor for advanced glycation end products” (RAGE). Increased serum HMGB1 levels have been reported in patients with Systemic Lupus Erythematosus and pre-eclampsia, as other alarmins are increased in patients with early abortions. Objectives To evaluate the serum levels of HMGB1 and soluble RAGE (sRAGE) in patients with obstetric and thrombotic APS. Methods 43 consecutive patients with APS, diagnosed according to the Sapporo criteria, were enrolled. The study cohort included both primary APS (=15) and APS associated with SLE (=28). In addition, 30 healthy subjects (HC) matched for age and sex were studied as controls. Serum levels of HMGB1 and sRAGE were analysed by Western blot. Results The clinical features of the enrolled patients (40 females and 3 males, mean age 40.98±13.48 years,) are reported in table 1. HMGB1 and sRAGE serum levels were significantly increased in APS patients in comparison with controls (p Conclusions In this study, we investigated for the first time the serum levels of HMGB1 and sRAGE in patients with APS, showing increased levels in both primary and secondary APS compared to controls. Larger studies are needed to assess whether monitoring serum HMGB1/sRAGE levels could be a useful tool for risk stratification in patients with APS. Disclosure of Interest None declared

https://ard.bmj.com/content/annrheumdis/77/Suppl_2/1272.full.pdf

AB0168 Elevated serum levels of hmgb1 and srage in patients with antiphospholipid syndrome

OBJECTIVES
Antiphospholipid syndrome (APS) is a prothrombotic condition defined by recurrent thrombosis, pregnancy complications and circulating antiphospholipid antibodies (aPL), including anti-β2-Glycoprotein I (β2-GPI). In clinical practice it is possible to find patients with APS persistently negative for the aPL tests according to Sydney criteria ("seronegative APS", SN-APS). Recently, several autoimmune responses have been described as a consequence of post-translational modifications of their target autoantigens. This study was undertaken to test carbamylated-β2-GPI (Carb-β2-GPI) as a new autoantigen of APS.


METHODS
β2-GPI was carbamylated by potassium cyanate and used to investigate its effect on monocyte-derived DC (moDC) phenotype and function. Sera from 114 SN-APS patients, 60 APS, 20 patients with Rheumatoid Arthritis, 20 NON-APS thrombosis and 50 healthy donors were analyzed for anti-Carb-β2-GPI by ELISA.


RESULTS
Carb-β2-GPI is able to activate moDCs, inducing up-regulation of CD80, CD86, and CD40, activation of ERK, p38 MAPK and NF-κB and IL-12p70 release.Serological results showed that both 37/114 SN-APS (32.45%) and 23/60 APS patients (38.33%) resulted positive for anti-Carb-β2-GPI. Interestingly, SN-APS patients tested positive for anti-Carb-β2-GPI showed a higher prevalence of thrombocytopenia (p= 0.04, likelihood positive ratio of 3.9).


CONCLUSION
Data obtained from both functional tests on moDCs and from immunologic approaches prompted identification of Carb-β2-GPI as a "new" antigenic target in APS. In particular, anti-Carb-β2-GPI revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients tested positive for anti-Carb-β2-GPI reported a high risk of thrombocytopenia, this test may be considered a suitable approach in the clinical evaluation of SN-APS.

Antonella Capozzi

Papers

Advances in the Pathophysiology of Thrombosis in Antiphospholipid Syndrome: Molecular Mechanisms and Signaling through Lipid Rafts

Ab0127 antigenic assessment for the β2gpiox-pf4 complex in a monocentric cohort of patients with aps, thrombosis during sars-cov-2 infection and vitt

the maturation and function of DCs glycoprotein I modulate 2 β Advanced glycation end products of human

AB0168 Elevated serum levels of hmgb1 and srage in patients with antiphospholipid syndrome

Carbamylation of β2-GPI generates new autoantigens for Antiphospholipid Syndrome: a new tool for diagnosis of "seronegative" patients.