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Antiphospholipid syndrome (APS) is an autoimmune disease with arteriovenous thrombosis and recurrent miscarriages as the main clinical manifestations. Due to the complexity of its mechanisms and the diversity of its manifestations, its diagnosis and treatment remain challenging issues. Antiphospholipid antibodies (aPL) not only serve as crucial "biomarkers" in diagnosing APS but also act as the "culprits" of the disease. Endothelial cells (ECs), as one of the core target cells of aPL, bridge the gap between the molecular level of these antibodies and the tissue and organ level of pathological changes. A more in-depth exploration of the relationship between ECs and the pathogenesis of APS holds the potential for significant advancements in the precise diagnosis, classification, and therapy of APS. Many researchers have highlighted the vital involvement of ECs in APS and the underlying mechanisms governing their functionality. Through extensive in vitro and in vivo experiments, they have identified multiple aPL receptors on the EC membrane and various intracellular pathways. This article furnishes a comprehensive overview and summary of these receptors and signaling pathways, offering prospective targets for APS therapy. [ABSTRACT FROM AUTHOR]

Objectives To investigate the impact of thrombocytopenia on survival in patients with APS. Methods Thrombocytopenia and other predictors of outcome were retrospectively evaluated in an aPL-positive and APS cohort with 38-year follow-up (1980–2018). Thrombocytopenia was defined as <150 × 109 platelets/l. Hazard ratios (HR) of mortality were calculated using Cox-regression models. Results Among 114 patients, 64% had primary APS, 25% secondary APS and 10% asymptomatic aPL. Mean follow-up was 19 (range 5–38) years. ANA [hazard ratio (HR) 1.8, 95% CI 0.8, 3.6, P  = 0.10], arterial thrombotic events (HR 7.0, 95% CI 1.4, 3.5, P  = 0.016), myocardial infarction (HR 8.3, 95% CI 1.1, 59, P  = 0.03), intracardiac thrombosis (HR 17, 95% CI 1, 279, P  = 0.04) and thrombocytopenia (HR 2.9, 95% CI 1.4, 6.1, P  = 0.004) were risk factors for all-cause mortality, but in multivariate analysis only thrombocytopenia (HR 2.7, 95% CI 1.3, 6.0, P  = 0.01) remained significant. Persistent (HR 4.4, 95% CI 2.1, 9.2, P  = 0.001) and low–moderate thrombocytopenia (HR 2.8, 95% CI 1.2, 6.4, P  = 0.01) were associated with a significant increase in mortality compared with acute (HR 1.6, 95% CI 0.5, 5.3, P  = 0.40) and severe (HR 2.1, 95% CI 0.5, 9.2, P  = 0.30) forms. APS patients with vs without thrombocytopenia were more frequently male (58 vs 24%, P  = 0.001) with arterial thrombosis (55 vs 32%, P  = 0.04), LA positivity (100 vs 87%, P  = 0.04), type I aPL profile (89% vs 71%, P  = 0.05) and anticoagulant treatment (89 vs 63%, P  = 0.01). Thrombosis caused 13% of deaths in thrombocytopenic patients and 1% in those without (P  = 0.01). Conclusion Thrombocytopenia is an aPL-related manifestation that identifies patients with severe disease phenotype and high thrombotic risk. Persistent low–moderate thrombocytopenia is associated with a reduced long-term survival. [ABSTRACT FROM AUTHOR]

Beta‐2‐glycoprotein I (β2GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of β2GPI can be recognized by the anti‐β2GPI antibody. Here, we prepared the anionic di‐oleoyl‐phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the β2GPI. The conformational changes of β2GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21–27 significantly increased after β2GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21–27 from the ring conformation. After β2GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1–20, 21–27, 196–205, 273–279 and 297–306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70–86, 153–162, 191–198, 196–205 and 273–279 indicated β2GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of β2GPI at sequences 21–27 and forms an intermediate conformation after 10 min of interaction. After a complete protein–lipid interaction, high negatively charged CL membrane induced a major conformation transition of β2GPI. [ABSTRACT FROM AUTHOR]

Protein disulphide isomerase (PDI) promotes platelet activation and constitutes a novel antithrombotic target. In this study, we reported that a PDI‐binding plant polyphenol, tannic acid (TA), inhibits PDI activity, platelet activation and thrombus formation. Molecular docking using plant polyphenols from dietary sources with cardiovascular benefits revealed TA as the most potent binding molecule with PDI active centre. Surface plasmon resonance demonstrated that TA bound PDI with high affinity. Using Di‐eosin‐glutathione disulphide fluorescence assay and PDI assay kit, we showed that TA inhibited PDI activity. In isolated platelets, TA inhibited platelet aggregation stimulated by either GPVI or ITAM pathway agonists. Flow cytometry showed that TA inhibited thrombin‐ or CRP‐stimulated platelet activation, as reflected by reduced granule secretion and integrin activation. TA also reduced platelet spreading on immobilized fibrinogen and platelet adhesion under flow conditions. In a laser‐induced vascular injury mouse model, intraperitoneal injection of TA significantly decreased the size of cremaster arteriole thrombi. No prolongation of mouse jugular vein and tail‐bleeding time was observed after TA administration. Therefore, we identified TA from natural polyphenols as a novel inhibitor of PDI function. TA inhibits platelet activation and thrombus formation, suggesting it as a potential antithrombotic agent. [ABSTRACT FROM AUTHOR]

Objective(s): Cardiovascular disease has an important role in mortality caused by lung injury. Emphysema is associated with impaired pulmonary gas exchange efficiency and airflow limitation associated with small airway inflammation. The aim was to evaluate the interactions between lung injury, inflammation, and cardiovascular disease. Since gallic acid has antioxidant and antiinflammatory effects, we hypothesized that gallic acid protects the lung and the related heart dysfunction in elastase-induced lung injury. Materials and Methods: Forty-eight Sprague-Dawley male rats were randomly divided into six groups: Control, Porcine pancreatic elastase (PPE), PPE+GA, and 3 groups for different doses of gallic acid (GA 7.5, GA 15, GA 30 mg/kg). PPE was injected intra-tracheally on days 1 and 10 of the test. In each group, electrocardiography, hemodynamic parameters, oxidative stress, and bronchoalveolar lavage fluid were examined. Results: PPE administration showed a decrease in HR and QRS voltage of electrocardiogram parameters, as well as in hemodynamic parameters (P<0.05, P<0.01, and P<0.001) and superoxide dismutase (SOD) (P<0.05). Tumor Necrosis Factor α (TNF-α) (P<0.001), interleukin 6 (IL-6) (P<0.001), interleukin 6 (MDA) (P<0.001), and the total number of white blood cells (P<0.001) showed an increase in PPE groups. Gallic acid preserved the values of hemodynamic properties, oxidative stress, inflammation, and electrocardiogram parameters in comparison to the PPE group. Conclusion: Briefly, this study showed the valuable effect of gallic acid in cardiac dysfunction related to elastase-induced lung injury. These findings suggested that gallic acid, as a natural antioxidant, has a potential therapeutic effect on preventing oxidative stress, inflammation, and subsequent cardiovascular disease. [ABSTRACT FROM AUTHOR]

Objective(s): Ventricular arrhythmias including ventricular tachycardia (VT) and ventricular fibrillation (VF) are the most important causes of mortality rate. Gallic acid (GA) has beneficial effects on cardiovascular diseases. The aim of this study was to evaluate the effects of GA on electrophysiological parameters such as QRS complex, heart rate (HR), PR interval parameters, and ventricular arrhythmia following chemical induction in rat. Materials and Methods: Seventy-two male rats were divided into 9 groups (n=8). Chronic groups pretreated by GA (10, 30, and 50 mg/kg, orally) and normal saline (N/S, 1 ml/kg, orally) for 10 days. At the start of the experiments (the first day) and on the final day of the experiments (tenth day), the electrocardiogram (lead II) was recorded. At acute group, GA (50 mg/kg), and anti-arrhythmic drugs such as propranolol, amiodarone, and verapamil injected via intravenous (IV). Then, arrhythmia induced by a CaCl2 2.5% solution (140 mg/kg, IV). Afterward, percentage of premature ventricular beats (PVB), VF, and VT were recorded at 1, and 3 min. Results: These findings showed that chronic and acute doses of GA have positive inotropic and antidysrhythmic effects by significant reduction of PVB, VT and VF on comparison with the control group. These actions are comparable to anti-arrhythmic drugs such as quinidine, propranolol, amiodarone, and verapamil. GA has not significant effect on chronotropic and dromotropic properties. Conclusion: Findings showed that GA has antiarrhythmic, and inotropic characteristics that suggested GA has effective for mild congestive heart failure, and cardiovascular disorders patients which susceptible to incidence of arrhythmias. [ABSTRACT FROM AUTHOR]

Introduction: Thrombocytopaenia and autoimmune haemolytic anaemia (AIHA) have considerable impact on prognosis in systemic lupus erythematosus (SLE). We investigated the frequencies of these haemocytopaenias, along with their associations and effect on outcome in a single-centre cohort of patients with SLE. Methods: Demographic characteristics, clinical features, autoantibody profiles, damage and mortality data were compared between patients with and without each haematological abnormality. Variables displaying significant differences between the groups were entered into logistic regression. Results: Ninety-three patients had AIHA and 215 had thrombocytopaenia. Both were associated with neuropsychiatric (NP) involvement, with each other, leucopaenia, antiphospholipid syndrome (APS) and antiphospholipid antibodies. More patients in both groups had organ damage, and their damage scores were higher. Association to NP damage was discernible. In addition, cardiovascular and renal damage and diabetes were more pronounced in patients with thrombocytopaenia. At logistic regression analysis, younger age, anticardiolipin antibody IgM positivity, leucopaenia and thrombocytopaenia were associated with AIHA whilst lupus anticoagulant activity, AIHA, leucopaenia, APS and NP involvement were associated with thrombocytopaenia. Among damage items, peripheral vascular damage, diabetes, NP damage, renal and ocular damage displayed significant associations with thrombocytopaenia, whereas none of the items did with AIHA. Patients with AIHA had significantly reduced survival rates at 10 and 20 years. Conclusions: We observed that AIHA and thrombocytopaenia were associated with severe lupus, affecting major organs and causing end organ damage. Thus, they may be considered as prognostic markers. Furthermore, AIHA and especially thrombocytopaenia may also be a marker for a subgroup of lupus patients who have or may develop APS. [ABSTRACT FROM AUTHOR]

Introduction: Recurrent miscarriage is the occurrence of three or more consecutive pregnancy loss before 20 weeks of gestation. Antiphospholipid Antibodies (APAs) are one of the causes responsible for recurrent pregnancy loss. Aim: Estimation of APAs, Anticardiolipin Antibodies (ACAs) and Beta-2 Glycoprotein 1 Antibodies (β-2 GP1 Abs) in recurrent pregnancy loss and also to assess which antibody has strong correlation with habitual abortion patients. Materials and Methods: This was a case-control study that included the total number of 45 women of reproductive age group from Obstetrics and Gynaecology Department, KGMU, Lucknow, Uttar Pradesh, India, out of which 30 women had been taken as case and 15 as control. Dilute Russell Viper Venom Test (DRVVT) was applied for Lupus Anticoagulant (LA) and Enzyme Linked Immunosorbent Assay (ELISA) for ACA, APA and β-2 GP1 Ab. Result: Majority of cases showed higher positivity for β-2 GP1 Abs 11/30 (36.7%) with lesser positivity for ACA 4/30 (13.4%) and APAs 4/30 (13.4%). All the antibodies were absent in control group. Majority of cases showed positivity for β-2 GP1 Ab out of which two cases also showed positivity for ACA, APA and LA. Conclusion: β-2 GP1 Abs are useful and a significant marker in recurrent pregnancy loss in women as compared to ACA and APAs. [ABSTRACT FROM AUTHOR]

Anti-beta-2-glycoprotein 1 (anti-β2GP1) antibodies are associated with increased thrombotic risk in patients with autoimmune disease. There is conflicting evidence on the effects of anti-β2GP1 antibodies on platelets, with both enhanced and inhibited aggregation previously reported. However, previous studies did not include isotype antibodies to ensure the observed effects were due to anti-β2GP1 antibodies. The aims of this study were to (1) investigate the effects of anti-β2GP1 antibodies on collagen-induced platelet aggregation in parallel with negative control (buffer normal saline) and isotype control antibodies and (2) determine the lupus anticoagulant (LA) activity of anti-β2GP1 antibodies used. Three animal-derived anti-human-β2GP1 antibodies (1.25, 2.5, and 5 μg/mL) incubated with healthy platelet-rich plasma were activated by collagen (2.5 μg/mL). Each anti-β2GP1 antibody demonstrated the inhibition of aggregation compared to negative control, but not to isotype control. No anti-β2GP1 antibody demonstrated LA activity, suggesting they were probably nonpathological. This study highlights both negative and isotype control markers are important to validate the effects of anti-β2GP1 antibodies. Assays to measure anti-domain I-β2GP1 antibodies are recommended to be used in conjunction with functional measures to further investigate the effects of anti-β2GP1 antibodies. [ABSTRACT FROM AUTHOR]

It is long known that antiphospholipid antibodies (aPL) induce proinflammatory and procoagulant cellular responses. The underlying signal transduction has been a major focus of research and is the topic of this review. An amazingly heterogeneous panel of signaling pathways has been described and it turns out that at least some of this heterogeneity can be explained by effects of distinct aPL species. On the one hand, there are antibodies against β2-glycoprotein I (β2GPI) which appear to exert their cellular effects only as a complex of β2GPI/anti-β2GPI. Their major targets are low-density lipoprotein-receptor related protein 8 (LRP8), annexin A2 (ANXA2), toll-like receptor 4 (TLR4), and possibly TLR2. The other relevant aPL species are antibodies against cardiolipin which are internalized into endosomes and induce cellular responses via activation of endosomal NADPH-oxidase. Their cell surface target is still unknown. Another important issue relates to the role of complement. It has been shown in vivo that certain pathogenic effects of aPL depend on complement activation, but the exact interplay with the signaling pathways described earlier needs to be elucidated. Thus, while there has been tremendous progress over the past decade, many open questions remain to be answered. [ABSTRACT FROM AUTHOR]

The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by an elevated risk for arterial and venous thrombosis and pregnancy-related morbidity. Since the discovery of the disease in 1980s, numerous studies in cell culture systems, in animal models, and in patient populations have been reported, leading to a deeper understanding of the pathogenesis of APS. These studies have determined that circulating autoantibodies, collectively called antiphospholipid antibodies (aPL), the majority of which recognize cell surface proteins attached to the plasmamembrane phospholipids, play a causal role in the development of the disease. The binding of aPL to the cell surface antigens triggers interaction of the complex with transmembrane receptors to initiate intracellular signaling in critical cell types, including platelets, monocytes, endothelial cells, and trophoblasts. Subsequent alteration of various cell functions results in inflammation, thrombus formation, and pregnancy complications. Apolipoprotein E receptor 2 (apoER2), a lipoprotein receptor family member, has been implicated as a mediator for aPL actions in platelets and endothelial cells. Nitric oxide (NO) is a signaling molecule known to exert potent antithrombotic, anti-inflammatory, and anti-atherogenic effects. NO insufficiency and oxidative stress have been linked to APS pathogenesis. This review will focus on the recent findings on how apoER2 and dysregulation of NO production contribute to aPL-mediated pathologies in APS. [ABSTRACT FROM AUTHOR]

Antiphospholipid antibody (aPL)‑mediated antiphospholipid syndrome (APS) is an autoimmune disease. Upon binding to aPL, the primary antigen of aPL, β2‑glycoprotein I (β2‑GP I), induces abnormal immune function, which further activates downstream signaling pathways in the cell and eventually leads to APS. The present study aimed to determine whether β2‑GP I antigen and anti‑β2‑glycoprotein I antibody (aβ2‑GP I), which belong to the aPL class of antibodies, may affect human chorionic epithelium cell (JEG‑3) proliferation, migration and invasion. Recombinant human (rh)β2‑GP I protein was expressed using a prokaryotic expression system and aβ2‑GP I antibody was purified from the blood serum of 10 patients with recurrent pregnancy loss. JEG‑3 cells were stimulated with rhβ2‑GP I and aβ2‑GP I separately or simultaneously, and serum immunoglobulin G of normal pregnant women was used as negative control. Using cell counting kit‑8, cell cycle and transwell assays in addition to EdU staining, it was determined that aβ2‑GP I/rhβ2‑GP I complex markedly increased JEG‑3 cell proliferation, migration and invasion. The results revealed that mRNA levels of inhibitor of nuclear factor (NF)‑κB kinase subunit (IKKβ), myeloid differentiation primary response protein MyD88 (MyD88), NF‑κB and NF‑κB inhibitor α (IκBα), as well as the protein levels of MyD88, IκBα and phospho(p)‑IκBα in JEG‑3 cells increased following incubation with the aβ2‑GP I/rhβ2‑GP I complex. The observed upregulation of p‑IκBα protein suggested that IκBα‑mediated inhibition of NF‑κB was weakened. Furthermore, JEG‑3 cells were transfected with PGMLV‑NF‑κB‑Lu vector. Luciferase activity in JEG‑3‑NFκB‑Luc1 and JEG‑3‑NFκB‑Luc2 cells was enhanced following treatment with aβ2‑GP I/rhβ2‑GP I complex. The present study demonstrated that aβ2‑GP I/rhβ2‑GP I complex activates NF‑κB through MyD88 signal transduction pathway, which further enhances JEG‑3 cell proliferation, migration and invasion. [ABSTRACT FROM AUTHOR]

BACKGROUND Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C. RESULTS Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant ( P < 0.05) alterations in abundance at different storage times. The proteins that showed significant changes in abundance included serum albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α2-macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and β2-glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40). CONCLUSION It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α2-macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE), glycoprotein I (gI), and Us9 promote efficient anterograde axonal transport of virus from the neuron cytoplasm to the axon terminus. HSV-1 and PRV gE and gI form a heterodimer that is required for anterograde transport, but an association that includes Us9 has not been demonstrated. NS-gE380 is an HSV-1 mutant that has five amino acids inserted after gE residue 380, rendering it defective in anterograde axonal transport. We demonstrated that gE, gI and Us9 form a trimolecular complex in Vero cells infected with NS-gE380 virus in which gE binds to both Us9 and gI. We detected the complex using immunoprecipitation with anti-gE or anti-gI monoclonal antibodies in the presence of ionic detergents. Under these conditions, Us9 did not associate with gE in cells infected with wild-type HSV-1; however, using a nonionic detergent, TritonX-100, an association between Us9 and gE was detected in immunoprecipitates of both wild-type and NS-gE380-infected cells. The results suggest that the interaction between Us9 and gE is weak and disrupted by ionic detergents in wild-type infected cells. We postulate that the tight interaction between Us9 and gE leads to the anterograde spread defect in the NS-gE380 virus. [ABSTRACT FROM AUTHOR]

Abstract: Antiphospholipid syndrome (APS) is a systemic autoimmune disease defined by thrombotic or obstetrical events and persistent antiphospholipid antibodies (aPLs). Chemokine-like factor-like MARVEL transmembrane domain-containing family (CMTM) is widely expressed in the immune system and may closely related to APS. This review aimed to systematically summarize the possible effects of CMTM on APS. Publications were collected from PubMed and Web of Science databases up to August 2020. CKLF, CKLFSF, CMTM, antiphospholipid syndrome, immune cells, and immune molecules were used as search criteria. Immune cells, including neutrophil, dendritic cells (DCs), T-cells, B-cells, and inflammatory cytokines, play an important role in the development of APS. Chemokine-like factor 1 (CKLF1) has a chemotactic effect on many cells and can affect the expression of inflammatory cytokines and adhesion molecules through the nuclear factor-kB (NF-kB) pathway or mitogen-activated protein kinase (MARK) pathway. CKLF1 can participate in the maturation of DCs, T lymphocyte activation, and the activation of neutrophils through the MAPK pathway. CMTM1 may act on Annexin A2 by regulating Ca2+ signaling. CMTM2 and CMTM6 are up-regulated in neutrophils of APS patients. Some CMTM family members influence the activation and accumulation of platelets. CMTM3 and CMTM7 are binding partners of B-cell linker protein (BLNK), thereby linking B cell receptor (BCR) and activating BLNK-mediated signal transduction in B cells. Moreover, CMTM3 and CMTM7 can act on DCs and B-1a cell development, respectively. CMTM may have potential effects on the development of APS by acting on immune cells and immune molecules. Thus, CMTM may act as a novel prognostic factor or immunomodulatory treatment option of APS., (Copyright © 2021 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

In the antiphospholipid syndrome (APS), patients produce antiphospholipid antibodies (aPL) that promote thrombosis and adverse pregnancy outcomes. Current therapy with anticoagulation is only partially effective and associated with multiple complications. We previously discovered that aPL recognition of cell surface β2-glycoprotein I (β2-GPI) initiates apolipoprotein E receptor 2 (apoER2)-dependent signaling in endothelial cells and in placental trophoblasts that ultimately promotes thrombosis and fetal loss, respectively. Here we sought to identify a monoclonal antibody (mAb) to β2-GPI that negates aPL-induced processes in cell culture and APS disease endpoints in mice. In a screen measuring endothelial NO synthase (eNOS) activity in cultured endothelial cells, we found that whereas aPL inhibit eNOS, the mAb 1N11 does not, and instead 1N11 prevents aPL action. Coimmunoprecipitation studies revealed that 1N11 decreases pathogenic antibody binding to β2-GPI, and it blocks aPL-induced complex formation between β2-GPI and apoER2. 1N11 also prevents aPL antagonism of endothelial cell migration, and in mice it reverses the impairment in reendothelialization caused by aPL, which underlies the non-thrombotic vascular occlusion provoked by disease-causing antibodies. In addition, aPL inhibition of trophoblast proliferation and migration is negated by 1N11, and the more than 6-fold increase in fetal resorption caused by aPL in pregnant mice is prevented by 1N11. Furthermore, the promotion of thrombosis by aPL is negated by 1N11. Thus, 1N11 has been identified as an mAb that attenuates APS-related pregnancy complications and thrombosis in mice. 1N11 may provide an efficacious, mechanism-based therapy to combat the often devastating conditions suffered by APS patients. [ABSTRACT FROM AUTHOR]

Objective. Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphati dylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression. Methods. RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR). Results. RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT. Conclusion. We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS. [ABSTRACT FROM AUTHOR]

Objective Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine whether the low-density lipoprotein receptor family member apolipoprotein E receptor 2 (ApoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL. Methods Placental and trophoblast ApoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG and aPL were purified from healthy individuals and APS patients, respectively. The role of ApoER2 in aPL-induced changes in trophoblast proliferation and migration and in kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of ApoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant ApoER2+/+ and ApoER2-/- mice injected with aPL or normal human IgG. Results We found that ApoER2 is abundant in human and mouse placental trophoblasts and in multiple trophoblast-derived cell lines, including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2-glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by epidermal growth factor in trophoblasts was mediated by ApoER2. Furthermore, in a murine passive-transfer model of pregnancy complications of APS, ApoER2-/- mice were protected from both aPL-induced fetal loss and aPL-induced IUGR. Conclusion ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS. [ABSTRACT FROM AUTHOR]

Anti-β glycoprotein I (anti-βGPI ) antibodies are important contributors to thrombosis, especially in patients with antiphospholipid syndrome (APS). However, the mechanism by which anti-βGPI antibodies are involved in the pathogenesis of thrombosis is not fully understood. In this report, we investigated the role of anti- βGPI antibodies in complexes with βGPI as mediators of platelet activation, which can serve as a potential source contributing to thrombosis. We examined the involvement of the apolipoprotein E receptor 2′ (apoER2′) and glycoprotein I ba (GP I ba) in platelet activation induced by the anti-βGPI /βGPI complex. The interaction between the anti-βGPI /βGPI complex and platelets was examined using in vitro methods, in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by measuring GPII b/III a activation and P-selectin expression and thromboxane B production as well as p38 mitogen-activated protein kinase phosphorylation. Our results revealed that the anti-βGPI /βGPI complex was able to activate platelets, and this activation was inhibited by either the anti-GP I bα antibody or the apoER2′ inhibitor. Results showed that the anti-βGPI /βGPI complex induced platelet activation via GPI ba and apoER2′, which may then contribute to the prothrombotic tendency in APS patients. [ABSTRACT FROM AUTHOR]

Objective Antiphospholipid antibodies (aPL), especially those targeting β2-glycoprotein I (β2GPI), are well known to activate endothelial cells, monocytes, and platelets, with prothrombotic implications. In contrast, the interaction of aPL with neutrophils has not been extensively studied. Neutrophil extracellular traps (NETs) have recently been recognized as an important activator of the coagulation cascade, as well as an integral component of arterial and venous thrombi. This study was undertaken to determine whether aPL activate neutrophils to release NETs, thereby predisposing to the arterial and venous thrombosis inherent in the antiphospholipid syndrome (APS). Methods Neutrophils, sera, and plasma were prepared from patients with primary APS (n = 52) or from healthy volunteers and characterized. No patient had concomitant systemic lupus erythematosus. Results Sera and plasma from patients with primary APS had elevated levels of both cell-free DNA and NETs, as compared to healthy volunteers. Freshly isolated neutrophils from patients with APS were predisposed to high levels of spontaneous NET release. Further, APS patient sera, as well as IgG purified from APS patients, stimulated NET release from control neutrophils. Human aPL monoclonal antibodies, especially those targeting β2GPI, also enhanced NET release. The induction of APS NETs was abrogated with inhibitors of reactive oxygen species formation and Toll-like receptor 4 signaling. Highlighting the potential clinical relevance of these findings, APS NETs promoted thrombin generation. Conclusion Our findings indicate that NET release warrants further investigation as a novel therapeutic target in APS. [ABSTRACT FROM AUTHOR]

Pathogenic antiphospholipid antibodies (aPL) are the driving factors of recurrent pregnancy loss and thrombosis that characterize antiphospholipid syndrome (APS). Current evidence indicates that aPL induce a procoagulant phenotype in the vasculature and abnormal cellular proliferation and differentiation in placental tissues to cause the typical clinical features; however, the molecular mechanisms underlying these processes remain incompletely understood. Inflammation serves as a necessary link between the observed procoagulant phenotype and actual thrombus development and is an important mediator of the placental injury in APS patients. However, the underlying mechanisms for these events have also not been fully elucidated. In this review, we will outline the available data that give us our current understanding of the pathophysiology of APS, especially as it relates to the development of thromboembolic and obstetric pathological phenomena in these patients. We will also describe the intracellular signaling pathways activated by aPL in various cellular subtypes and outline the current evidence linking these pathways to clinical phenotypes. Finally, we will discuss the implications of distinct molecular patterns defining clinical phenotypes of APS patients. [ABSTRACT FROM AUTHOR]

The association between antiphospholipid antibodies (aPL) and clinical problems goes beyond what is stated in the antiphospholipid syndrome (APS) classification criteria, namely thrombosis and pregnancy morbidity, and thrombocytopenia is the most common non-criteria hematologic manifestation of aPL with a frequency ranging from 20 to 50 %. Thrombocytopenia is rarely severe, and hemorrhage is far less common than thrombosis. However, when anticoagulation is considered, it may constitute a clinical problem with increased bleeding risk. Furthermore, thrombocytopenia represents a risk factor for thrombosis in aPL-positive patients. Therefore, it is important to understand the pathogenesis and the clinical associations of thrombocytopenia to build the right medical approach in aPL-positive patients. In this paper, we review the literature on aPL/APS-associated thrombocytopenia and briefly discuss the other conditions that can result in thrombocytopenia as they have commonalities with APS and their recognition is important to establish the most appropriate treatment strategy. [ABSTRACT FROM AUTHOR]

β2 glycoprotein I (β2 GPI) is the most common antigen for autoimmune antibodies in antiphospholipid syndrome ( APS). Thrombosis is a clinical feature of APS. We created a molecule (A1-A1) that consists of two identical β2 GPI-binding modules from ApoE receptor 2 (Apo ER2). A1-A1 binds to β2 GPI/antibody complexes, preventing their association with Apo ER2 and anionic phospholipids, and reducing thrombus size in the mouse model of APS. Here, we describe a mutant of A1-A1 (mA1-A1 ND) with improved affinity for β2 GPI. mA1-A1 ND inhibits the binding of β2 GPI to cardiolipin in the presence of anti-β2 GPI antibodies, and inhibits the binding to phospholipids in plasma samples of APS patients, affecting the clotting time. Reduction of the clotting time demonstrates the presence of soluble β2 GPI/antibody complexes in patients' plasma. These complexes either already exist in patients' plasma or form rapidly in the proximity to phospholipids. All members of the low-density lipoprotein receptor family bind β2 GPI. Modeling studies of A1 in a complex with domain V of β2 GPI (β2 GPI- DV) revealed two possible modes of interaction of a ligand-binding module from lipoprotein receptors with β2 GPI- DV. In both orientations, the ligand-binding module interferes with binding of β2 GPI to anionic phospholipids; however, it interacts with two different but overlapping sets of lysine residues in β2 GPI- DV, depending on the orientation. [ABSTRACT FROM AUTHOR]

Quinine, a quinoline derivative, is an ancient antipyretic drug with antimalarial properties that has been phased out by more effective synthetic candidates. In previous studies we discovered that hydroxychloroquine (HCQ), a synthetic antimalarial with structural similarities to quinine, reduced the binding of antiphospholipid (aPL) immune complexes to phospholipid bilayers. We performed ellipsometry and atomic force microscopy (AFM) studies to measure the effect of quinine on dissociation of anti-β2-glycoprotein I (anti-β2GPI) immune complexes. We found that quinine desorbed pre-formed β2GPI-aPL immunoglobulin (Ig)G complexes from phospholipid bilayers at significantly lower molar concentrations than HCQ. Quinine also inhibited the formation of immune complexes with a higher efficacy than HCQ at equivalent drug concentrations of 0.2 mg/ml (0.192 ± 0.025 µg/cm2 for quinine vs. 0.352 ± 0.014 µg/cm2 for HCQ, p < 0.001). Furthermore, AFM imaging experiments revealed that addition of quinine disintegrated immune complexes bound to planar phospholipid layers. The desorptive and inhibitory effects of the old drug, quinine, toward β2GPI-aPL IgG complexes and β2GPI were significantly more pronounced compared to the synthetic antimalarial, HCQ. The results suggest that the quinoline core of the molecule is a critical domain for this activity and that side chains may further modulate this effect. The results also indicate that there may yet be room for considering new activities of very old drugs in devising clinical trials on potential non-anticoagulant treatments for antiphospholipid syndrome (APS). [ABSTRACT FROM AUTHOR]

Several studies reported on the association between antiphospholipid syndrome (APS) and venous thrombosis. In contrast, little is known about cardiovascular (CV) risk in APS. We performed a meta-analysis on the impact of APS on major markers of CV risk. Studies on the relationship between APS and common carotid artery intima-media thickness (CCA-IMT), internal carotid artery IMT (ICA-IMT), carotid bifurcation IMT (BIF-IMT), prevalence of carotid plaques, flow-mediated dilation (FMD), nitrate-mediated dilation (NMD), and ankle-brachial index (ABI) were systematically searched in PubMed, Web of Science, Scopus, and EMBASE databases. Twenty case-control studies (668 cases, 678 controls) were included. Compared to controls, APS patients showed a higher CCA-IMT (mean difference [MD] 0.11 mm; 95% CI 0.07, 0.14), ICA-IMT (MD 0.08 mm; 95% CI 0.05, 0.11), BIF-IMT (MD 0.09 mm; 95% CI 0.06, 0.12) and a higher frequency of carotid plaques (OR 3.87; 95% CI 1.61, 9.31). Moreover, a lower FMD was found in APS subjects than in controls (MD -4.49%; 95% CI -6.20, -2.78), with no differences in NMD (MD -1.80%; 95% CI -4.01, 0.42). Finally, an increased prevalence of pathological ABI was found in APS patients compared to controls (OR 7.26; 95% CI 1.77, 29.71). Despite heterogeneity among studies, APS appears significantly associated with markers of subclinical atherosclerosis and CV risk. These findings can be useful to plan adequate prevention strategies and therapeutic approaches. [ABSTRACT FROM AUTHOR]

Antiphospholipid syndrome is an autoimmune condition, characterised by the persistent presence of antiphospholipid antibodies and either thrombosis or obstetric morbidity. The cornerstone of therapy is long-term anticoagulation to reduce morbidity and mortality; however, better understanding of the immunological pathways may direct us to develop future therapeutic strategies. We provide an overview of the current understanding of the immunopathogenesis of this perplexing condition and its associated morbidities and current evidence for some of the immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a life-threatening disease that can be induced by dasatinib, a dual Src and BCR-ABL tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). Today, key questions remain regarding the mechanisms involved in the long-term development of dasatinib-induced PAH. Here, we demonstrated that chronic dasatinib therapy causes pulmonary endothelial damage in humans and rodents. We found that dasatinib treatment attenuated hypoxic pulmonary vasoconstriction responses and increased susceptibility to experimental pulmonary hypertension (PH) in rats, but these effects were absent in rats treated with imatinib, another BCR-ABL tyrosine kinase inhibitor. Furthermore, dasatinib treatment induced pulmonary endothelial cell apoptosis in a dose-dependent manner, while imatinib did not. Dasatinib treatment mediated endothelial cell dysfunction via increased production of ROS that was independent of Src family kinases. Consistent with these findings, we observed elevations in markers of endothelial dysfunction and vascular damage in the serum of CML patients who were treated with dasatinib, compared with CML patients treated with imatinib. Taken together, our findings indicate that dasatinib causes pulmonary vascular damage, induction of ER stress, and mitochondrial ROS production, which leads to increased susceptibility to PH development., Introduction Pulmonary arterial hypertension (PAH) is a severe progressive cardiopulmonary disorder that carries a high mortality rate, for which there is no cure at this time (1). Pulmonary endothelial cell [...]