Familial lupus anticoagulant

The antiphospholipid antibody syndrome (APS) is defined by widespread arterial and venous thromboses associated with elevated plasma levels of antiphospholipid antibodies (APLA). The primary antiphospholipid antibody syndrome (PAPS) appear to be a fairly homogeneous disease, and HLA, family and other studies provide new insights into this cause of thrombosis and vascular disease. We describe two patients with PAPS (lupus anticoagulant positive), whose family members were analyzed for clinical and laboratory abnormalities associated with APS. Familial screening seems to be important, in order to prevent the thrombotic events. Low dose aspirin is the first line treatment in asymptomatic subjects with APLA, previous or present thrombosis requiring long-term, possibly life-long anticoagulation.     

Primary antiphospholipid syndrome: an unusual cause of adrenal insufficiency

We describe a boy, 10 years and 5 months old, who developed acute adrenal gland insufficiency which was confirmed by hormonal investigation. Abdominal magnetic resonance imaging showed unilateral enlargement of the right adrenal gland, whereas the other gland seemed normal - no cause was apparent. Three months later the patient presented with thrombosis in the right femoral vein and in the veins of the right leg. Autoantibodies against cardiolipin were strongly positive, while antinuclear antibodies and antibodies against double-stranded deoxyribonucleic acid were absent. There was no evidence of antiphospholipid syndrome associated with drugs, connective tissue disorders, or malignancies, strongly suggesting the diagnosis of primary antiphospholipid syndrome. The development of adrenal insufficiency has been reported in primary antiphospholipid syndrome due to adrenal hemorrhage following vascular occlusion of adrenal vessels or secondary to anticoagulant therapy. It was interesting to note that in our patient adrenal gland insufficiency preceded other clinical evidence of the syndrome by 3 months. The primary antiphospholipid syndrome should be considered a possible cause of Addison's disease when the etiology is not obvious.     

Anti-beta2-glycoprotein I antibodies recognizing platelet factor 4-heparin complex in antiphospholipid syndrome in patient substantiated with mouse model

The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies associated with arterial and/or venous thrombosis, and recurrent abortion accompanied often by thrombocytopenia. These antibodies are heterogeneous and react against phospholipid-binding proteins such as beta2-glycoprotein I (beta2GPI) and prothrombin. The recognition of anti-beta2-glycoprotein I (anti-beta2GPI) by platelet factor 4-heparin complex (PF4-Hc) has been previously evoked and partially confirmed by the present inhibition studies. Further, the anti-beta2-glycoprotein I antibodies were purified from a patient with primary antiphospholipid syndrome using Affi-gel-10-beta2GPI immunoaffinity chromatography. The purified anti-beta2GPI IgM as well as patient serum equally recognized PF4-Hc in ELISA mode. In order to substantiate this data and to better understand we studied an animal model using mouse active immunization with the purified human anti-beta2GPI. The mice showed a significant decrease in their platelet count. In addition the ELISA responses of the immunized mice sera were positive against both beta2GPI and PF4-Hc, substantiating the double recognition. Despite many previous reported animal model studies, this is the first time we have shown the specific recognition of anti-beta2GPI antibodies by PF4-Hc, the results in the induced mice correlating the data observed with some patients.     

Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation

The antiphospholipid syndrome (APS) is defined by thrombosis and recurrent pregnancy loss in the presence of antiphospholipid (aPL) antibodies and is generally treated with anticoagulation therapy. Because complement activation is essential and causative in aPL antibody-induced fetal injury, we hypothesized that heparin protects pregnant APS patients from complications through inhibition of complement. Treatment with heparin (unfractionated or low molecular weight) prevented complement activation in vivo and in vitro and protected mice from pregnancy complications induced by aPL antibodies. Neither fondaparinux nor hirudin, other anticoagulants, inhibited the generation of complement split products or prevented pregnancy loss, demonstrating that anticoagulation therapy is insufficient protection against APS-associated miscarriage. Our data indicate that heparins prevent obstetrical complications in women with APS because they block activation of complement induced by aPL antibodies targeted to decidual tissues, rather than by their anticoagulant effects.     

Antibody profile of pregnant women with antiphospholipid syndrome and pregnancy outcome after treatment with low dose aspirin and low-weight-molecular heparin

The aim of the research was to show our diagnostic and therapeutic experience with antiphospholipid syndrome (APS) in pregnant women. 36 pregnant women suspect on APS were included in the study: 32 with primary antiphospholipd syndrome (PAPS) and 4 with secondary antiphospholipid syndrome (SAPS). All pregnant women received low-molecular-weight-heparin (LMWH) and low dose aspirin (LDA) therapy. Control group represented 26 women with SAPS and previous bad reproductive anamnesis. Average pregnancy lasted 37.06 +/- 0.707 weeks. LMWH and LDA therapy was successful in 97.22%. Lupus anticoagulant (LA) was found to be more frequent in PAPS group (71.87%). Anticardiolipin antibodies (aCL) were found to be more frequent in SAPS (26.66%). For three patients (3.37%), PAPS was diagnosed due to a fact that they had positive antibeta2-glycoproteinl (antibeta-GP1). To make APS diagnosis, it is of great importance to search for all antiphospholipid antibodies. LMWH and low dose of acetylsalicylic acid should be the first choice therapy.     

Self-interaction of soluble and surface-bound beta2-glycoprotein I and its enhancement by lupus anticoagulants

Antiphospholipid antibodies found in antiphospholipid syndrome are autoantibodies to phospholipid-binding proteins, such as beta2-glycoprotein I (beta2GPI). We have previously reported that among these antibodies, the so-called lupus anticoagulants (LAs) augment beta2GPI binding to the phospholipid membrane surface, which is associated with the pathological action of LAs. However, the molecular mechanisms underlying this augmentation are uncertain. Here we show that beta2GPI, which is monomeric in solution, self-interacts at the interface of soluble and surface-bound molecules. In addition, this self-interaction is enhanced by LA-positive, but not LA-negative, anti-beta2GPI monoclonal antibodies. This study suggests that beta2GPI self-interaction upon surface binding could be involved in the LA-induced potentiation of beta2GPI binding to the phospholipid surface.     

Possible pathogenetic role of antiphospholipid antibodies in a clinical case of human immunodeficiency virus infection with peripheral polyneuropathy and arterial thrombosis

Human immunodeficiency virus (HIV) infection can be associated with many autoimmune disorders such as antiphospholipid antibody syndrome. The most common neurologic complication is represented by peripheral neuropathies, but its pathogenesis is still unknown. We report the clinical case of a 44-year-old woman with HIV infection, peripheral polyneuropathy and arterial thrombosis, in which high serum levels of antiphospholipid antibodies were repeatedly documented. We suggest that the evaluation of serum antiphospholipid antibodies levels in HIV infection is fundamental both from an exploratory point of view and for starting the best treatment.     

The Role of Zinc in Thrombosis and Pulmonary Embolism in the Course of Antiphospholipid Syndrome (APS)—Short Review

Antiphospholipid antibodies may occur in the course of various diseases, but its presence is not necessarily associated with clinical symptoms. Zinc has multiple biological roles. For example, it stabilizes the cell’s membrane and regulates its functions by influencing the synthesis of phospholipids and its distribution. The present review focuses on the possible associations between zinc and antiphospholipid antibodies and with the symptoms of antiphospholipid syndrome.     

Antiphospholipid syndrome

Antiphospholipid syndrome is diagnosed when arterial or venous thrombosis or recurrent miscarriages occur in a person in whom laboratory tests for antiphospholipid antibodies (anticardiolipin antibodies and/or lupus anticoagulant and/or anti-beta 2-glycoprotein I) are positive. Despite the strong association between antiphospho-lipid antibodies and thrombosis, their pathogenic role in the development of thrombosis has not been fully elucidated. Novel mechanisms involving both the complement pathway and micro-particles have been described. The knowledge of these new pathogenic approaches might identify novel therapeutic targets and therefore may improve the management of these patients.     

Single-step autoantibody profiling in antiphospholipid syndrome using a multi-line dot assay

Introduction  

Diagnosis of antiphospholipid syndrome (APS) still remains a laboratory challenge due to the great diversity of antiphospholipid antibodies (aPL) and their significance regarding APS-diagnostic criteria.     

A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

Background  

The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β₂GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL.     

Antiphospholipid reactivity against cardiolipin metabolites occurring during endothelial cell apoptosis

We have recently shown that cardiolipin (CL) and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. In this study, we investigate the immunoreactivity to CL derivatives occurring during endothelial apoptosis in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). We compared the serum immunoreactivity to CL with that of its derivatives monolysocardiolipin (MCL), dilysocardiolipin (DCL), and hydrocardiolipin (HCL) by means of both enzyme-linked immunosorbent assay and thin-layer chromatography (TLC) immunostaining. In addition, we investigated the composition of phospholipid extracts from the plasma membrane of apoptotic endothelial cells and the binding of patients' sera to the surface of the same cells by using high-performance TLC and immunofluorescence analysis. The average reactivity to MCL was comparable with that of CL and significantly higher than that for DCL and HCL in patients studied, both in the presence or in the absence of beta2-glycoprotein I. Of relevance for the pathogenic role of these autoantibodies, immunoglobulin G from patients' sera showed an increased focal reactivity with the plasma membrane of endothelial cells undergoing apoptosis. Interestingly, the phospholipid analysis of these light membrane fractions showed an accumulation of both CL and MCL. Our results demonstrated that a critical number of acyl chains in CL derivatives is important for the binding of antiphospholipid antibodies and that MCL is an antigenic target with immunoreactivity comparable with CL in APS and SLE. Our finding also suggests a link between apoptotic perturbation of CL metabolism and the production of these antibodies.     

Vascular involvement in rheumatic diseases: 'vascular rheumatology'

The vasculature plays a crucial role in inflammation, angiogenesis, and atherosclerosis associated with the pathogenesis of inflammatory rheumatic diseases, hence the term 'vascular rheumatology'. The endothelium lining the blood vessels becomes activated during the inflammatory process, resulting in the production of several mediators, the expression of endothelial adhesion molecules, and increased vascular permeability (leakage). All of this enables the extravasation of inflammatory cells into the interstitial matrix. The endothelial adhesion and transendothelial migration of leukocytes is a well-regulated sequence of events that involves many adhesion molecules and chemokines. Primarily selectins, integrins, and members of the immunoglobulin family of adhesion receptors are involved in leukocyte 'tethering', 'rolling', activation, and transmigration. There is a perpetuation of angiogenesis, the formation of new capillaries from pre-existing vessels, as well as that of vasculogenesis, the generation of new blood vessels in arthritis and connective tissue diseases. Several soluble and cell-bound angiogenic mediators produced mainly by monocytes/macrophages and endothelial cells stimulate neovascularization. On the other hand, endogenous angiogenesis inhibitors and exogenously administered angiostatic compounds may downregulate the process of capillary formation. Rheumatoid arthritis as well as systemic lupus erythematosus, scleroderma, the antiphospholipid syndrome, and systemic vasculitides have been associated with accelerated atherosclerosis and high cardiovascular risk leading to increased mortality. Apart from traditional risk factors such as smoking, obesity, hypertension, dyslipidemia, and diabetes, inflammatory risk factors, including C-reactive protein, homocysteine, folate deficiency, lipoprotein (a), anti-phospholipid antibodies, antibodies to oxidized low-density lipoprotein, and heat shock proteins, are all involved in atherosclerosis underlying inflammatory rheumatic diseases. Targeting of adhesion molecules, chemokines, and angiogenesis by administering nonspecific immunosuppressive drugs as well as monoclonal antibodies or small molecular compounds inhibiting the action of a single mediator may control inflammation and prevent tissue destruction. Vasoprotective agents may help to prevent premature atherosclerosis and cardiovascular disease.     

Induction of decreased fecundity by tetanus toxoid hyper-immunization in C57BL/6 mice depends on the applied adjuvant

It has already been shown that tetanus toxoid (TTd) hyper-immunization is a suitable experimental method for creating the animal model of antiphospholipid syndrome (APS) in BALB/c mice. The severity of APS pathology in BALB/c mice mainly correlates to the affinity of anti-β(2) glycoprotein I (β(2)GPI) antibodies. In this study we have investigated reproductive pathology induced in C57BL/6 mice by TTd hyper-immunization using a combination of different pretreatments (complete Freund's adjuvant or glycerol) and adjuvants (alhydrogel or glycerol). A decrease in fecundity was recorded in only C57BL/6 mice immunized with alhydrogel adjuvant, irrespective of the kind of applied pretreatment; it was associated with an increase in abundance of low affinity anti-β(2)GPI IgG antibodies and Th1 prevalence.     

The role of beta2-glycoprotein I (beta2GPI) in the activation of plasminogen

Beta2-glycoprotein I (beta2GPI) is a glycoprotein of unknown physiological function. It is the main target antigen for antiphospholipid antibodies in patients with antiphospholipid syndrome (APS). beta2GPI binds with high affinity to the atherogenic lipoprotein Lp(a) which shares structural homology with plasminogen, a key molecule in the fibrinolytic system. Impaired fibrinolysis has been described in APS. The present work reports the interaction between beta2GPI and Glu-Plasminogen which may explain the recently described proteolytic effect of plasmin on beta2GPI. In the process of Glu-Plasminogen activation, we found an increase in plasmin generation both at fibrin and cellular surface level as a function of the concentration of beta2GPI added, suggesting an important role as a cofactor in the trimolecular complex beta2GPI-Plasminogen-tPA. This phenomenon represents a novel regulatory step both in the positive feedback mechanism for extrinsic fibrinolysis and in antithrombotic regulation. IgG anti-beta2GPI antibodies recognized the beta2GPI at the endothelial surface inducing its activation with an increase of ICAM-I and a decrease in the expression of thrombomodulin favoring a pro-thrombotic state in the vascular endothelium. The interference in the plasmin conversion by anti-beta2GPI antibodies could generate thrombosis as observed in APS.     

Phospholipid membranes form specific nonbilayer molecular arrangements that are antigenic.

Hexagonal phase (H(II))-preferring lipids such as phosphatidate, cardiolipin, and phosphatidylserine form nonbilayer molecular arrangements in lipid bilayers. While their presence in biological membranes has not been established, in vitro studies suggest that alterations in membrane properties modify their function. In this study, antiphospholipid monoclonal antibodies were developed against nonbilayer structures. One of the monoclonal antibodies identifies nonplanar surfaces in liposomes and in membranes of cultured cells. These results are the first evidence that natural membranes maintain a fragile balance between bilayer and nonbilayer lipid arrangements. Therefore, these antibodies can be used to evaluate the role of H(II)-preferring lipids in the modulation of membrane activities. Our studies demonstrated that nonplanar surfaces are highly immunogenic. Although these structures are normally transient, their formation can be stabilized by temperature variations, drugs, antibiotics, apolar peptides, and divalent cations. Our studies demonstrated that abnormal exposure of nonbilayer arrangements may induce autoimmune responses as found in the antiphospholipid syndrome.     

Artemisinin analogue SM934 protects against lupus-associated antiphospholipid syndrome via activation of Nrf2 and its targets

Kidney is a major target organ in both antiphospholipid syndrome(APS) and systemic lupus erythematosus(SLE). The etiology of antiphospholipid syndrome nephropathy associated lupus nephritis(APSN-LN) is intricate and remains largely unrevealed.We proposed in present work, that generation of antiphospholipid antibodies(a PLs), especially those directed towards the oxidized neoepitopes, are largely linked with the redox status along with disease progression. Moreover, we observed that compromised antioxidative capacity coincided with turbulence of inflammatory cytokine profile in the kidney of male NZW×BXSB F1 mice suffered from APSN-LN. SM934 is an artemisinin derivative that has been proved to have potent immunosuppressive properties. In current study, we elaborated the therapeutic benefits of SM934 in male NZW×BXSB F1 mice,a murine model develops syndrome resembled human APS associated with SLE, for the first time. SM934 treatment comprehensively impeded autoantibodies production, inflammatory cytokine accumulation and excessive oxidative stress in kidney.Among others, we interpreted in present work that both anti-inflammatory and antioxidative effects of SM934 is closely correlated with the enhancement of Nrf2 signaling and expression of its targets. Collectively, our finding confirmed that therapeutic strategy simultaneously exerting antioxidant and anti-inflammatory efficacy provide a novel feasible remedy for treating APSN-LN.     

Antibodies to lipids and liposomes: immunology and safety

Naturally occurring antibodies to phospholipids and cholesterol are widespread; they occur commonly during the course of acute infections; they are not causally related to the anti-phospholipid syndrome; they have been associated with other clinical entities only as an epiphenomenon; and they have not been implicated as causing any clinical syndrome or disease. There are theoretical and experimental reasons to believe that normal cells and tissues are protected from binding of antibodies to bilayer lipids by steric hindrance due to adjacent larger molecules, such as large or charged adjacent glycolipids or proteins on the cell surface. There are also reasons to believe that certain natural antibodies to lipids can even serve useful normal functions. Antibodies to liposomal lipids induced by liposomes containing lipid A appear to have characteristics that are similar or identical to naturally occurring antibodies to lipids, and it is therefore believed that such antibodies would not cause adverse clinical effects. Numerous Phase I and II human clinical trials of experimental vaccines containing liposomes and lipid A have shown a high level of safety.     

Antibodies to Lipids and Liposomes: Immunology and Safety

Naturally occurring antibodies to phospholipids and cholesterol are widespread; they occur commonly during the course of acute infections; they are not causally related to the anti-phospholipid syndrome; they have been associated with other clinical entities only as an epiphenomenon; and they have not been implicated as causing any clinical syndrome or disease. There are theoretical and experimental reasons to believe that normal cells and tissues are protected from binding of antibodies to bilayer lipids by steric hindrance due to adjacent larger molecules, such as large or charged adjacent glycolipids or proteins on the cell surface. There are also reasons to believe that certain natural antibodies to lipids can even serve useful normal functions. Antibodies to liposomal lipids induced by liposomes containing lipid A appear to have characteristics that are similar or identical to naturally occurring antibodies to lipids, and it is therefore believed that such antibodies would not cause adverse clinical effects. Numerous Phase I and II human clinical trials of experimental vaccines containing liposomes and lipid A have shown a high level of safety.     

High affinity binding of beta 2-glycoprotein I to human endothelial cells is mediated by annexin II

Beta(2)-glycoprotein I (beta(2)GPI) is an abundant plasma phospholipid-binding protein and an autoantigen in the antiphospholipid antibody syndrome. Binding of beta(2)GPI to endothelial cells targets them for activation by anti-beta(2)GPI antibodies, which circulate and are associated with thrombosis in patients with the antiphospholipid antibody syndrome. However, the binding of beta(2)GPI to endothelial cells has not been characterized and is assumed to result from association of beta(2)GPI with membrane phospholipid. Here, we characterize the binding of beta(2)GPI to endothelial cells and identify the beta(2)GPI binding site. (125)I-beta(2)GPI bound with high affinity (K(d) approximately 18 nm) to human umbilical vein endothelial cells (HUVECs). Using affinity purification, we isolated beta(2)GPI-binding proteins of approximately 78 and approximately 36 kDa from HUVECs and EAHY.926 cells. Amino acid sequences of tryptic peptides from each of these were identical to sequences within annexin II. A role for annexin II in binding of beta(2)GPI to cells was confirmed by the observations that annexin II-transfected HEK 293 cells bound approximately 10-fold more (125)I-beta(2)GPI than control cells and that anti-annexin II antibodies inhibited the binding of (125)I-beta(2)GPI to HUVECs by approximately 90%. Finally, surface plasmon resonance studies revealed high affinity binding between annexin II and beta(2)GPI. These results demonstrate that annexin II mediates the binding of beta(2)GPI to endothelial cells.