thromboplastin and Antiphospholipid-Syndrome

Antiphospholipid syndrome (APS) or 'Hughes syndrome' is a prothrombotic disease characterized by thrombosis and pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). More than three decades have passed, and experts are still uncovering new pieces of this disease complex pathogenesis and management.. We searched in literature using MEDLINE and PubMed databases focusing on the latest development on disease pathogenesis, risk assessment of thrombosis and treatment of APS.. The phosphatidylinositol 3-kinase (PI3K)-AKT-mTORC pathway was most recently identified to have a crucial role in activating inflammation among endothelial vessel wall causing vascular lesions in APS. Additionally, new variables are being implemented to assess the risk of thrombosis in patients with APS. Global APS Score (GAPSS) utilizes cardiovascular risk factors and new autoimmune antibodies as part of the score assessment and is the most valid so far. It can be a promising tool in the future for prediction of thrombosis. Anticoagulation remains the cornerstone in APS; however, many new potential therapeutic agents are developing and are currently under investigation.. The most recent advances in pathogenesis, risk stratification and treatment provide a platform for high yield studies with the ultimate goal of providing the optimal management to patients with APS.

Topics: Adrenal Cortex Hormones; Animals; Annexin A2; Anticoagulants; Antiphospholipid Syndrome; Drugs, Investigational; Female; Humans; Hydroxychloroquine; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunoglobulins, Intravenous; Immunosuppressive Agents; MAP Kinase Signaling System; Mice; Phosphatidylinositol 3-Kinases; Plasmapheresis; Pregnancy; Pregnancy Complications, Cardiovascular; Risk Assessment; Rituximab; Secondary Prevention; Thromboplastin; Thrombosis; TOR Serine-Threonine Kinases

Antiphospholipid syndrome (APS) is a disorder characterized by the association of arterial or venous thrombosis and/or pregnancy morbidity with the presence of antiphospholipid antibodies (anticardiolipin antibodies, lupus anticoagulant antibodies, and/or anti-β2-glycoprotein I antibodies). Thrombosis is the major manifestation in patients with aPLs, but the spectrum of symptoms and signs associated with aPLs has broadened considerably, and other manifestations, such as thrombocytopenia, non-thrombotic neurological syndromes, psychiatric manifestations, livedo reticularis, skin ulcers, hemolytic anemia, pulmonary hypertension, cardiac valve abnormality, and atherosclerosis, have also been related to the presence of those antibodies. Several studies have contributed to uncovering the basis of antiphospholipid antibody pathogenicity, including the targeted cellular components, affected systems, involved receptors, intracellular pathways used, and the effector molecules that are altered in the process. Therapy for thrombosis traditionally has been based on long-term oral anticoagulation; however, bleeding complications and recurrence despite high-intensity anticoagulation can occur. The currently accepted first-line treatment for obstetric APS (OAPS) is low-dose aspirin plus prophylactic unfractionated or low-molecular-weight heparin (LMWH). However, in approximately 20% of OAPS cases, the final endpoint, i.e. a live birth, cannot be achieved. Based on all the data obtained in different research studies, new potential therapeutic approaches have been proposed, including the use of new oral anticoagulants, statins, hydroxychloroquine, coenzyme Q10, B-cell depletion, platelet and TF inhibitors, peptide therapy or complement inhibition among others. Current best practice in use of these treatments is discussed.

Topics: Animals; Anticoagulants; Antiphospholipid Syndrome; Biological Products; Humans; Hydroxychloroquine; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunotherapy; Rituximab; Thromboplastin; Ubiquinone

Topics: Animals; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Disease Models, Animal; Factor XI; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunity, Innate; Lupus Coagulation Inhibitor; Mice; Oxidation-Reduction; Thromboplastin; Thrombosis

The use of high intensity anticoagulation in the prevention of recurrence of arterial thrombosis related to the Antiphospholipid Syndrome (APS) is still controversial. This paper reports a debate that took place at the CORA meeting (Controversies in Rheumatology and Autoimmunity), held in Florence in March 2011. Major points of discussion were: 1) the paucity of prospective randomized clinical trials; retrospective studies were the main source supporting the use of high intensity anticoagulation; 2) heterogeneity in antiphospholipid antibodies (aPL) definition, due to the lack of standardization of aPL assays and to the failure to distinguish patients with a high risk profile ("triple positive") from those a low risk profile; 3) bleeding is a major concern about high intensity anticoagulation; however, studies are not concordant in reporting an increased risk compared to the standard regimen; 4) practical issues consist of difficulties in keeping a stable PT-INR over 3 and the possibility for interference by aPL on the thromboplastins used for PT-INR measurement. In conclusion, there is currently a lack of consensus on the use of high intensity anticoagulation for the secondary prophylaxis of arterial thrombosis. However, such a treatment may be particularly recommended in those APS patients who have a high risk aPL profile and other concomitant cardiovascular risk factors, provided that the potential benefit outweighs the risk of bleeding.

Topics: Animals; Antibodies, Antiphospholipid; Anticoagulants; Antiphospholipid Syndrome; Arteries; Clinical Protocols; Consensus Development Conferences as Topic; Hemorrhage; Humans; Practice Guidelines as Topic; Randomized Controlled Trials as Topic; Recurrence; Thromboplastin; Thrombosis

β(2)glycoprotein I (β(2)GPI) is the major antigen in the antiphospholipid syndrome. It has been shown that β(2)GPI can adapt to different conformations, a circular, a S-shaped and a J-shaped conformation. In literature anticoagulant properties of β(2)GPI have been indicated, though there is no consensus on how β(2)GPI exerts a certain action. This article will first review existing data on the conformation of β(2)GPI. In addition, we will investigate whether the conformation of β(2)GPI plays a role in in the proposed anticoagulant activity of β(2)GPI. We investigated the effect of native β(2)GPI and phospholipid-bound β(2)GPI on thrombin generation (TG). Native β(2)GPI was found to have no significant effect on the TG regardless of the concentration of tissue factor. On the contrary, β(2)GPI preincubated with phospholipids significantly inhibited TG triggered with low TF concentration, suggesting an effect on the intrinsic pathway. This indicates that native β(2)GPI in circulation obtains its anticoagulant activity in the presence of anionic phospholipids such as activated blood cells thereby serving as an inhibitory modulator in hemostasis.

Topics: Amino Acid Sequence; Animals; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Blood Coagulation; Humans; Molecular Sequence Data; Phospholipids; Protein Conformation; Structure-Activity Relationship; Thrombin; Thromboplastin

Antiphospholipid syndrome (APS) is an autoimmune disorder defined by the presence of antiphospholipid antibodies in plasma of patients with vascular thrombosis and/or pregnancy morbidity. APS is considered the major cause of brain infarction in young adults and is a generally accepted cause of recurrent pregnancy loss. Antiphospholipid antibodies are a heterogeneous group of autoantibodies strongly related to the clinical manifestations of APS and with a widely recognized pathogenic role in thrombosis. In this article, recent clinical and pathophysiological advances in APS are discussed.

Topics: Abortion, Habitual; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoimmunity; Biomarkers; Female; Humans; Male; Molecular Targeted Therapy; NF-kappa B; P-Selectin; p38 Mitogen-Activated Protein Kinases; Pregnancy; Pregnancy Complications; Risk Assessment; Signal Transduction; Thromboplastin; Thrombosis

Recurrent fetal loss affects 1-5% of women of childbearing age. Immunological mechanisms may account for 40% of recurrent miscarriages, and in particular, the antiphospholipid syndrome (APS) appears to be implicated in 7-25% of the cases. Because antiphospholipid (aPL) antibodies have thrombogenic properties, fetal loss in patients with APS has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. Thrombosis and inflammation are linked in many clinical conditions. Tissue factor (TF), the major cellular initiator of the coagulation protease cascade, plays important roles in both thrombosis and inflammation, and its expression is increased in patients with APS. Here we describe how TF, acting as a proinflammatory molecule, induces trophoblast injury and fetal death in a mouse model of APS. Importantly, we will discuss how TF contributes to C5a-induced oxidative burst in neutrophils leading to trophoblasts and fetal injury in APS. The finding that TF is an important effector in aPL-induced inflammation may allow the development of new therapies to abrogate the inflammatory loop caused by tissue factor and improve pregnancy outcomes in patients with aPL antibodies. Statins downregulate TF-induced inflammation and rescued the pregnancies in aPL-treated mice, suggesting they may be a good treatment for women with aPL-induced pregnancy complications.

Topics: Abortion, Habitual; Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Complement Activation; Disease Models, Animal; Embryo, Mammalian; Factor VIIa; Female; Fetal Death; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mice; Neutrophil Activation; Pregnancy; Receptor, PAR-2; Thromboplastin; Thrombosis

Antiphospholipid syndrome (APS) is a systemic autoimmune vascular disease characterized by recurrent thrombotic episodes and/or obstetric complications. Management of this disease has been restricted mainly to anticoagulation; however, in recent years, significant advancement has been made in elucidating the pathophysiology of the disease including antiphospholipid antibody (aPL)-induced activation of the platelets, endothelial cells, monocytes, complement and coagulation cascade. Stemming from these advances, potential targeted therapeutic approaches have been proposed.. We utilized a computer-assisted search of the literature (MEDLINE, National Library of Medicine, Bethesda, MD, USA) up until September 2009 using the keywords: antiphospholipid syndrome, antiphospholipid antibodies, anticardiolipin antibodies, lupus anticoagulant, anti beta-2 glycoprotein antibodies, complement system, tissue factor, p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor kappa B, toll-like receptors, annexin, Rituximab, statins and tumour necrosis factor.. Several study groups have separately demonstrated the importance of inflammatory mediators in the pathogenesis of APS. It was also established that tissue factor, MAPK, nuclear factors kappa B, and the complement system are integral to the disease process. Toll-like receptors and annexin have as well been associated with the disease pathophysiology. Some study groups proposed new targeted therapeutic strategies some of which have shown promising results in preclinical studies. These include Rituximab, complement inhibition, anti-cytokine therapy, p38 MAPK inhibitors, nuclear factor inhibitors and tissue factor inhibitors.. As more insight is being gained into the pathophysiology of APS, newer therapeutic strategies are being proposed that might lead to safer and more efficacious treatment modalities in the future.

Topics: Annexin A2; Anti-Inflammatory Agents; Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antiphospholipid Syndrome; Complement Activation; Humans; Hydroxychloroquine; Immunologic Factors; Monocytes; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Platelet Activation; Rituximab; Thromboplastin

Antiphospholipid syndrome (APS) is an acquired autoimmune disorder defined by the presence of an antiphospholipid antibody (aPL) and the occurrence of at least one associated clinical condition that includes venous thrombosis, arterial thrombosis or pregnancy morbidity. The aPL detected in APS have long been thought to have a direct prothrombotic effect in vivo. However, the pathophysiology underlying their coagulopathic effect has not been defined. Emerging data suggest a role for the procoagulant protein tissue factor (TF). In this review we provide an overview of TF, describe mouse models used in the evaluation of the role of TF in thrombosis, as well as summarize recent work on TF and APS.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Disease Models, Animal; Female; Humans; Mice; Pregnancy; Pregnancy Complications; Thromboplastin; Thrombosis

The antiphospholipid syndrome (APS) is an autoimmune disorder presenting with tissue injury in various organs attributed to large or small vessel thrombosis or, in some instances, possible nonthrombotic inflammatory mechanisms, associated with in vitro evidence of antibodies to certain proteins, or proteinphospholipid complexes. Although the pathophysiology, diagnosis, and management of APS may seem clear and straightforward from a distance, closer inspection reveals a more complex, incomplete, and uncertain image. This article reviews the evolution of APS from the first description of lupus anticoagulant to the current criteria used to guide clinical research, critiques laboratory methods used to identify autoantibodies, comments on prognosis and management, and summarizes insights into the pathophysiology of this elusive disorder.

Topics: Antibodies, Anticardiolipin; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Clinical Laboratory Techniques; Female; Fetal Death; Humans; Lupus Coagulation Inhibitor; Pregnancy; Thromboplastin

Although, the main physiological role of monocytes is attributed to innate immunity (that is, phagocytosis) and the development of tissue macrophages and dendritic cells, the pathophysiological role of these goes far behind these (simplistic) limits. Indeed, monocytes constitute a major source of blood tissue factor, a key element of the extrinsic coagulation cascade. Monocytes actively bind to platelets, thus forming very prothrombotic monocyte-platelet aggregates. Additionally, these cells link inflammation and the procoagulant state observed in various prothrombotic conditions. However, monocytes are also crucial for successful thrombus recanalisation. In this article, we review the available data on potential mechanisms that link monocytes with thrombosis-related processes.

Topics: Animals; Antiphospholipid Syndrome; Blood Platelets; Cell Adhesion; Cells, Cultured; Coronary Disease; Cytokines; Female; Humans; Inflammation; Male; Mice; Models, Biological; Monocytes; Neovascularization, Physiologic; Platelet Aggregation; Rabbits; Risk Factors; Swine; Thrombophilia; Thromboplastin; Thrombosis

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.

Topics: Abortion, Habitual; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoantigens; beta 2-Glycoprotein I; Blood Platelets; Complement Activation; Endothelial Cells; Female; Fibrinolysis; Humans; Inflammation Mediators; Monocytes; Placenta; Pregnancy; Pregnancy Complications, Hematologic; Thrombin; Thrombophilia; Thromboplastin; Trophoblasts; Venous Thrombosis

The antiphospholipid syndrome (APS) is characterized by clinical manifestations such as venous and arterial thrombosis, thrombocytopenia and/or recurrent pregnancy loss, as well as the persistent presence of laboratory markers of antiphospholipid (aPL) antibodies detected in laboratory assays. Though it is generally accepted that aPL antibodies, such as anticardiolipin (aCL), anti-beta2 glycoprotein I (anti-beta2GPI), and lupus anticoagulants (LA) contribute to the pathogenesis of APS, precise mechanism(s) are yet to be fully described. It is probable that aPL antibodies bind to a range of cellular targets (e.g., platelets, endothelial cells, and monocytes), leading to thrombosis and obstetric complications. There is now increasing evidence that alterations to the tissue factor (TF) pathway of blood coagulation contribute toward hypercoagulability in patients with aPL antibodies. This article reviews current evidence that suggests changes and/or interference to the major pathway of blood coagulation may represent a novel mechanism that contributes to the development of APS.

Topics: Abortion, Habitual; Antibodies, Antiphospholipid; Antibody Specificity; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Endothelial Cells; Female; Humans; Immunoglobulin G; Lipoproteins; Lupus Coagulation Inhibitor; Models, Biological; Pregnancy; Pregnancy Complications, Hematologic; Thrombin; Thrombophilia; Thromboplastin; Venous Thrombosis

Fetal loss in patients with antiphospholipid antibodies (aPL) has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically complement activation with generation of the anaphylotoxin C5a, is an essential mediator of fetal injury. We have analysed the role of tissue factor (TF) in a mouse model of aPL-induced pregnancy loss. TF is the major cellular activator of the coagulation cascade but also has cell signaling activity. Mice that received aPL-IgG showed strong TF staining throughout the decidua and on embryonic debris. This TF staining was not associated with either fibrin staining or thrombi in deciduas. The absence of fibrin deposition and thrombi suggests that TF-dependent activation of coagulation does not mediate aPL-induced pregnancy loss.We found that either blockade of TF with a monoclonal antibody in wild type mice or a genetic reduction of TF prevented aPL-induced inflammation and pregnancy loss indicated a pathogenic role for TF in aPL-induced pregnancy complications. In response to aPL-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells, but not fetal-derived cells (trophoblasts), was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL. The identification of TF, acting as an important pro-inflammatory mediator in aPL-induced fetal injury, provides a new target for therapy to prevent pregnancy loss in the aPL syndrome.

Topics: Animals; Antibodies, Antiphospholipid; Antigen-Antibody Reactions; Antiphospholipid Syndrome; Disease Models, Animal; Female; Fetal Death; Humans; Mice; Pregnancy; Thromboplastin

Antiphospholipid (aPL) antibodies are clinically important acquired risk factors for thrombosis and pregnancy loss and are thought to have a direct prothrombotic effect in vivo. Data suggest that a major mechanism by which aPL antibodies contribute to thrombophilia is the upregulation of tissue factor (TF) (CD142) on blood cells and vascular endothelium. TF is the physiological trigger of normal blood coagulation and thrombosis in many hypercoagulable conditions. This article reviews the physiology of TF, the molecular regulation of TF expression and the effects of aPL antibodies on intravascular TF regulation and expression. Inhibition of TF and the pathways by which aPL antibodies induce TF expression are potentially attractive therapeutic targets in the antiphospholipid syndrome.

Topics: Antibodies, Antiphospholipid; Antigen-Antibody Reactions; Antiphospholipid Syndrome; Gene Expression Regulation; Humans; Signal Transduction; Thromboplastin

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). Among the thrombogenic mechanisms proposed, it has been suggested that aPL can stimulate tissue factor (TF) expression by endothelial cells (ECs) and monocytes. Moreover, our in vivo studies have shown that APS patients (particularly those with thrombosis) have increased monocyte TF expression. Yet, the molecular mechanism(s) by which aPL induce TF expression has not been completely underscored. In a recent study, we have demonstrated that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK-1/ERK proteins, and the p38 MAP kinase-depenent nuclear translocation and activation of NFkappaB/Rel proteins. Understanding the intracellular mechanism(s) of aPL-mediated monocyte activation may help to establish new therapeutic approaches, such as selective inhibition of MAP kinases, to reverse the prothrombotic state in APS. Furthermore, the contribution of TF to a protrombotic state in the APS provides a renewed focus on antithrombotic therapies in current use, including the oral anticoagulation and, more recently, the use of statins, which have been proven to be effective in the inhibition of EC and monocyte TF-expression.

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Thromboplastin; Thrombosis

Antiphospholipid syndrome (APS) is characterized by recurrent thrombosis or pregnancy morbidity associated with antiphospholipid antibodies (aPL). Impaired fibrinolysis is a contributing factor for the development of thrombosis, and the effect of aPL in the fibrinolytic system has been investigated. Impaired release of tPA and enhanced release of PAI-1 after endothelial activation is reported in patients with APS. Elevated Lipoprotein (a) levels have been found in APS, which results in inhibition of fibrinolytic activity. Phospholipid-bound beta(2)-glycoprotein I (beta(2)GPI) is a major autoantigen for aPLs. beta(2)GPI exerts both anti-coagulant and pro-coagulant properties mainly by interacting with other phospholipid-binding proteins such as coagulation factors and protein C. Dramatic increase in the affinity of beta(2)GPI to the cell surface is induced by binding of pathogenic anti-beta(2)GPI antibodies, which may modify the physiological function of beta(2)GPI and may affect the coagulation/fibrinolysis balance on the cell surface. Using chromogenic assays for measuring fibrinolytic activity, we demonstrated that addition of monoclonal anticardiolipin antibody (aCL) decreases the activity of extrinsic/intrinsic fibrinolysis. Significantly lower activity of intrinsic fibrinolysis was also demonstrated in the euglobulin fractions from APS patients. Endothelial cells and monocytes are activated by aPLs in vitro, resulting in production of tissue factor (TF), a major initiator of the coagulation system. Recently, aPLs are reported to induce thrombocytes to produce thromboxane. The importance of apoE receptor 2 on platelets for the binding of artificially dimerized beta(2)GPI was suggested. By investigating aPL-inducible genes in peripheral blood mononuclear cells, we found that the mitogen-activated protein kinase (MAPK) pathway was up-regulated. Using a monocyte cell line, phosphorylation of p38 MAPK, NF-kappaB translocation to the nuclear fraction, and up-regulated TF mRNA expression were demonstrated after treatment with monoclonal aCL. These phenomena were observed only in the presence of beta(2)GPI. Moreover, a specific p38 MAPK inihibitor SB203580 decreased aCL/beta(2)GPI-induced TF mRNA expression. Thus, aCL/beta(2)GPI plays dual roles in the pathogenesis of APS, firstly by deranging the fibrinolytic system and secondly by activating monocytes, endothelial cells and thrombocytes to produce TF or thromboxane.

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Fibrinolysis; Glycoproteins; Monocytes; p38 Mitogen-Activated Protein Kinases; Thromboplastin; Thrombosis

The expression of tissue factor (TF) activity to flowing blood is the trigger for physiological coagulation as well as many types of thrombosis. A growing body of evidence suggests that increased tissue factor activity is a significant contributor towards the hypercoagulability associated with the antiphospholipid syndrome (APS). The increase in tissue factor activity appears to be due to increased transcription and translation of nascent tissue factor molecules but is not due to de-encryption of existing tissue factor molecules on cells. Autoantibodies and/or immune complexes circulating in APS patients appear to enhance the expression of tissue factor activity on monocytes and endothelial cells. Anti-beta2-glycoprotein I (beta2GPI) autoantibodies have been specifically implicated in the antibody-mediated enhancement of tissue factor activity. The presence of antibodies against tissue factor pathway inhibitor (TFPI) in certain APS patients suggests that negative regulation of tissue factor activity might also be impaired in these patients. Given a mechanism involving increased tissue factor activity in APS-associated thrombosis, agents specifically targeting tissue factor activity may be a novel and efficacious therapy that is safer than current approaches to the management of APS.

Topics: Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Female; Glycoproteins; Humans; Lipoproteins; Male; Models, Biological; Monocytes; Pregnancy; Thromboplastin; Thrombosis

The association of antiphospholipid (aPL) antibodies with thrombosis in patients with antiphospholipid syndrome (APS) is well documented in humans and in animal studies. However, the mechanisms by which aPL antibodies induce thrombosis are the subject of much current study. It has been suggested that aPL may activate endothelial cells (ECs), thus creating a hypercoagulable state that precedes and contributes to thrombosis in patients with APS. Several studies have shown that aPL upregulate ECs' adhesion molecules (CAMs): intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (E-sel) or induce tissue factor (TF) in monocytes in vitro. Similarly, the incubation of EC with antibodies reacting with beta2glycoprotein I (beta2GPI) has been shown to induce EC activation with concomitant upregulation of CAMs, IL-6 production and alteration of prostaglandin metabolism. Our group has shown that aPL-mediated upregulation of adhesion molecules on ECs correlates with an increased adhesion of leukocytes to endothelium in the microcirculation of mouse cremaster muscle, a n indication of EC activation in vivo, andwith enhanced thrombosis in vivo. In another series of studies, investigators have shown that upregulation of expression of adhesion molecules by some murine monoclonal anti-beta2glycoprotein I (anti-beta2GPI) antibodies correlated with fetal resorption in mice in vivo. More recently, one study showed that the anti-hypercholesterolaemic drug fluvastatin inhibited the aPL-mediated enhanced adhesion of monocytes to ECs in vitro. Data from our laboratories indicate that fluvastatin also reverses thrombus formation and activation of EC induced by aPL in an in vivo mouse model. As additional support for the hypothesis that aPL antibodies activate ECs and may create an hypercoagulable state in APS patients, two recent studies indicated that levels of soluble ICAM-1 and VCAM-1 were significantly increased in the plasma of patients with APS and recurrent thrombosis. Furthermore, studies utilizing knockout mice and specific monoclonal anti-VCAM-1 antibodies have demonstrated that expression of ICAM-1, P-selectin, E-selectin and VCAM-1 are important in in vivo aPL-mediated thrombosis and EC activation in mice. Recent data suggests that aPL antibodies also induce expression of TF not only in monocytes but in ECs. Hence, the interference of aPL with the TF mechanism may be another important mechanism by which these antibod

Topics: Animals; Antibodies, Anticardiolipin; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cell Adhesion Molecules; Endothelium, Vascular; Humans; Mice; Thromboplastin; Thrombosis

Human tissue factor pathway inhibitor (TFPI) is a modular protein comprised of three Kunitz type domains flanked by peptide segments that are less structured. The sequential order of the elements are: an N-terminal acidic region followed by the first Kunitz domain (K1), a linker region, a second Kunitz domain (K2), a second linker region, the third Kunitz domain (K3), and the C-terminal basic region. The K1 domain inhibits factor VIIa complexed to tissue factor (TF) while the K2 domain inhibits factor Xa. No direct protease inhibiting functions have been demonstrated for the K3 domain. Importantly, the Xa-TFPI complex is a much more potent inhibitor of the VIIa-TF than TFPI by itself. Furthermore, the C-terminal basic region of TFPI is required for rapid physiologic inhibition of coagulation and is needed for the inhibition of smooth muscle cell proliferation. Although a number of additional targets for attachment have been reported, the C-terminal basic region appears to play an important role in binding of TFPI to cell surfaces. A primary site of TFPI synthesis is endothelium and the endothelium-bound TFPI contributes to the antithrombotic potential of the vascular endothelium. Further, increased levels of plasma TFPI under septic conditions may represent endothelial dysfunction. We have proposed that the extravascular cells that synthesize TF also synthesize TFPI providing dual components necessary for the regulation of clotting in their microenvironment. Like the TF synthesis in these cells is augmented by serum, so is the case with the TFPI gene expression. TFPI gene knock out mice reveal embryonic lethality suggesting a possible role of this protein in early development. Since TF-induced coagulation is thought to play a significant role in many disease states, including disseminated intravascular clotting, sepsis, acute lung injury and cancer, recombinant TFPI may be a beneficial therapeutic agent in these disease states to attenuate pathologic clotting. The purpose of this review is to outline recent developments in the field related to the structural specificity and biology of TFPI.

Topics: Acute Disease; Amino Acid Sequence; Amino Acids; Antiphospholipid Syndrome; Blood Coagulation; Cardiovascular Diseases; Endothelium, Vascular; Humans; Lipoproteins; Lung Diseases; Models, Biological; Models, Molecular; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Protein Conformation; Protein Structure, Tertiary; Sepsis; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Thrombophilia; Thromboplastin

Topics: Animals; Anticoagulants; Antiphospholipid Syndrome; Endothelium, Vascular; Hemostatics; Humans; Mice; Monocytes; Thromboplastin; Thrombosis

Expression of tissue factor activity on cells in contact with flowing blood is the trigger for physiological coagulation as well as many types of thrombosis. A number of older observations and considerable recent data suggest that increased tissue factor activity is an important cause of hypercoagulability in the antiphospholipid syndrome. Potential mechanisms contributing to upregulation of the tissue factor pathway include increased expression of tissue factor due to increased transcription, increased functional activity of tissue factor molecules due to de-encryption and decreased activity of tissue factor pathway inhibitor. Autoantibodies and/or immune complexes appear to play a major role in enhanced tissue factor activity, although increased levels of inflammatory cytokines may also contribute. Anti-beta 2-glycoprotein I autoantibodies have been specifically implicated in the antibody-mediated enhancement of tissue factor activity.

Topics: Antiphospholipid Syndrome; Humans; Thromboplastin; Thrombosis

For many years, the laboratory investigation of patients with thrombophilia has lagged behind that of patients with bleeding diathesis. Improved understanding of the mechanisms that control and regulate coagulation, and the resultant recognition of new defects, have greatly stimulated clinical laboratory interest in this area. Assays to detect resistance to activated protein C; deficiencies of antithrombin, protein C, and protein S; and the presence of antiphospholipid antibodies are widely available and should form part of the investigation of patients that present with idiopathic thrombosis. Such a work-up will likely provide an explanation for thrombosis in 40 to 60% of patients. Abnormalities of fibrinogen and fibrinolysis may explain still more, although such defects are currently considered rare. In addition, presently unrecognized defects almost certainly exist, and the identification of such individuals will undoubtedly improve our understanding of the hemostatic mechanism. Laboratory tests to define the hypercoagulable state are continually being developed. They include whole blood coagulation and platelet function tests and novel activation markers. However, acceptance of these approaches by clinical laboratories has been slow.

Topics: Afibrinogenemia; Antiphospholipid Syndrome; Antithrombins; Blood Coagulation Tests; Factor V; Fibrinolysis; Homocysteine; Humans; Lipoproteins; Lupus Coagulation Inhibitor; Monocytes; Platelet Activation; Protein C; Protein S; Protein S Deficiency; Prothrombin; Thrombomodulin; Thrombophilia; Thromboplastin

It is widely hypothesized that autoantibodies directly contribute to the prothrombotic state in the antiphospholipid syndrome (APS). The discovery that antiphospholipid autoantibodies are specific for phospholipid-binding plasma proteins (beta2-glycoprotein I, prothrombin, etc.) has allowed a much more precise investigation of the interactions of autoantibodies and antigens, and the effects of these interaction on hemostasis. Recent studies suggest that two types of interactions may be important in the pathophysiology of APS: (1) antibody cross-linking of membrane bound antigens may alter the kinetics of phospholipid-dependent reactions; and (2) antibody cross-linking of antigens bound to cell surface receptors may trigger signal transduction and cellular activation. In light of these findings, previous reports implicating various mechanisms of autoantibody-mediated thrombosis are being re-evaluated.

Topics: Antibodies, Anticardiolipin; Antigen-Antibody Reactions; Antiphospholipid Syndrome; Autoantibodies; Autoantigens; Autoimmune Diseases; beta 2-Glycoprotein I; Cell Adhesion; Epitopes; Glycoproteins; Hemostasis; Humans; Lupus Coagulation Inhibitor; Membrane Lipids; Models, Immunological; Monocytes; Phospholipids; Plasminogen Activator Inhibitor 1; Thrombophilia; Thromboplastin; Thrombosis

Antiphospholipid-protein antibodies (APA) are a family of immunoglobulins which have been defined by varying laboratory test systems. Lupus anticoagulants (LA) and anticardiolipin antibodies (ACA) are the two most prominent members of this family of antibodies. LA are detected utilizing various phospholipid (PL) dependent tests of coagulation (e.g., activated partial thromboplastin time [APTT], Kaolin Clotting Time [KCT], dilute Russell Viper Venom Time [dRVVT]). Originally, LA were thought to be a laboratory nuisance since the vast majority of individuals with LA did not bleed. Paradoxically, patients with LA were found to have an increased incidence of thromboembolic events and also recurrent spontaneous abortions (RSA). Thus, the laboratory detection of LA has become part of the work up of patients with thromboembolic disorders and RSA. ACA are detected using solid phase assay systems (radioimmunoassay or ELISA). The presence of ACA has the same clinical implications as that of LA. Although originally it was suggested ACA and LA were the same antibody, it is now well accepted that they, in many instances, are different antibodies. Therefore, it is critical for laboratories to evaluate patient samples for both LA and ACA. In approximately 60% of circumstances, both antibodies will be found. In the remaining cases, there will be discordance between the two test systems. The question of whether APA are causative, coincidental, or a consequence of the clinical complications of RSA and thrombosis remains controversial. Recent evidence based on prospective clinical studies and analysis of markers of in vivo coagulation suggests APA are causative.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abortion, Habitual; Adult; Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoimmune Diseases; beta 2-Glycoprotein I; Cattle; Eicosanoids; Endothelium, Vascular; Female; Glycoproteins; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; Pregnancy; Thromboplastin; Thrombosis

Antiphospholipid antibodies are a diverse group of immunoglobulins initially thought to have specificity to phospholipid epitopes. It is apparent that autoimmune anticardiolipin antibodies require a serum cofactor beta-2-glycoprotein I (beta 2GPI) for their binding to phospholipids. Lupus anticoagulant also may bind to phospholipids by beta 2GPI or by prothrombin. The description of binding to protein-phospholipid epitopes may explain several perplexing features of these antibodies both in vitro and in vivo. Antiphospholipid antibodies have a well-established association with clinical disease--in particular thrombosis, thrombocytopenia and recurrent fetal loss. The mechanism of the predisposition to thrombosis seen with these antibodies is poorly understood. It has been suggested that they may cause endothelial dysfunction by causing increased tissue factor expression, by inhibiting prostacyclin secretion or by inhibiting fibrinolysis. Various platelet-activating activities have also been described. The evidence that antiphospholipid antibodies promote thrombosis by effects on endothelium or platelets is inconclusive. Inhibition of protein C activation, or of activated protein C action, has been demonstrated in vitro. A poor correlation between thrombosis in vivo and these inhibitory effects has been found. Beta-2-glycoprotein I has been identified as a cofactor for binding to phospholipid by thrombogenic anticardiolipin antibodies. That beta 2GPI may be a natural anticoagulant of importance remains to be proved. Inhibition by antiphospholipid antibodies of this anticoagulant function could explain the propensity to thrombosis seen in association with these antibodies.

Topics: Antibodies, Anticardiolipin; Antibodies, Antiphospholipid; Antibody Specificity; Antiphospholipid Syndrome; Antithrombin III; Autoimmune Diseases; beta 2-Glycoprotein I; Endothelium, Vascular; Epoprostenol; Fibrinolysis; Glycoproteins; Humans; Lupus Coagulation Inhibitor; Platelet Activation; Protein C; Risk; Thromboplastin; Thrombosis

Antiphospholipid antibodies are autoantibodies that can be detected in plasma or serum with phospholipid-dependent coagulation tests or solid-phase immunoassays. The presence of these autoantibodies is strongly associated with an increased risk for arterial and venous thrombosis, recurrent fetal loss and thrombocytopenia. This paradoxical association of the in vitro prolongation of clotting assays and in vivo thrombosis has stimulated the search for the real antigen to which the autoantibodies are directed. A large number of potential pathological mechanisms have been proposed, and although disturbance of a certain metabolic pathway by the antibodies can explain a thrombotic tendency in one patient, no general pathological mechanism explaining thrombosis in the whole patient population has been found. This suggests that the antiphospholipid antibodies are a heterogeneous group of autoantibodies and is supported by the recent observations that antiphospholipid antibodies are not directed against phospholipids alone but against a combination of phospholipids and phospholipid-binding proteins. Both the phospholipid and the protein are part of the antigen. For the detection of antiphospholipids in an ELISA set-up, beta 2-glycoprotein I is the protein cofactor. In the coagulation tests, beta 2-glycoprotein, as well as prothrombin, can act as cofactor. However, the presence of these two proteins as a part of the epitope of the antiphospholipid antibodies does not explain the thrombotic tendency in the patient group. We have found that more physiologically relevant cofactors such as protein C and protein S, for which it is known that a partial deficiency is correlated with a thrombotic tendency, can also act as cofactors for the binding of antiphospholipid antibodies. It is concluded that antiphospholipid antibodies are a heterogeneous group of autoantibodies with varying affinity for different protein-phospholipid complexes and that inhibition of the biological activity of the protein part of the complex determines the pathological capacity of the antibodies.

Topics: Abortion, Habitual; Antibodies, Antiphospholipid; Antibody Specificity; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Endothelium, Vascular; Epoprostenol; Female; Glycoproteins; Humans; Lupus Coagulation Inhibitor; Monocytes; Phospholipids; Platelet Aggregation; Pregnancy; Protein C; Thrombocytopenia; Thromboembolism; Thrombomodulin; Thromboplastin; von Willebrand Factor

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoimmune Diseases; Blood Platelets; Eicosanoids; Endothelium, Vascular; Female; Fetal Diseases; Fibrinolysis; Humans; Lupus Coagulation Inhibitor; Male; Pregnancy; Pregnancy Complications; Protein C; Protein S; Thromboplastin

To determine if proinflammatory and prothrombotic biomarkers are differentially upregulated in persistently antiphospholipid antibody (aPL)-positive patients, and to examine the effects of fluvastatin on these biomarkers.. Four groups of patients (age 18-65) were recruited: (a) primary antiphospholipid syndrome; (b) systemic lupus erythematosus (SLE) with antiphospholipid syndrome (APS) (SLE/APS); (c) persistent aPL positivity without SLE or APS (Primary aPL); and (d) persistent aPL positivity with SLE but no APS (SLE/aPL). The frequency-matched control group, used for baseline data comparison, was identified from a databank of healthy persons. Patients received fluvastatin 40 mg daily for 3 months. At 3 months, patients stopped the study medication and they were followed for another 3 months. Blood samples for 12 proinflammatory and prothrombotic biomarkers were collected monthly for 6 months.. Based on the comparison of the baseline samples of 41 aPL-positive patients with 30 healthy controls, 9/12 (75%) biomarkers (interleukin (IL)-6, IL1β, vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF)-α, interferon (IFN)-α, inducible protein-10 (IP10), soluble CD40 ligand (sCD40L), soluble tissue factor (sTF) and intracellular cellular adhesion molecule (ICAM)-1) were significantly elevated. Twenty-four patients completed the study; fluvastatin significantly and reversibly reduced the levels of 6/12 (50%) biomarkers (IL1β, VEGF, TNFα, IP10, sCD40L and sTF).. Our prospective mechanistic study demonstrates that proinflammatory and prothrombotic biomarkers, which are differentially upregulated in persistently aPL-positive patients, can be reversibly reduced by fluvastatin. Thus, statin-induced modulation of the aPL effects on target cells can be a valuable future approach in the management of aPL-positive patients.

Topics: Adult; Antiphospholipid Syndrome; Biomarkers; Cell Adhesion Molecules; Cytokines; Fatty Acids, Monounsaturated; Female; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Inflammation Mediators; Intercellular Adhesion Molecule-1; Lupus Erythematosus, Systemic; Male; Middle Aged; Pilot Projects; Prospective Studies; Thromboplastin; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

Antiphospholipid syndrome (APS) is defined by the association of autoantibodies to certain phospholipid-binding proteins with arterial or venous thrombosis ('AT' or 'VT', respectively), and/or pregnancy-related morbidity (PM). Antiphospholipid antibodies (aPLA) promote activation of several cell types including monocytes, resulting in procoagulant tissue factor (TF) expression that may contribute to the vascular complications. Since TF synthesis by monocytes is frequently accompanied by release of TF-bearing microparticles, we hypothesized that plasma microparticle TF activity (MP-TF) may be elevated in APS patients and contribute to thrombosis and/or PM. Platelet-poor plasma specimens were obtained from 30 patients with definite APS and 72 patients with asymptomatic aPLA from the Antiphospholipid Syndrome Collaborative Registry (APSCORE). MP-TF was measured by an in-house factor Xa generation assay. The two groups were well matched for gender, age, ethnicity, proportions with underlying SLE, and aPLA profiles. MP-TF (median and (IQR)) in asymptomatic aPLA subjects was 0.09 pg/mL (0.05-0.14) compared to 0.13 pg/mL (0.10-0.17) in APS (p < 0.001). No differences in MP-TF levels were observed between APS subjects with PM, thrombosis, or PM+thrombosis. Similarly, among subjects with either APS or asymptomatic aPLA, MP-TF did not differ in the presence or absence of underlying SLE. Prospective studies will be required to determine if plasma MP-TF activity is causally related to thrombotic or gestational complications in APS.

Topics: Adult; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cell-Derived Microparticles; Female; Humans; Male; Middle Aged; Pregnancy; Pregnancy Complications; Thromboplastin; Thrombosis

Fluvastatin has been shown to revert proinflammatory/prothrombotic effects of antiphospholipid antibodies (aPL) in vitro and in mice. Here, we examined whether fluvastatin affects the levels of proinflammatory/prothrombotic markers in antiphospholipid syndrome (APS) patients. Vascular endothelial growth factor (VEGF), soluble tissue factor (sTF), tumor necrosis factor-alpha (TNF-alpha), soluble intercellular adhesion molecule-1 (sICAM-1), sE-selectin (E-sel), C-reactive protein (CRP), and soluble vascular cell adhesion molecule (sVCAM-1), were measured in the sera of 93 APS patients and 60 controls and in the sera of nine patients with APS before and after 30 days of treatment with fluvastatin. Elevated levels of VEGF, sTF, and TNF-alpha were found in APS patients. Fluvastatin significantly reduced those markers in the majority of treated subjects. The data from this study show that statins may be beneficial in aPL-positive patients and warrant larger clinical trials to confirm the efficacy of the drug for the treatment of APS clinical manifestations.

Topics: Adult; Aged; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Biomarkers; C-Reactive Protein; E-Selectin; Fatty Acids, Monounsaturated; Female; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Intercellular Adhesion Molecule-1; Male; Middle Aged; Pilot Projects; Thromboplastin; Time Factors; Treatment Outcome; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Contraindications; Endothelium, Vascular; Fatty Acids, Monounsaturated; Fibrinolytic Agents; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Thromboplastin; Up-Regulation

In a first study, we performed a cross-sectional analysis of urinary excretion of isoprostanes, IPF(2alpha-III) and (VI), and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody-positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day(-1), vitamin C at 2000 mg day(-1)) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti-beta(2) glycoprotein 1 (beta(2)GP(1)) or control IgG, either with or without vitamin E. APL-positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL-negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF(2alpha-III) (rho 0.79; P < 0.003) and IPF(2alpha-VI) (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti-beta(2)GP(1) antibodies dose-dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL-positive patients, oxidative stress contributes to activate the clotting system via over-expression of monocyte TF. We suggest that anti-beta(2)GP(1) antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.

Topics: Adult; Antibodies, Antiphospholipid; Antioxidants; Antiphospholipid Syndrome; Cross-Sectional Studies; Dose-Response Relationship, Drug; Female; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; Monocytes; Oxidative Stress; Superoxides; Thromboplastin

Lupus anticoagulants (LA) rarely affect routine prothrombin time assays because the high phospholipid (PL) content in thromboplastin reagents tends to overwhelm the antibodies. Dilution of thromboplastin to create a dilute prothrombin time (dPT) screening test renders the assay sensitive to the presence of LA. Technical and diagnostic performances are enhanced if recombinant thromboplastins are employed in place of tissue-derived reagents. Presence of an LA cannot be deduced from an elevated screening test alone since other coagulation disturbances can prolong clotting times. Confirmatory testing with less dilute or undiluted thromboplastin reveals the PL-dependent nature of LA by reducing the clotting time relative to that of the screening test. Where appropriate, such as a known or suspected coagulation factor deficiency, mixing tests are valuable in correcting factor deficiencies and evidencing inhibitory properties of LA, to increase diagnostic specificity. Although LA testing is commonly restricted to dilute Russell's viper venom time and activated partial thromboplastin time, dPT is sensitive to LA unreactive in those assays, and inclusion in routine testing increases detection rates of clinically significant antibodies.

Topics: Antiphospholipid Syndrome; Blood Coagulation Tests; Humans; Lupus Coagulation Inhibitor; Partial Thromboplastin Time; Phospholipids; Prothrombin Time; Thromboplastin

In this study we analyzed whether anti-β2-GPI antibodies from patients with APS induce the endothelial cell expression of Tissue Factor (TF) by a LRP6 signal transduction pathway involving lipid rafts. HUVEC were stimulated with affinity purified anti-β2-GPI antibodies. Both LRP6 and β-catenin phosphorylation, as well as TF expression, were evaluated by western blot. Results demonstrated that triggering with affinity purified anti-β2-GPI antibodies induced LRP6 phosphorylation with consequent β-catenin activation, leading to TF expression on the cell surface. Interestingly, the lipid rafts affecting agent methyl-β-cyclodextrin as well as the LRP6 inhibitor Dickkopf 1 (DKK1) partially reduced the anti-β2-GPI antibodies effect, indicating that the anti-β2-GPI effects on TF expression may depend on a signalling transduction pathway involving both lipid rafts and LRP6. An interaction between β2-GPI, LRP6 and PAR-2 within these microdomains was demonstrated by gradient fractionation and coimmunoprecipitation experiments. Thus, anti-β2-GPI antibodies react with their target antigen likely associated to LRP6 and PAR-2 within plasma membrane lipid rafts of the endothelial cell. Anti-β2-GPI binding triggers β-catenin phosphorylation, leading to a procoagulant phenotype characterized by TF expression. These findings deal with a novel signal transduction pathway which provides new insight in the APS pathogenesis, improving the knowledge of valuable therapeutic target(s).

Topics: Antiphospholipid Syndrome; beta 2-Glycoprotein I; beta Catenin; Endothelial Cells; Humans; Low Density Lipoprotein Receptor-Related Protein-6; Membrane Microdomains; Signal Transduction; Thromboplastin

Tissue factor (TF) is a procoagulant protein associated with increased risk of thrombotic events in antiphospholipid syndrome (t-APS). The mechanisms by which TF levels are increased in APS have not yet been established. The aim of this study was to investigate whether TF mRNA expression is associated with TF levels and thrombosis in APS. We compared levels of circulating TF and high sensitivity C-reactive protein (hs-CRP) between t-APS and controls (individuals without thrombosis). The association between TF mRNA expression, quantified by real time quantitative polymerase chain reaction, and t-APS was accessed using regression analysis. We included 41 controls and 42 t-APS patients, mean age was 41 years old (SD 14) in both groups. Hs-CRP and TF levels were higher in t-APS patients (mean hs-CRP levels 0.81 mg/dL [SD 1.88] and median TF levels 249.0 pg/mL [IQR 138.77-447.61]) as compared to controls (mean hs-CRP levels 0.24 mg/dL [SD 0.26] and median TF levels 113.0 pg/mL [IQR 81.17-161.53]; P = 0.02 and P < 0.0001, respectively). There was no correlation between TF mRNA expression and TF levels in t-APS (r - 0.209, P = 0.19). TF mRNA expression was not associated with t-APS (adjusted OR 1.16; 95%CI 0.72-1.87). Despite circulating TF levels being higher in patients with t-APS than in controls, TF mRNA expression was similar between groups. The results demonstrate that TF mRNA expression is not associated with levels of circulating TF and hypercoagulability in t-APS.

Topics: Adult; Antiphospholipid Syndrome; Female; Gene Expression; Humans; Male; Middle Aged; RNA, Messenger; Thromboplastin; Thrombosis

Antibodies specific for cardiolipin (CL)-β

Topics: Animals; Antibodies, Antinuclear; Antiphospholipid Syndrome; beta 2-Glycoprotein I; DNA; Gene Expression; Humans; Lupus Erythematosus, Systemic; Mice; Monocytes; Signal Transduction; Thromboplastin; Toll-Like Receptor 9

Antiphospholipid syndrome (APS) is associated with greater atherothrombotic risk and endothelial dysfunction, suggesting that endothelial glycocalyx is impaired in this disease.. The aim was to investigate the endothelial glycocalyx and the relationship between glycocalyx markers, endothelial dysfunction parameters and atherosclerotic markers in APS.. A total of 15 primary arterial APS patients and healthy controls were included in the study. Glycocalyx was assessed in both groups by sublingual sidestream dark field imaging and syndecan-1 plasma level. Endothelial function was evaluated by brachial artery flow-mediated dilatation (FMD) and early atherosclerosis by carotid intima media thickness (IMT). Thrombotic profile was also performed by measuring the plasma level of the tissue factor (TF).. APS patients had significantly increased syndecan-1 plasma level 38.6 ± 5.0 pg/ml vs. 19.1 ± 3.5 pg/ml;. This preliminary study supports, for the first time, that in APS patients endothelial glycocalyx is impaired, which could lead to thrombosis, endothelial dysfunction and early atherosclerosis.

Topics: Adolescent; Adult; Aged; Antiphospholipid Syndrome; Atherosclerosis; Autoantibodies; Biomarkers; Brachial Artery; Carotid Arteries; Carotid Intima-Media Thickness; Case-Control Studies; Cross-Sectional Studies; Endothelium, Vascular; Female; Glycocalyx; Humans; Male; Middle Aged; Risk Factors; Syndecan-1; Thromboplastin; Thrombosis; Vasodilation; Young Adult

Anti-phospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity. It is well known that in these patients thrombosis may be the result of a hypercoagulable state related to anti-β2-glycoprotein I (β2-GPI) antibodies. Moreover, platelets may play a role in thrombotic manifestations by binding of anti-β2-GPI antibodies. Platelets express tissue factor (TF), the major initiator of the clotting cascade, after activation. We primarily analyzed whether anti-β2-GPI antibodies may trigger a signal transduction pathway leading to TF expression in human platelets. Platelets from healthy donors were incubated with affinity purified anti-β2-GPI antibodies for different times. Platelet lysates were analyzed for phospho-interleukin-1 receptor-associated kinase 1 (IRAK), phospho-p65 nuclear factor kappaB (NF-κB) and TF by Western blot. IRAK phosphorylation was observed as early as 10 min of anti-β2-GPI treatment, with consequent NF-κB activation, whereas TF expression, detectable at 45 min, was significantly increased after 4 h of anti-β2-GPI treatment. Virtually no activation was observed following treatment with control immunoglobulin IgG. We then analyzed TF expression in platelets from 20 APS patients and 20 healthy donors. We observed a significant increase of TF in APS patients versus control subjects (P < 0·0001). This work demonstrates that anti-β2-GPI antibodies may trigger in vitro a signal transduction pathway in human platelets, which involves IRAK phosphorylation and NF-κB activation, followed by TF expression. Furthermore, ex vivo, platelets of APS patients showed a significantly increased expression of TF. These findings support the view that platelets may play a role in the pathogenesis of APS, with consequent release of different procoagulant mediators, including TF.

Topics: Adult; Antibody Formation; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Blood Coagulation; Blood Platelets; Cells, Cultured; Female; Humans; Interleukin-1 Receptor-Associated Kinases; Male; Middle Aged; NF-kappa B; Phosphorylation; Signal Transduction; Thromboplastin; Transgenes

Antiphospholipid antibodies (aPL) promote endothelial dysfunction, inflammation and procoagulant state. We investigated the effect of hydroxychloroquine (HCQ) on prothrombotic state and endothelial function in mice and in human aortic endothelial cells (HAEC). Human aPL were injected to C57BL/6 mice treated or not with HCQ. Vascular endothelial function and eNOS were assessed in isolated mesenteric arteries. Thrombosis was assessed both in vitro by measuring thrombin generation time (TGT) and tissue factor (TF) expression and in vivo by the measurement of the time to occlusion in carotid and the total thrombosis area in mesenteric arteries. TGT, TF, and VCAM1 expression were evaluated in HAEC. aPL increased VCAM-1 expression and reduced endothelium dependent relaxation to acetylcholine. In parallel, aPL shortened the time to occlusion and extended thrombus area in mice. This was associated with an overexpression of TF and an increased TGT in mice and in HAEC. HCQ reduced clot formation as well as TGT, and improved endothelial-dependent relaxations. Finally, HCQ increased the p-eNOS/eNOS ratio. This study provides new evidence that HCQ improves procoagulant status and vascular function in APS by modulating eNOS, leading to an improvement in the production of NO.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Female; Humans; Hydroxychloroquine; Male; Mice; Nitric Oxide Synthase Type III; Thrombin; Thromboplastin; Thrombosis; Vascular Cell Adhesion Molecule-1

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thromboembolism and/or pregnancy morbidity in the presence of Antiphospholipid antibodies, mainly anti-β2 glycoprotein I (anti-β2GPI). The autoantibodies lead to monocyte and endothelial cell activation and subsequent secretion of tissue factor (F3) and proinflammatory cytokines, like interleukins 6 (IL6) and 8 (IL8). The etiology of the syndrome remains largely unknown, with the contribution of environmental, genetic and epigenetic factors considered significant.. We aimed to identify epigenetic changes and factors potentially implicated in the pathophysiology of APS. To this end, we compared DNA methylation levels of the IL8 and F3 genes between healthy donors (HDs) and APS patients, using whole blood as a source.. Methylation was significantly reduced in the IL8 promoter and significantly increased in the F3 gene body in APS patients compared to HDs and correlated with specific clinical parameters. In an ex vivo model partially mimicking APS, stimulation of monocytes with a mixture of β2GPI, anti-β2GPI and CXCL4 also induces DNA methylation changes in the above genes, along with increase of their expression. Stimulation of human umbilical vein endothelial cells (HUVECs) with the same mixture also results in transcriptional upregulation of epigenetic factors, including MΕCP2, DNMT3, TET1, HDAC9 and ARID5B.. The above data support that epigenetic alterations could be implicated in the pathophysiology of APS and prompt further investigation of their potential diagnostic or therapeutic utility.

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cells, Cultured; CpG Islands; DNA Methylation; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Promoter Regions, Genetic; Thromboplastin

HCQ has been described as having a beneficial effect in patients with APS but its mechanism of action is unclear. We hypothesized that HCQ may have effects on subnormal angiogenesis, inflammation and haemostatic biomarkers seen in APS. The aim of our study was to assess laboratory markers [annexin A5 (AnxA5) anticoagulant activity, tissue factor (TF) levels, thromboelastography (TEG), CRP, Bb, C3a and VEGF] in HCQ-naïve patients with aPL at baseline and after commencing HCQ.. Twenty-two patients with aPL [20 female, 2 male, median age 55 (range 18-70) years] had blood taken pre- and 3 months after starting HCQ 200 mg daily.. Soluble TF levels were significantly reduced comparing baseline and 3 months after HCQ commencement [401.8 (152.8) vs 300.9 (108) pg/ml (P = 0.010)]. No significant changes were found in the following [reported as pre- and post-HCQ commencement, mean (s.d.)]: AnxA5 anticoagulant ratio [187.1 (29.5) vs 193 (31) (P = 0.157)], anti-domain1 β2 glycoprotein1 IgG activity [1.8 (2) vs 1.2 (1.4) μg/ml (P = 0.105)], complement C3a-des-Arg [147.8 (84.5) vs 154.4 (88.1) ng/ml (P = 0.905)], complement Bb [1.3 (0.7) vs 1.1 (0.7) μg/ml (P = 0.422)], VEGF [68.8 (40) vs 59.4 (19.6) pg/ml (P = 0.454)] and CRP [7 (3.5) vs 7 (3.9) μg/ml (P = 0.917)]. TEG results including TEG reaction time, achievement of clot firmness, TEG maximum amplitude and TEG percentage lysis 30 and 60 min after maximum amplitude showed no significant difference.. HCQ significantly reduced soluble TF levels in patients with aPL. No significant change was observed in AnxA5 activity, anti-domain 1 IgG activity, TEG, CRP, complement Bb and C3a-des-Arg, and VEGF. Further studies of a larger patient cohort are needed.

Topics: Adolescent; Adult; Aged; Annexin A5; Antibodies, Antinuclear; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Antirheumatic Agents; beta 2-Glycoprotein I; C-Reactive Protein; Complement C3a; Complement System Proteins; Hemostasis; Humans; Hydroxychloroquine; Immunoglobulin G; Middle Aged; Neovascularization, Physiologic; Prospective Studies; Thrombelastography; Thromboplastin; Treatment Outcome; Vascular Endothelial Growth Factor A; Young Adult

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS.. We studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity.. aPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression.. We demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.

Topics: 5'-Nucleotidase; Abortion, Spontaneous; Adult; Animals; Antibodies, Antiphospholipid; Antigens, CD; Antiphospholipid Syndrome; Apyrase; Complement C3d; Disease Models, Animal; Female; Humans; Immunization, Passive; Inflammation; Inflammation Mediators; Lipid Peroxidation; Mice; Mice, Knockout; Mice, Transgenic; Pregnancy; Pregnancy Complications; Thromboplastin; Tumor Necrosis Factor-alpha

The mechanisms behind the severe hypercoagulable state in antiphospholipid syndrome (APS) have not yet been fully elucidated. Knowledge on the etiology of thrombosis in APS is needed to improve treatment. We performed a case control study to evaluate the association of the levels of circulating tissue factor (TF) with thrombotic APS and unprovoked venous thromboembolism (VTE), as compared with controls without a history of thrombosis. Study participants were selected in the same geographic area. Linear regression was used to evaluate possible determinants of TF levels among controls and logistic regression was used to evaluate the association between TF, unprovoked VTE and t-APS. TF levels were grouped into three categories based on: below 50th percentile [reference], between 50-75th percentiles [second category] and 75th percentile [third category]. Two hundred and eighty participants were included in the study; 51 patients with unprovoked VTE, 111 patients with t-APS and 118 control individuals. The levels of TF were not associated with an increased risk of unprovoked VTE, as compared with controls. The adjusted odds ratio for t-APS was 2.62 (95%CI 1.03 to 6.62) with TF levels between 50-75th percentiles and 8.62 (95%CI 3.76 to 19.80) with TF levels above the 75th percentile, as compared with the reference category (below the 50th percentile). In the subgroup analysis, higher levels of TF were associated with both arterial and venous thrombosis in APS and with both primary and secondary APS. Circulating TF is associated with thrombotic complications related to APS, but not with the risk of unprovoked VTE.

Topics: Adult; Antiphospholipid Syndrome; Blood Coagulation; Female; Humans; Logistic Models; Male; Middle Aged; Odds Ratio; Thromboplastin; Thrombosis; Venous Thromboembolism; Young Adult

Thrombosis and complement activation are pathogenic features of antiphospholipid syndrome (APS). Their molecular link is Plasma carboxypeptidase-B, also known as thrombin activatable fibrinolysis inhibitor (TAFIa), which plays a dual role: anti-fibrinolytic, by cleaving carboxyl-terminal lysine residues from partially degraded fibrin, and anti-inflammatory, by downregulating complement anaphylatoxins C3a and C5a.. To investigate the levels of TAFI (proenzyme) and TAFIa (active enzyme) in relation to complement activation, fibrin clot permeability and fibrinolytic function in clinical and immunological subsets of 52 APS patients and 15 controls.. TAFI (p<0.001), TAFIa (p<0.05) and complement factor C5a (p<0.001) were increased, while fibrin permeability (p<0.01) was decreased and clot lysis time (CLT) was prolonged (p<0.05) in APS patients compared to controls. Furthermore, TAFIa was increased (p<0.01) in samples from APS patients affected by arterial thrombosis compared to other APS-phenotypes. Positive associations were found between TAFI and age, fibrinogen and C5a, and between TAFIa and age, fibrinogen and thrombomodulin.. TAFI and TAFIa levels were increased in patients with APS as a potential response to complement activation. Interestingly, TAFI activation was associated with arterial thrombotic APS manifestations. Thus, TAFIa may be considered a novel biomarker for arterial thrombosis in APS.

Topics: Adult; Antiphospholipid Syndrome; Carboxypeptidase B2; Complement Activation; Complement C5a; Female; Fibrin; Humans; Male; Thromboplastin

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-β2-glycoprotein I (anti-β2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of β2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

Topics: Animals; Antibodies, Antiphospholipid; Antibodies, Monoclonal, Murine-Derived; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Adhesion; Endothelium, Vascular; Female; Humans; Immunity, Innate; Leukocytes; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Models, Biological; Thromboplastin; Thrombosis; Toll-Like Receptor 4

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β2GPI/β2GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β2GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β2GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β2GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β2GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.

Topics: Animals; Antibodies; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Curcumin; E-Selectin; Immunoglobulin G; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-6; Macrophages, Peritoneal; Male; Mice, Inbred BALB C; NF-kappa B; Phosphorylation; Proline; Real-Time Polymerase Chain Reaction; Thiocarbamates; Thromboplastin; Thrombosis; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Humans; Leukocytes; Thromboplastin; Thrombosis; Venous Thrombosis

The objective of this paper is to elucidate the not yet known plasma molecule candidates involved in the induction of tissue factor (TF) expression mediated by β2GPI-dependent anticardiolipin antibody (aCL/β2GPI) on monocytes.. Human serum incubated with FLAG-β2GPI was applied for affinity chromatography with anti- FLAG antibody. Immunopurified proteins were analyzed by a liquid chromatography coupled with mass spectrometry (LC-MS). TF mRNA induced by the identified molecules on monocytes was also analyzed.. Apolipoprotein B100 (APOB) was the only identified serum molecule in the MS search. Oxidized LDL, containing APOB as well as ox-Lig1 (a known ligand of β2GPI), was revealed as a β2GPI-binding molecule in the immunoprecipitation assay. TF mRNA was markedly induced by oxidized LDL/β2GPI complexes with either WBCAL-1 (monoclonal aCL/β2GPI) or purified IgG from APS patients. The activities of lipoprotein-associated phospholipase A2, one of the component molecules of oxidized LDL, were significantly higher in serum from APS patients than in those from controls.. APOB (or oxidized LDL) was detected as a major β2GPI binding serum molecule by LC-MS search. Oxidized LDL/aCL/β2GPI complexes significantly induced TF expressions on monocytes. These data suggest that complexes of oxidized LDL and aCL/β2GPI may have a crucial role in the pathophysiology of APS.

Topics: Animals; Antibodies, Anticardiolipin; Antiphospholipid Syndrome; Apolipoprotein B-100; beta 2-Glycoprotein I; HEK293 Cells; Humans; Lipoproteins, LDL; Mice; RAW 264.7 Cells; Thromboplastin

In this part of the study, where we determined the causes of preeclampsia and other obstetric complications, we focused on the role of tissue factor (TF) in the activation of these pathophysiological processes. Recent findings attribute a significant part of the activation of coagulation creation of autoantibodies. Once this mechanism is activated, the antibodies induce expression of tissue factor (TF, CD142) on monocytes and vascular endothelial cells.. We have proposed a monitor activation model of the coagulation system in preeclampsia and other pregnancy complications using TF expression on monocytes by flow cytometry and simultaneous determination the TF-induced thrombin generation in plasma. To determine expression of tissue factor (CD142) on monocytes, we proposed a method of multicolor flow cytometry using anti CD45 PerCP, anti CD14 APC, anti CD16b FITC, and anti CD142 PE antibodies and the corresponding isotype controls.. We verified the model on patients with severe antiphospholipid syndrome, which is a high expression of antibodies, in particular against beta-2GPI.. We demonstrated complete inhibition of TF expression on monocytes and a significant reduction of thrombin generation in plasma.

Topics: Adult; Antiphospholipid Syndrome; Blood Coagulation; Female; Flow Cytometry; Humans; Monocytes; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Thromboplastin

The antiphospholipid syndrome is characterized by venous or arterial thrombosis and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies. These antibodies cause activation of endothelial and other cell types leading to the release of microparticles with procoagulant and pro-inflammatory properties. The aims of this study were to characterize the levels of endothelial cell, monocyte or platelet derived, and tissue factor-bearing microparticles in patients with antiphospholipid antibodies, to determine the association of circulating microparticles with anticardiolipin and anti-β2-glycoprotein antibodies, and to define the cellular origin of microparticles that express tissue factor. Microparticle content within citrated blood from 47 patients with antiphospholipid antibodies and 144 healthy controls was analyzed within 2hours of venipuncture. Levels of Annexin-V, CD105 and CD144 (endothelial derived), CD41 (platelet derived) and tissue factor positive microparticles were significantly higher in patients than controls. Though levels of CD14 (monocyte-derived) microparticles in patient plasma were not significantly increased, increased levels of CD14 and tissue factor positive microparticles were observed in patients. Levels of microparticles that stained for CD105 and CD144 showed a positive correlation with IgG (R=0.60, p=0.006) and IgM anti-beta2-glycoprotein I antibodies (R=0.58, p=0.006). The elevation of endothelial and platelet derived microparticles in patients with antiphospholipid antibodies and their correlation with anti-β2-glycoprotein I antibodies suggests a chronic state of vascular cell activation in these individuals and an important role for β2-glycoprotein I in development of the pro-thrombotic state associated with antiphospholipid antibodies.

Topics: Adult; Antibodies, Anticardiolipin; Antibodies, Antiphospholipid; Antigens, CD; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Blood Platelets; Cadherins; Cell-Derived Microparticles; Endoglin; Endothelial Cells; Female; Flow Cytometry; Humans; Lipopolysaccharide Receptors; Male; Middle Aged; Partial Thromboplastin Time; Receptors, Cell Surface; Thromboplastin; Thrombosis

to analyse the effects of immunoglobulin(Ig)G and IgM anti-beta-2 glycoprotein-1 (anti-2GP1) on the expression of tissue factor (TF), thrombomodulin (TM), and plasminogen activator inhibitor-1(PAI-1) of endothelial cells in the messenger RNA level.. laboratory experimental study in human umbilical vein endothelial cells (HUVEC) was done at Cipto Mangunkusumo Hospital/Faculty of Medicine, Universitas Indonesia. Samples are purified IgG anti-2GP1 from six antiphospholipid syndrome (APS) patients serum and IgM anti-2GP1 from six APS patients serum. For controls, purified IgG from six normal human serum (IgM-NHS) and purified IgM from six normal human serum (IgM-NHS) were used. HUVEC were treated with purified IgG anti-2GP1, IgM anti-2GP1, IgG-NHS, IgM-NHS for four hours of incubation. We measured TF, TM, and PAI-1 of HUVEC in mRNA relative expression levels (before and after treatment) by real time reverse transcription polymerase chain reaction.. the mean value of TF, TM, and PAI-1 mRNA levels in HUVEC after treated with IgG anti-2GP1 compared to Ig-NHS were 3.14 (0.93)-, 0.31 (0.13)-, 5.33 (2.75)-fold respectively. In other hand, after treated with IgM anti-2GP1 compared to IgM-NHS, mRNA levels of TF, TM, and PAI-1 were 4.33 (1.98)-, 0.33 (0.22)-, 5.47 (2.64)-fold respectively. Before and after treatment with IgG anti-2GP1 showed significant differences of TF mRNA levels {1.09 (0.76) versus 3.14 (0.93), p=0.003}, TM mRNA levels {0.91 (0.11) versus 0.31(0.13), p=0.001}, and PAI-1 mRNA levels 0.93 (0.13) versus 5.33 (2.75), p=0.013}. Before and after treatment with IgM anti-2GP1 showed significant differences of TF mRNA levels {1.03 (0.11) versus 4.33 (1.98), p=0.008}, TM mRNA levels {0.93 (0.08) versus 0.33 (0.22, p=0.003}, and PAI-1 mRNA levels {1.02 (0.10) versus 5.47 (2.64), p=0.01}.. IgG anti-2GP1 and IgM anti-2GP1 increased TF and PAI-1 mRNA levels. However, IgG anti-2GP1 and IgM anti-2GP1 decreased TM mRNA levels. It proved that the mechanism of thrombosis in APS occurs through coagulation activation, reduction of fibrinolysis activity, and reduction of anticoagulant activity.

Topics: Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cells, Cultured; Human Umbilical Vein Endothelial Cells; Humans; Immunoglobulin G; Immunoglobulin M; Indonesia; Plasminogen Activator Inhibitor 1; RNA, Messenger; Thrombomodulin; Thromboplastin

Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. β2 glycoprotein I (β2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, β2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to β2GPI and its relationship with atherosclerotic process.. Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte β2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to β2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte β2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte β2GPI and TF with IMT or FMD was detected. β2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to β2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm.. A significant percentage of patients with SLE and PAPS show a β2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

Topics: Adult; Aged; Antibody Specificity; Antiphospholipid Syndrome; Atherosclerosis; Autoantibodies; Autoantigens; beta 2-Glycoprotein I; Blood Pressure; Cardiovascular Diseases; Carotid Intima-Media Thickness; Cell Division; Endothelium, Vascular; Female; Hemorheology; Humans; Interferon-gamma Release Tests; Lipoproteins, HDL; Lupus Erythematosus, Systemic; Male; Middle Aged; Monocytes; Risk Factors; T-Cell Antigen Receptor Specificity; Thromboplastin; Vasodilation; Young Adult

CD36, known as a scavenger receptor, is a transmembrane glycoprotein expressed on monocytes, platelets and endothelial cells, recognizes multiple ligands, including phosphatidylserine, and regulates atherogenesis and thrombosis. The objective of this study is to investigate the possible involvement of CD36 in the pathophysiology of thrombosis in patients with antiphospholipid syndrome (APS).. First, rs3765187, a missense mutation linked to CD36 deficiency, was investigated by TaqMan polymerase chain reaction (PCR) genotyping method in 819 Japanese, including 132 patients with APS, 265 with systemic lupus erythematosus (SLE) in the absence of APS, and 422 healthy subjects. Then, the involvement of CD36 in antiphospholipid antibody (aPL)-induced tissue factor (TF) expression was examined using CD36-null mice or anti-CD36. Purified IgG from patients with APS and a monoclonal phosphatidylserine-dependent antiprothrombin antibody were used in these experiments. TF expression was tested by real-time PCR and flow cytometry.. Minor allele carrier of rs3765187 was less frequent in patients with APS (3.8% p=0.032), but not in patients with SLE in the absence of APS (7.9% p=0.32), compared with healthy subjects (10.2%). The aPL-induced TF expression was significantly suppressed on peritoneal macrophages from CD36-null mice compared to wild type and significantly inhibited by anti-CD36 on human monocytes.. The gene mutation linked to CD36 deficiency was less frequent in patients with APS. The deficient or suppressed CD36 function significantly reduced aPL-induced TF expression in vitro. Taken together, in a susceptible background CD36 scavenger receptor function may be involved in the thrombotic pathophysiology in patients with APS.

Topics: Adolescent; Adult; Aged; Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Case-Control Studies; CD36 Antigens; Female; Flow Cytometry; Genotype; Humans; Japan; Lupus Erythematosus, Systemic; Macrophages, Peritoneal; Male; Mice; Mice, Knockout; Middle Aged; Monocytes; Mutation, Missense; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Thromboplastin; Thrombosis; Young Adult

The aim of this study was to investigate the effects of phosphatidylserine-dependent antiprothrombin antibody (aPS/PT) on the expression of tissue factor (TF) and the signal transduction pathway in procoagulant cells.. Peripheral blood mononuclear cells (PBMCs) from a healthy donor, murine monocyte RAW264.7 cells and human umbilical vein endothelial cells (HUVECs) were treated with either IgG fractions obtained from APS patients who were positive for aPS/PT or a murine monoclonal aPS/PT antibody, 231D, in the presence of prothrombin. The levels of TF mRNA were measured using real-time PCR. TF function, as measured by procoagulant activity, was determined with a clotting assay. 231D binding on the surface of treated cells was determined by flow cytometric analysis. Screening for phosphorylation of intracellular signalling proteins was conducted using an array assay. Phosphorylation of p38 MAPK was quantitatively analysed with ELISA, and SB203580 was used as a specific inhibitor of p38 MAPK. Specific siRNA for p38 MAPK was used for the knockdown assay.. The IgG fractions from APS patients and 231D induced TF mRNA overexpression and shortening of coagulation time in cells in the presence of prothrombin. The 231D moiety induced phosphorylation of p38 MAPK after binding to the cell surface of RAW264.7 cells. SB203580 or p38 siRNA significantly hampered TF overexpression.. Expression of TF in procoagulant cells was induced by aPS/PT via p38MAPK phosphorylation. This phenomenon may be correlated with the thrombogenicity of APS.

Topics: Adult; Aged; Animals; Antiphospholipid Syndrome; Blood Coagulation; Cells, Cultured; Endothelium, Vascular; Female; Gene Expression Regulation; Humans; Immunoglobulin G; Leukocytes, Mononuclear; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Middle Aged; Monocytes; p38 Mitogen-Activated Protein Kinases; Phosphatidylserines; Phosphorylation; Protein Kinase Inhibitors; Prothrombin; RNA, Messenger; RNA, Small Interfering; Thromboplastin; Umbilical Veins; Up-Regulation

Our previous study demonstrated that Toll-like receptor 4 (TLR4) could act as a co-receptor with annexin A2 (ANX2) mediating anti-β2-glycoprotein I/β2-glycoprotein I (anti-β(2)GPI/β(2)GPI)-induced tissue factor (TF) expression in human acute monocytic leukemia cell line THP-1. In the current study, we further explored the roles of TLR4 and its adaptors, MyD88 and TRIF, in anti-β(2)GPI/β(2)GPI-induced the activation of human blood monocytes and THP-1 cells and the relationship among TLR4, β(2)GPI and ANX2 in this process. The results showed that treatment of monocytes or THP-1 cells with anti-β(2)GPI/β(2)GPI complex could increase TF, MyD88, TRIF as well as TNF-α (tumor necrosis factor alpha) expression. These effects were blocked by addition of TAK-242, a blocker of signaling transduction mediated by the intracellular domain of TLR4. Moreover, TLR4/β(2)GPI/ANX2 complex could be detected in THP-1 cell lysates. Overall, our results indicate that anti-β(2)GPI/β(2)GPI complex induced TF and TNF-α expression involving both TLR4/MyD88 and TLR4/TRIF signaling pathways and TLR4 and its adaptors might be molecular targets for therapy of antiphospholipid syndrome (APS).

Topics: Adaptor Proteins, Vesicular Transport; Annexin A2; Antigen-Antibody Complex; Antiphospholipid Syndrome; Base Sequence; beta 2-Glycoprotein I; Cell Line; DNA Primers; Gene Expression; Humans; In Vitro Techniques; Monocytes; Myeloid Differentiation Factor 88; RNA, Messenger; Signal Transduction; Sulfonamides; Thromboplastin; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Antiphospholipid antibodies (aPL) have been shown to induce tissue factor (TF) expression in monocytes and endothelial cells. However, the underlying signal transduction has been more or less elusive in the past. We have recently shown that aPL enter the lysosomal route in monocytes and dendritic cells, and subsequently activate endosomal NADPH-oxidase (NOX). The generation of superoxide which is dismutated to hydrogen peroxide upregulates the intracellular toll like receptors (TLR) 7 and 8, and leads to robust production of inflammatory cytokines. Here we show that induction of TF by aPL follows the same signaling pathway. Inhibition of endosomal NOX by the anion channel blocker niflumic acid or capture of superoxide by the radical scavenger N-acetylcysteine blocks TF induction by aPL. Furthermore, monocytes from mice deficient in NOX2 do not increase TF surface expression in response to aPL, while cells from mice deficient in glutathione peroxidase-1 (GPx-1) show an increased response. Unexpectedly, also induction of TF by tumour necrosis factor (TNF)α and lipopolysaccharide (LPS) was strongly dependent on the activation of endosomal NOX. While TNFα apparently depends alm ost fully on endosomal NOX, signalling of LPS is only partially dependent on this pathway. These data provide further insight into the well-known role of reactive oxygen species in the induction of TF expression and suggest that endosomal signalling may represent a central coordinating point in this process.

Topics: Acetylcysteine; Animals; Antiphospholipid Syndrome; Endosomes; Endothelial Cells; Female; Free Radical Scavengers; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen Peroxide; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Monocytes; NADPH Oxidase 2; NADPH Oxidases; Niflumic Acid; Superoxides; Thromboplastin; Tumor Necrosis Factor-alpha

In clinical practice it is possible to find patients with clinical signs suggestive of anti-phospholipid syndrome (APS) who are persistently negative for the routinely used anti-phospholipid antibodies (aPL). Therefore, the term proposed for these cases was seronegative APS (SN-APS). We investigated the clinical usefulness of thin-layer chromatography (TLC) immunostaining in detecting serum aPL in patients presenting clinical features of SN-APS. Sera from 36 patients with SN-APS, 19 patients with APS, 18 patients with systemic lupus erythematosus (SLE), 20 anti-hepatitis C virus (HCV)-positive subjects and 32 healthy controls were examined for aPL using TLC immunostaining. Anti-β(2) -glycoprotein-I, anti-annexin II, anti-annexin V and anti-prothrombin antibodies were tested by enzyme-linked immunosorbent assays (ELISA). Eahy926, a human-derived endothelial cell line, was incubated with immunoglobulin (Ig)G fraction from SN-APS patients and analysis of phospho-interleukin (IL)-1 receptor-associated kinase (IRAK) and phospho-nuclear factor (NF)-κB was performed by Western blot, vascular cell adhesion molecule 1 (VCAM-1) expression by cytofluorimetric analysis and supernatants tissue factor (TF) levels by ELISA. TLC immunostaining showed aPL in 58·3% of SN-APS patients: anti-cardiolipin in 47·2%, anti-lyso(bis)phosphatidic acid in 41·7% and anti-phosphatidylethanolamine in 30·5%. Six of 36 patients showed anti-annexin II. Incubation of Eahy926 cells with IgG from SN-APS induced IRAK phosphorylation, NF-κB activation, VCAM-1 surface expression and TF cell release. TLC immunostaining could identify the presence of aPL in patients with SN-APS. Moreover, the results suggest the proinflammatory and procoagulant effects in vitro of these antibodies.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Case-Control Studies; Cell Line; Chromatography, Thin Layer; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Interleukin-1 Receptor-Associated Kinases; Male; Middle Aged; NF-kappa B; Phosphorylation; Thromboplastin; Vascular Cell Adhesion Molecule-1; Young Adult

Complement activation plays a role in pathogenesis of the antiphospholipid syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6(-/-)) mice.. C6(-/-) mice or the wild-type C3H/HeJ (C6(+/+)) mice were injected twice with IgG-APS (n = 2) or IgM-APS (n = 1) isolated from APS patients or with the corresponding control immunoglobulins (Igs) of normal human serum, (NHS) (IgG-NHS or IgM-NHS). Then, the sizes of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages.. Thrombus sizes were significantly larger in C6(+/+) treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6(+/+) mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6(-/-) mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p < 0.001), compared to their C6(+/+) counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6(-/-) mice treated with IgG-APS or IgM-APS were diminished when compared to C3H/HeJ (C6(+/+)) mice treated with the same Igs. All mice injected with IgG-APS and IgM-APS had medium-high titers of anticardiolipin (aCL) and anti-β(2)glycoprotein I (aβ(2)GPI) antibodies.. These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations.

Topics: Adult; Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Carotid Arteries; Complement C6; Female; Femoral Vein; Humans; Immunoglobulin G; Immunoglobulin M; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C3H; Mice, Knockout; Middle Aged; Thrombophilia; Thromboplastin; Thrombosis; Up-Regulation

Our previous study has demonstrated that the Toll-like receptor 4 (TLR4) signaling pathways contribute to the induction of tissue factor (TF) expression by anti-β(2)-glycoprotein I/β(2)-glycoprotein I (anti-β(2)GPI/β(2)GPI) in human acute monocytic leukemia cell line THP-1. In this study, we focused on the identification of the downstream targets of the TLR4 pathways. When THP-1 cells were treated with anti-β(2)GPI/β(2)GPI complex, enhanced TF expression was observed, along with induced phosphorylation of p38, ERK1/2 and JNK1/2 MAPKs. When the activity of MAPKs was blocked by their corresponding inhibitors (SB203580: p38; U0126: ERK; SP600125: JNK), the expression of TF was reduced significantly. Furthermore, the anti-β(2)GPI/β(2)GPI-induced phosphorylation of p38, ERK1/2 and JNK1/2 was inhibited significantly by TAK-242, a blocker of the signaling transduction mediated by the intracellular domain of TLR4; sc-204013, a specific inhibitor of IRAKs, was also able to partially inhibit the phosphorylation of the MAPKs. Our results demonstrated that MAPKs (p38, ERK1/2 and JNK1/2) were the crucial downstream targets of the anti-β(2)GPI/β(2)GPI-triggered TLR4 signaling pathways in THP-1 cells. This essential role of MAPKs may also promote better understanding of the pathogenesis of antiphospholipid syndrome (APS).

Topics: Antigen-Antibody Complex; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Line, Tumor; Enzyme Activation; Gene Expression Regulation, Leukemic; Humans; Interleukin-1 Receptor-Associated Kinases; Leukemia, Monocytic, Acute; MAP Kinase Signaling System; Sulfonamides; Thromboplastin; Toll-Like Receptor 4

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterised by thrombosis, obstetric complications and the presence of anti-phospholipid antibodies such as anti-β2GPI-Abs. These antibodies may set off the coagulation cascade via several mechanisms, including the induction of tissue factor (TF) expression. Vitamin D has recently emerged as an immunomodulator that might exert an anti-thrombotic effect. Therefore, we studied serum vitamin D levels in a cohort of APS patients, as well as the effect of vitamin D in an in vitro model of APS-mediated thrombosis.. Serum vitamin D levels were measured in 179 European APS patients and 141 healthy controls using the LIAISON chemiluminescent immunoassay, and the levels were evaluated in conjunction with a wide spectrum of clinical manifestations. In an vitro model, anti-β2GPI antibodies were purified from four patients with APS to evaluate the expression of TF in activated starved human umbilical vein endothelial cells. The effect of vitamin D (1,25-dihydroxyvitamin D, 10 nm) on anti-β2GPI-Abs mediated TF expression was analysed by immunoblot.. Vitamin D deficiency (serum level ≤15 ng/ml) was documented in 49.5% of our APS patients versus 30% of controls (p<0.001) and was significantly correlated with thrombosis (58% vs 42%; p<0.05), neurological and ophthalmic manifestations, pulmonary hypertension, livedo reticularis and skin ulcerations. In vitro vitamin D inhibited the expression of TF induced by anti-β2GPI-antibodies.. Vitamin D deficiency is common among APS patients and is associated with clinically defined thrombotic events. Vitamin D inhibits anti-β2GPI-mediated TF expression in vitro. Thus, vitamin D deficiency might be associated with decreased inhibition of TF expression and increased coagulation in APS. Evaluation of vitamin D status and vitamin D supplementation in APS patients should be considered.

Topics: Adult; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Case-Control Studies; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Female; Humans; Male; Middle Aged; Thromboplastin; Thrombosis; Vitamin D; Vitamin D Deficiency; Vitamins

Our previous study demonstrated that annexin A2 (ANX2) on cell surface could function as a mediator and stimulate tissue factor (TF) expression of monocytes by anti-β₂-glycoprotein I/β₂-glycoprotein I complex (anti-β₂GPI/β₂GPI). However, ANX2 is not a transmembrane protein and lacks the intracellular signal transduction pathway. Growing evidence suggests that Toll-like receptor 4 (TLR-4) might act as an 'adaptor' for intracellular signal transduction in anti-β₂GPI/β₂GPI-induced TF expressing cells. In the current study, we investigated the roles of TLR-4 and its related molecules, myeloid differentiation protein 2 (MD-2) and myeloid differentiation factor 88 (MyD88), in anti-β₂GPI/β₂GPI-induced TF expressing human monocytic-derived THP-1 (human acute monocytic leukaemia) cells. The relationship of TLR-4 and ANX2 in this process was also explored. Along with TF, expression of TLR-4, MD-2 and MyD88 in THP-1 cells increased significantly when treated by anti-β₂GPI (10 µg/ml)/β₂GPI (100 µg/ml) complex. The addition of paclitaxel, which competes with the MD-2 ligand, could inhibit the effects of anti-β₂GPI/β₂GPI on TLR-4, MD-2, MyD88 and TF expression. Both ANX2 and TLR-4 in THP-1 cell lysates could bind to β₂GPI that had been conjugated to a column (β₂GPI-Affi-Gel). Furthermore, TLR-4, MD-2, MyD88 and TF expression was remarkably diminished in THP-1 cells infected with ANX2-specific RNA interference (RNAi) lentivirus (LV-RNAi-ANX2), in spite of treatment with a similar concentration of anti-β₂GPI/β₂GPI complex. These results indicate that TLR-4 and its signal transduction pathway contribute to anti-β₂GPI/β₂GPI-induced TF expression in THP-1 cells, and the effects of TLR-4 with ANX2 are tightly co-operative.

Topics: Annexin A2; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Line; Humans; Lymphocyte Antigen 96; Monocytes; Myeloid Differentiation Factor 88; Paclitaxel; Thromboplastin; Toll-Like Receptor 4

Numerous mechanisms have been proposed to explain the thrombotic/proinflammatory tendency of antiphospholipid syndrome (APS) patients. Prothrombotic monocyte activation by antiphospholipid antibodies involves numerous proteins and intracellular pathways. The anti-inflammatory, anticoagulant and immunoregulatory effects of statins have been aimed as a therapeutic tool in APS patients. This study delineates the global effects of fluvastatin on the prothrombotic tendency of monocytes from APS patients.. Forty-two APS patients with thrombosis and 35 healthy donors were included in the study. APS patients received 20 mg/day fluvastatin for 1 month. Blood samples were obtained before the start, at the end and 2 months after the end of treatment.. After 1 month of treatment, monocytes showed a significant inhibition of tissue factor, protein activator receptors 1 and 2, vascular endothelial growth factor and Flt1 expression that was related to the inhibition of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B/Rel DNA-binding activity. Proteomic analysis showed proteins involved in thrombotic development (annexin II, RhoA and protein disulphide isomerase) with altered expression after fluvastatin administration. In-vitro studies indicated that the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by fluvastatin might inhibit protein prenylation and MAPK activation.. The data from this study support the belief that fluvastatin has multiple profound effects in monocyte activity, which might contribute to thrombosis prevention in APS patients.

Topics: Adult; Antiphospholipid Syndrome; Case-Control Studies; Cells, Cultured; Fatty Acids, Monounsaturated; Female; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Male; Middle Aged; Monocytes; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proteomics; Receptors, Proteinase-Activated; Thromboplastin; Thrombosis; Vascular Endothelial Growth Factor A

Antiphospholipid syndrome (APS) is characterized by thromboembolic phenomena and recurrent fetal loss associated with elevated circulating anti-phospholipid/beta2glycoprotein-I(β2GPI)-binding-antibodies(Abs). Individual APS patients harbor diverse clusters of circulating anti-β2GPI Abs, targeting different epitopes on the β2GPI molecule. Our novel approach was to construct a peptide composed of β2GPI-ECs-binding-site (phospholipids-membrane), named "EMBI". EMBI exert dual activities: a) At first EMBI prevented β2GPI ECs binding, thus reduced by 89% the binding of β2GPI/anti-β2GPI to the cells in comparison with 9.3% inhibition by EMBI scrambled form (scEMBI). b) Longer exposure of ECs to EMBI resulted in intracellular EMBI penetration which did not prevent β2GPI/anti-β2GPI binding to HUVEC. Surprisingly, β2GPI/anti-β2GPI did not activate ECs harboring EMBI, illustrated by prevention of E-selectin and tissue factor (TF) expression. The inhibition of TF mRNA transcription was illustrated by quantitative RT-PCR. EMBI decreased the expression of phosphorylated JNK1/2, p38, HSP27 and enhanced phosphorylation of glycogen synthase kinase-3β (pGSK3β). Knocking down the GSK3β expression by siRNA-GSK3β, reduced the TF expression by β2GPI/anti-β2GPI-exposed-HUVEC. In-vivo, EMBI significantly decreased the percentage of fetal loss in naïve mice infused with anti-β2GPI Abs, p<0.04. Thus, the dual activity of EMBI may introduce EMBI as a potential novel candidate peptide, to treat patients with APS.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Disease Models, Animal; E-Selectin; Endothelial Cells; Enzyme Inhibitors; Female; Fetal Death; Gene Expression Regulation, Enzymologic; Glycogen Synthase Kinase 3; Humans; Male; Mice; Mice, Inbred BALB C; Peptides; Phospholipids; Phosphorylation; Protein Binding; Protein Conformation; RNA, Messenger; Thromboplastin

Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes.. To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients.. An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP-1 and MDA-MB-231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR-20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT-PCR) in patients with APS and SLE.. Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR-17∼92, for example miR-19b and miR-20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR-20a binds to TF mRNA. Finally, we measured levels of miR-19b and miR-20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF.. Down-regulation of miR-19b and miR-20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.

Topics: Adult; Aged; Antiphospholipid Syndrome; Blotting, Western; Case-Control Studies; Cell Line; Female; Humans; Lupus Erythematosus, Systemic; Male; MicroRNAs; Middle Aged; Real-Time Polymerase Chain Reaction; Thromboplastin

Our previous study has shown that Toll-like receptor 4 (TLR4) and its signalling pathway contribute to anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI)-induced tissue factor (TF) expression in human acute monocytic leukaemia cell line THP-1 and annexin A2 (ANX2) is involved in this pathway. However, its downstream molecules have not been well explored. In this study, we have established that interleukin-1 receptor-associated kinases (IRAKs) and tumour necrosis factor receptor-associated factors (TRAFs) are crucial downstream molecules of anti-β₂GPI/β₂GPI-induced TLR4 signaling pathway in THP-1 cells and explored the potential mechanisms of their self-regulation. Treatment of THP-1 cells with anti-β₂GPI/β₂GPI complex induced IRAKs and TRAFs expression and activation. Anti-β₂GPI/β₂GPI complex firstly induced expression of IRAK4 and IRAK1, then IRAK1 phosphorylation and last IRAK3 upregulation. In addition, anti-β₂GPI/β₂GPI complex simultaneously and acutely enhanced mRNA levels of TRAF6, TRAF4 and zinc finger protein A20 (A20), while chronically increased A20 protein level. Moreover, anti-β₂GPI/β₂GPI complex-induced IRAKs and TRAFs expression and activation were attenuated by knockdown of ANX2 by infection with ANX2-specific RNA interference lentiviruses (LV-RNAi-ANX2) or by treatment with paclitaxel, which inhibits TLR4 as an antagonist of myeloid differentiation protein 2 (MD-2) ligand. Furthermore, both IRAK1/4 inhibitor and a specific proteasome inhibitor MG-132 could attenuate TRAFs expression as well as TF expression induced by anti-β₂GPI/β₂GPI complex. In conclusion, our results indicate that IRAKs and TRAFs play important roles in anti-β₂GPI/β₂GPI-stimulated TLR4/TF signaling pathway in THP-1 cells and contribute to the pathological processes of antiphospholipid syndrome (APS).

Topics: Annexin A2; Antibodies; Antigen-Antibody Complex; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation; Humans; Interleukin-1 Receptor-Associated Kinases; Leupeptins; Monocytes; Paclitaxel; RNA, Small Interfering; Signal Transduction; Thromboplastin; TNF Receptor-Associated Factor 4; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

Prothrombin (PT) is one of the most important antigenic targets for aPL antibodies; however, the prothrombotic mechanism of anti-PT (aPT) antibodies in APS is not fully clarified. Considering that some autoantibodies possess the enzymatic activity, the aim of this study was to test the hypothesis that some aPT antibodies in APS may display prothrombinase activity.. Six APS patient-derived PT-reactive monoclonal antibodies (mAbs) were analysed for prothrombinase activity on PT. One mAb with prothrombinase activity was examined for its proteolytic activity on PT. In addition, IgG was purified from plasma samples positive with IgG aPT antibodies, and their prothrombinase activity analysed.. Initial analysis of six mAbs revealed that, upon incubation with PT, IS6 mAb displayed prothrombinase activity and catalysed the proteolysis of PT to fragments. Analysis of plasma samples revealed that 9/21 (42.8%) APS patients had IgG antibodies against PT, based on a cut-off value equal to mean + 3 S.D. of the level in 21 normal controls. Importantly, of those samples positive for IgG aPT antibodies, two polyclonal IgG (P1 and P2) also displayed prothrombinase activity.. In this study, we showed that some aPT antibodies displayed prothrombinase activity. Such catalytic aPT antibodies may contribute to thrombosis in APS.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Antiphospholipid Syndrome; Autoantibodies; Case-Control Studies; Catalysis; Child; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Male; Middle Aged; Prothrombin; Thrombin; Thromboplastin; Young Adult

To investigate the expression of protease-activated receptors (PARs), their potential regulation by anticardiolipin antibodies (aCL), and their association with the expression of other molecules relevant to thrombosis in monocytes obtained from 62 patients with primary antiphospholipid syndrome (APS).. Monocytes were isolated from peripheral blood mononuclear cells by magnetic depletion of nonmonocytes. Expression of tissue factor (TF) and PARs 1-4 genes was measured by quantitative real-time reverse transcription-polymerase chain reaction. Cell surface TF and PARs 1-4 expression was analyzed by flow cytometry. For in vitro studies, purified normal monocytes were incubated with purified APS patient IgG, normal human serum IgG, or lipopolysaccharide, in the presence or absence of specific monoclonal antibodies anti-PAR-1 (ATAP2) or anti-PAR-2 (SAM11) to test the effect of blocking the active site of PAR-1 or PAR-2.. Analysis of both mRNA and protein for the 4 PARs revealed significantly increased expression of PAR-2 as compared with the control groups. PAR-1 was significantly overexpressed in APS patients with thrombosis and in the control patients with thrombosis but without APS. PAR-3 expression was not significantly altered. PAR-4 expression was absent in all groups analyzed. In addition, we demonstrated a correlation between the levels of PAR-2 and the titers of IgG aCL, as well as parallel behavior of TF and PAR-2 expression. In vitro, IgG from APS patients significantly increased monocyte expression of PAR-1 and PAR-2. Inhibition studies suggested that there was direct cross-talk between TF and PAR-2, such that inhibition of PAR-2 prevented the aCL-induced expression of TF.. These results provide the first demonstration of increased expression of PARs in monocytes from patients with APS. Thus, PAR antagonists might have therapeutic potential as antithrombotic agents in APS.

Topics: Adult; Antibodies, Anticardiolipin; Antibodies, Monoclonal; Antiphospholipid Syndrome; Female; Flow Cytometry; Humans; Immunoglobulin G; Male; Middle Aged; Monocytes; Receptor Cross-Talk; Receptors, Proteinase-Activated; Reverse Transcriptase Polymerase Chain Reaction; RNA; Thromboplastin; Thrombosis

A major mechanism of hypercoagulability in the antiphospholipid syndrome (APS) is antiphospholipid Ab-mediated upregulation of tissue factor (TF) on monocytes via activation of TLRs, p38 MAPK, and NF-kappaB pathways. We examined whether monocyte signaling pathways are differentially activated by IgG from patients with vascular thrombosis (VT) alone compared with IgG from patients with pregnancy morbidity (PM) alone. We purified IgG from 49 subjects. A human monocyte cell line and ex vivo healthy monocytes were treated with 100 microg/ml IgG for 6 h, and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-kappaB. To further investigate intracellular signaling pathways induced by these IgGs, specific inhibitors of p38 MAPK, NF-kappaB, TLR4, and TLR2 were used to determine their effect on TF activity. Only IgG from patients with VT but no PM (VT+/PM-) caused phosphorylation of NF-kappaBand p38 MAPK and upregulation of TF activity in monocytes. These effects were not seen with IgG from patients with PM alone (VT-/PM+), anti-phospholipid Ab-positive patients without APS, or healthy controls. TF upregulation caused by the VT+/PM- samples was reduced by inhibitors of p38 MAPK, NF-kappaB, and TLR4. The effects of VT+/PM- IgG on signaling and TF upregulation were concentrated in the fraction that bound beta-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-kappaB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4.

Topics: Adult; Antiphospholipid Syndrome; Blotting, Western; Female; Humans; Immunoglobulin G; Male; Middle Aged; Monocytes; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pregnancy; Pregnancy Complications; Signal Transduction; Thromboplastin; Toll-Like Receptor 4

Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

Topics: Animals; Annexin A2; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Aorta; Blotting, Western; Cardiovascular Diseases; Carotid Arteries; Cells, Cultured; E-Selectin; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; In Vitro Techniques; Intercellular Adhesion Molecule-1; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Thromboplastin; Umbilical Veins; Vascular Cell Adhesion Molecule-1

Among the heterogeneous antiphospholipid antibodies, many studies suggest that those directed to beta2-glycoprotein I (beta2GPI) are the major pathogenic antibodies in antiphospholipid syndrome (APS). They have been shown to activate the coagulation pathway via several mechanisms, activate platelets via thrombin formation, and suppress fibrinolysis. Additionally, we propose another possible mechanism that involves certain chemokines and results in platelet activation. This hypothesis is based on the observations that anti-beta2GPI antibodies stimulated monocytes to secrete inflammatory cytokines such as IL-1beta and TNF-alpha, which in turn stimulated vascular endothelial cells to express chemokines such as CX3CL1 and CCL5. CX3CL1 increased the ability of normal platelets to adhere to collagen at a high shear rate, while CCL5 induced platelet aggregation. Expression of tissue factor, IL-1beta, and TNF-alpha by monocytes stimulated with anti-beta2GPI antibodies, as well as CX3CL1 and CCL5 by vascular endothelial cells stimulated with IL-1beta or TNF-alpha were all suppressed by the NF-kappaB-specific inhibitor DHMEQ. These results suggest that the NF-kappaB pathway may be a potential therapeutic target relating to both the coagulation pathway and platelet activity.

Topics: Antiphospholipid Syndrome; Autoantibodies; Benzamides; beta 2-Glycoprotein I; Blood Platelets; Cell Line; Cells, Cultured; Chemokine CCL5; Chemokine CX3CL1; Chemokines; Cyclohexanones; Disease Susceptibility; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Leukocytes, Mononuclear; Platelet Activation; Platelet Aggregation; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; Thrombosis

It has been shown that stimulation of endothelial cells and monocytes by antiphospholipid antibodies leads to a prothrombotic state involving upregulation of tissue factor (TF). We examined the in vitro effects of IgG fractions from patients with antiphospholipid syndrome (APS) and of a beta-2-glycoprotein 1-independent human monoclonal antiphospholipid antibody (HL-5B) on human umbilical vein endothelial cells (HUVEC) in comparison to untreated cell controls and to exposure to monoclonal IgG control antibody. We also examined the effect of recombinant monocyte chemoattractant protein-1 (MCP-1) on peripheral blood monocytes. Stimulation of endothelial cells with APS IgG fractions or HL-5B resulted in time-dependent upregulation of MCP-1 mRNA and protein expression. Stimulation with HL-5B also led to time-dependent upregulation of interleukin (IL)-8 and intracellular adhesion molecule-1 (ICAM-1) mRNA and IL-8 protein expressions. Stimulation of monocytes with recombinant MCP-1 resulted in an upregulation of TF mRNA and TF protein. In conclusion these results might represent a mechanism for antiphospholipid antibody-mediated thrombosis in APS patients.

Topics: Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antiphospholipid Syndrome; Cells, Cultured; Chemokine CCL2; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Immunoblotting; Immunoglobulin G; Intercellular Adhesion Molecule-1; Interleukin-8; Monocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Time Factors; Umbilical Veins

Antiphospholipid antibodies (aPL) are closely related to the development of thrombosis, but the exact mechanism(s) leading to thrombotic events remains unknown. In this study, using proteomic techniques, we evaluated changes in protein expression of monocytes from patients with antiphospholipid syndrome (APS) related to the pathophysiology of the syndrome.. Fifty-one APS patients were included. They were divided into 2 groups: patients with previous thrombosis, and patients with recurrent spontaneous abortion. As controls, we studied patients with thrombosis but without aPL, and age- and sex-matched healthy subjects.. The proteins that were more significantly altered among monocytes from APS patients with thrombosis (annexin I, annexin II, protein disulfide isomerase, Nedd8, RhoA proteins, and Hsp60) were functionally related to the induction of a procoagulant state as well as to autoimmune-related responses. Proteins reported to be connected to recurrent spontaneous abortion (e.g., fibrinogen and hemoglobin) were also determined to be significantly deregulated in APS patients without thrombosis. In vitro treatment with IgG fractions purified from the plasma of APS patients with thrombosis changed the pattern of protein expression of normal monocytes in the same way that was observed in vivo for monocytes from APS patients with thrombosis.. For the first time, proteomic analysis has identified novel proteins that may be involved in the pathogenic mechanisms of APS, thus providing potential new targets for pathogenesis-based therapies for the disease.

Topics: Abortion, Habitual; Adolescent; Adult; Aged; Analysis of Variance; Antibodies, Antiphospholipid; Anticoagulants; Antiphospholipid Syndrome; Autoantibodies; Autoimmunity; Blotting, Western; Cells, Cultured; Electrophoresis, Gel, Two-Dimensional; Female; Flow Cytometry; Humans; Male; Middle Aged; Monocytes; Proteomics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Thromboplastin; Thrombosis

Women with antiphospholipid syndrome (APS), a condition characterized by the presence of antiphospholipid antibodies (aPL), often suffer pregnancy-related complications, including miscarriage. We have previously shown that C5a induction of tissue factor (TF) expression in neutrophils contributes to respiratory burst, trophoblast injury, and pregnancy loss in mice treated with aPL. Here we analyzed how TF contributes to neutrophil activation and trophoblast injury in this model. Neutrophils from aPL-treated mice expressed protease-activated receptor 2 (PAR2), and stimulation of this receptor led to neutrophil activation, trophoblast injury, and fetal death. An antibody specific for human TF that has little impact on coagulation, but potently inhibits TF/Factor VIIa (FVIIa) signaling through PAR2, inhibited aPL-induced neutrophil activation in mice that expressed human TF. Genetic deletion of the TF cytoplasmic domain, which allows interaction between TF and PAR2, reduced aPL-induced neutrophil activation in aPL-treated mice. Par2-/- mice treated with aPL exhibited reduced neutrophil activation and normal pregnancies, which indicates that PAR2 plays an important role in the pathogenesis of aPL-induced fetal injury. We also demonstrated that simvastatin and pravastatin decreased TF and PAR2 expression on neutrophils and prevented pregnancy loss. Our results suggest that TF/FVIIa/PAR2 signaling mediates neutrophil activation and fetal death in APS and that statins may be a good treatment for women with aPL-induced pregnancy complications.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Disease Models, Animal; Factor VIIa; Female; Fetal Death; Gene Deletion; Gene Expression Regulation; Humans; Immunoglobulin G; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Activation; Neutrophils; Phagocytosis; Pregnancy; Reactive Oxygen Species; Receptor, PAR-1; Receptor, PAR-2; Respiratory Burst; Signal Transduction; Simvastatin; Thromboplastin

Fetal loss induced by antiphospholipid antibodies (aPLs) in mice is a complement-driven inflammatory condition. Engagement of the complement receptor C5aR on neutrophils induces expression of the principal initiator of the blood clotting mechanism, tissue factor (TF), and blocking this downstream event of complement activation prevents antibody-induced fetal loss. In this issue of the JCI, the study by Redecha et al. clarifies that in mice, the contribution of TF to this pathogenic mechanism is independent of its role in coagulation and thrombosis, but involves inflammatory signaling through the receptor PAR2 (see the related article beginning on page 3453). The study not only sheds light on a critical effector mechanism of aPL-induced fetal loss, but also suggests that treatment with statins, which decrease TF and PAR2 expression, may hold promise as a therapeutic approach to antiphospholipid syndrome-associated pregnancy complications.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Female; Fetal Death; Humans; Mice; Pregnancy; Pregnancy Complications, Hematologic; Receptor, PAR-2; Thromboplastin; Thrombosis

Inhibition of tissue factor pathway inhibitor type 1 (TFPI) is one of the mechanisms by which lupus anticoagulants (LA) may upregulate tissue factor (TF) activity. We wanted to examine whether purified immunoglobulin G (IgG) from patients with LA may interfere with the ability of TFPI to inhibit ex vivo TF-induced thrombin generation. The endogenous thrombin potential (ETP) in pooled normal plasma (PNP) supplemented with IgG from either patients with LA or controls was determined in the absence or presence of recombinant TFPI (rTFPI). In the presence of a heparin neutralizer, the ETP was also determined in plasmas from patients with LA and controls before and after heparin injection in order to quantify the anticoagulant effect of heparin-releasable TFPI in vivo. Compared with IgG from controls (n = 14), IgG from patients with LA (n = 28) induced a wide range of enhancing or inhibitory effects on the ETP in PNP. The response to rTFPI in PNP with IgG from patients with LA correlated inversely with thrombin generation (r(s) = 0.637, P = 0.0003). Correspondingly, the relative inhibition of ETP in postheparin plasmas was smaller for patients (n = 11) than for controls (n = 9) (32% vs. 68%, P = 0.007). Our findings support the hypothesis that TFPI anticoagulant activity is inhibited in some patients with LA.

Topics: Adult; Aged; Anticoagulants; Antiphospholipid Syndrome; Blood Coagulation; Case-Control Studies; Female; Humans; Immunoglobulin G; Lipoproteins; Lupus Coagulation Inhibitor; Male; Middle Aged; Recombinant Proteins; Statistics, Nonparametric; Thrombin; Thrombin Time; Thromboplastin

A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS-/-) mice and the association between tlr4 gene polymorphisms and APS in patients.. IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS-/- mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin.. IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-beta(2)glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS-/- mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS-/- mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls.. These findings in LPS-/- mice and the reduction in the "protective" polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Topics: Adult; Animals; Antibodies, Anticardiolipin; Antibodies, Antiphospholipid; Anticoagulants; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Adhesion; Endothelial Cells; Female; Humans; Immunoglobulin G; Leukocytes; Male; Mice; Mice, Inbred C3H; Polymorphism, Genetic; Thrombophilia; Thromboplastin; Thrombosis; Toll-Like Receptor 4

Topics: Animals; Antiphospholipid Syndrome; Humans; Mice; Platelet Aggregation; Thromboplastin; Vascular Cell Adhesion Molecule-1

It has been shown that endothelial cell (EC) activation and tissue factor (TF) upregulation in EC and monocytes by antiphospholipid antibodies (aPL Abs) leads to a prothrombotic state and involves translocation of nuclear factor-kappa B (NF-kappaB). Here we examined the effects of an NF-kappaB inhibitor on aPL-induced thrombosis, TF activity, and EC in vivo. We treated CD1 mice with IgG from a patient with antiphospholipid syndrome (IgG-APS) or with control IgG (IgG-NHS). The adhesion of leukocytes (number of white blood cells) to EC in cremaster muscle (as an indication of EC activation) as well as the size of an induced thrombus in the femoral vein of the mice were examined. Some mice in each group were infused with 10 microM MG132 (an inhibitor of NF-kappaB). TF activity was determined using a chromogenic assay in homogenates of carotid arteries and in peritoneal cells of mice. In vivo, IgG-APS increased significantly the number of white blood cells adhering to ECs (4.7 +/- 2.2) when compared to control mice (1.5 +/- 0.8), and these effects were significantly reduced when mice were pretreated with MG132 (0.8 +/- 0.2). IgG-APS increased significantly the thrombus size and MG132 inhibited that effect (93%). Treatment of the mice with IgG-APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid arteries. Pretreatment of the mice with MG132 abrogated those effects significantly. Mice injected with IgG-APS or with IgM-APS with or without the inhibitor had medium-high titers of anticardiolipin antibodies in serum at the time of the surgical procedures. The data show that prothrombotic and proinflammatory properties of IgG-APS and IgM-APS are downregulated in vivo by an NF-kappaB inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cell Adhesion; Endothelial Cells; Humans; Leupeptins; Male; Mice; Middle Aged; NF-kappa B; Thromboplastin; Thrombosis

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL). In patients with primary APS, expression of tissue factor (TF) on the surface of monocytes is increased, which may contribute to thrombosis in these patients. However, the intracellular mechanisms involved in aPL-mediated up-regulation of TF on monocytic cells are not understood. This study was undertaken to investigate the intracellular signals induced by aPL that mediate TF activation in monocytes from APS patients.. We analyzed, both in vivo and in vitro, aPL interactions with proteins that have signaling functions, including mitogen-activated protein kinases (MAP kinases) and NF-kappaB/Rel proteins.. In vivo studies demonstrated significantly higher levels of both TF messenger RNA and TF protein in monocytes from APS patients compared with controls. At the molecular level, increased proteolysis of IkappaBalpha and activation of NF-kappaB were observed. Constitutive activation of both p38 and ERK-1 MAP kinases was also found. Treatment of normal monocytes with aPL activated ERK-1 and p38 MAP kinases, as well as the IkappaB/NF-kappaB pathway, in a dose-dependent manner. NF-kappaB activation and IkappaBalpha degradation induced by aPL were inhibited by the NF-kappaB inhibitor SN50 and the p38 MAP kinase inhibitor SB203580, thus suggesting crosstalk between these pathways. However, the MEK-1/ERK inhibitor PD98059 did not affect aPL-induced NF-kappaB binding activity. TF expression induced by aPL was significantly inhibited by combined treatment with the 3 inhibitors.. Our results suggest that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK-1/ERK proteins, and the p38 MAP kinase-dependent nuclear translocation and activation of NF-kappaB/Rel proteins.

Topics: Adult; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Female; Humans; Male; MAP Kinase Kinase 1; MAP Kinase Kinase Kinase 1; Middle Aged; Monocytes; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Transport; Thromboplastin

The association of thrombosis and gestational morbidity with antiphospholipid antibodies is termed antiphospholipid syndrome (APS). Annexin 2 (A2) is a profibrinolytic endothelial cell surface receptor that binds plasminogen, its tissue activator (tPA), and beta(2)-glycoprotein I (beta2GPI), the main antigen for antiphospholipid antibodies. Here, we evaluate A2 as a target antigen in APS. Serum samples from 434 individuals (206 patients with systemic lupus erythematosus without thrombosis, 62 with APS, 21 with nonautoimmune thrombosis, and 145 healthy individuals) were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblot for antiphospholipid and A2 antibodies. Anti-A2 antibodies (titer > 3 SDs) were significantly more prevalent in patients with APS (22.6%; venous, 17.5%; arterial, 34.3%; and mixed thrombosis, 40.4%) than in healthy individuals (2.1%, P < .001), patients with nonautoimmune thrombosis (0%, P = .017), or patients with lupus without thrombosis (6.3%, P < .001). Anti-A2 IgG enhanced the expression of tissue factor on endothelial cells (6.4-fold +/- 0.13-fold SE), blocked A2-supported plasmin generation in a tPA-dependent generation assay (19%-71%) independently of beta2GPI, and inhibited cell surface plasmin generation on human umbilical vein endothelial cells (HUVECs) by 34% to 83%. We propose that anti-A2 antibodies contribute to the prothrombotic diathesis in antiphospholipid syndrome.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Annexin A2; Antiphospholipid Syndrome; Autoantibodies; Case-Control Studies; Endothelial Cells; Endothelium, Vascular; Female; Fibrinolysin; Humans; Immunoassay; Male; Middle Aged; Pregnancy; Pregnancy Complications; Thromboplastin; Thrombosis

Antiphospholipid syndrome (APS), characterized by circulating antiphospholipid (aPL) antibodies, is a major cause of early pregnancy failure and placental insufficiency. In this study, we examined whether impaired endometrial differentiation before conception contributes to the high incidence of pregnancy complications in APS. Timed secretory endometrial biopsies were obtained from a cohort of women with recurrent pregnancy loss (RPL). Real-time quantitative (RTQ)-PCR was used to determine the expression levels of transcripts that encode for decidual markers, proinflammatory cytokines and complement regulatory proteins. Expression of decidual markers such as prolactin (PRL), tissue factor (TF) and signal transducer and activator of transcription 5 (Stat5), but not insulin-like growth factor-binding protein 1 (IGFBP-1), was significantly lower in samples obtained from aPL(+) patients (n = 24) when compared with aPL(-) group (n = 58) (P < 0.05). The abundance of transcripts encoding for interferon gamma (IFNgamma), tumour necrosis factor alpha (TNFalpha) or Stat1 did not differ significantly between both groups (P >/= 0.05). However, analysis of transcripts that encode for complement regulatory proteins showed a marked decrease in decay-accelerating factor (DAF/CD55) levels in aPL(+) patients (P = 0.005), which was mimicked at protein level as demonstrated by immunohistochemistry. In summary, patients with RPL have distinct endometrial gene expression profiles depending on the presence or absence of circulating aPL antibodies. In APS, impaired endometrial differentiation and lower DAF/CD55 expression before conception may compromise implantation and predispose to complement-mediated pregnancy failure.

Topics: Abortion, Habitual; Adult; Antibodies, Antiphospholipid; Antigens, Differentiation; Antiphospholipid Syndrome; Case-Control Studies; CD55 Antigens; CD59 Antigens; Complement System Proteins; Endometrium; Female; Gene Expression; Humans; Immunohistochemistry; Insulin-Like Growth Factor Binding Protein 1; Interferon-gamma; Membrane Cofactor Protein; Pregnancy; Prolactin; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; Tumor Necrosis Factor-alpha

Prothrombin is now accepted as one of the target antigens recognised by antiphospholipid (aPL) antibodies. However, it is not clear whether anti-prothrombin antibodies are pathogenic in vivo and if so, the possible mechanism(s) involved. Here, we examined the pathogenic effects of the IS6 monoclonal anti-prothrombin antibody isolated from a patient with Antiphospholipid Syndrome (APS). IS6 antibody was purified from hybridoma supernatant. Its pathogenic potentials were studied in an in vivo model of induced thrombosis. The expression of tissue factor (TF) and E-selectin on human umbilical vein endothelial cells (HUVEC) was determined by cyto-enzyme-linked immunosorbent assay. Transcription of TF mRNA was determined by quantitative real time-polymerase chain reaction (RT-PCR). In vivo, the thrombus size increased significantly when treated with IS6 compared with control-treated mice (5388 +/- 1035 microm2 vs. 2845 +/- 1711 microm2). In vitro, IS6 induced significant expression of TF and E-selectin on HUVEC, when compared with control preparation (3.1- and 5.1-fold increase compared with the control-treated cells). RT-PCR analysis of TF mRNA transcription showed a 2.5-fold increase of IS6-treated cells over the value obtained with control-treated cells. Taken together, these data show that IS6 monoclonal anti-prothrombin antibody promotes thrombosis and this is associated with TF and E-selectin expression.

Topics: Adolescent; Animals; Antibodies, Monoclonal; Antiphospholipid Syndrome; Cells, Cultured; E-Selectin; Endothelium, Vascular; Female; Humans; Male; Mice; Mice, Inbred Strains; Prothrombin; RNA, Messenger; Thromboplastin; Thrombosis; Umbilical Veins; Up-Regulation

One of the described mechanisms leading to thrombosis in antiphospholipid syndrome (APS) is overexpression of tissue factor (TF) in the monocytes and endothelial cells of patients with antiphospholipid antibodies (aPL). Vascular endothelial growth factor (VEGF) may stimulate monocyte TF expression through its receptor, the tyrosine kinase Flt-1.. This study aimed to analyze the following in monocytes of 55 primary APS patients: VEGF and Flt-1 expression levels, their potential regulation by aPL, and the association of VEGF and Flt-1 expression with the increased TF expression found in APS patients.. Purified monocytes from APS patients showed higher levels of VEGF and Flt-1 than healthy donors, which further correlated with immunoglobulin G (IgG) anticardiolipin titers and TF expression rank. Moreover, monocyte VEGF and Flt-1 levels were significantly higher in patients with than in patients without previous thrombosis. In vitro, IgG from APS patients increased monocyte VEGF and Flt-1 expression in a dose-dependent manner. VEGF and Flt-1 expression was significantly inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; this suggests the involvement of this kinase in the aPL-induced VEGF and Flt-1 upregulation.. Our data show, for the first time in vivo, that monocytes from primary APS patients have an increased expression of VEGF and Flt-1. Furthermore, in vitro results indicated that this cytokine is produced by monocytes when treated with aPL, and that the p38 MAPK signaling pathway plays an important role. Thus, VEGF might act as a regulatory factor in aPL-mediated monocyte activation and TF expression, thereby contributing to the proinflammatory-prothrombotic phenotype of APS patients.

Topics: Adolescent; Adult; Aged; Antiphospholipid Syndrome; Cells, Cultured; Enzyme Inhibitors; Female; Gene Expression Regulation; Humans; Imidazoles; Male; MAP Kinase Signaling System; Middle Aged; Monocytes; p38 Mitogen-Activated Protein Kinases; Pyridines; Thromboplastin; Thrombosis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Neutrophils and complement are key sentinels of innate immunity and mediators of acute inflammation. Recent studies have suggested that inflammatory processes modulate thrombogenic pathways. To date, the potential cross-talk between innate immunity and thrombosis and the precise molecular pathway by which complement and neutrophils trigger the coagulation process have remained elusive. In this study, we demonstrate that antiphospholipid Ab-induced complement activation and downstream signaling via C5a receptors in neutrophils leads to the induction of tissue factor (TF), a key initiating component of the blood coagulation cascade. TF expression by neutrophils was associated with an enhanced procoagulant activity, as verified by a modified prothrombin time assay inhibited by anti-TF mAb. Inhibition studies using the complement inhibitor compstatin revealed that complement activation is triggered by antiphospholipid syndrome (APS) IgG and leads to the induction of a TF-dependent coagulant activity. Blockade studies using a selective C5a receptor antagonist and stimulation of neutrophils with recombinant human C5a demonstrated that C5a, and its receptor C5aR, mediate the expression of TF in neutrophils and thereby significantly enhance the procoagulant activity of neutrophils exposed to APS serum. These results identify a novel cross-talk between the complement and coagulation cascades that can potentially be exploited therapeutically in the treatment of APS and other complement-associated thrombotic diseases.

Topics: Antiphospholipid Syndrome; Blood Coagulation; Blotting, Western; Flow Cytometry; Humans; Immunity, Innate; Immunoglobulin G; Immunohistochemistry; Neutrophils; Receptor Cross-Talk; Receptor, Anaphylatoxin C5a; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Thrombosis

The presence of antiphospholipid antibodies (APLA) is associated with an increased risk of recurrent thrombosis and pregnancy loss. APLA are able to activate endothelial cells (EC) and induce an increase in the expression of inflammatory marker proteins, such as leukocyte adhesion molecules, tissue factor or the monocyte chemoattractant protein-1 (MCP-1). Our objective was to investigate the effect of statins on EC activation induced by APLA in vitro. IgG was purified from the plasma of six patients with APLA and from healthy controls. EC were incubated with patient IgG or with control IgG, in the presence or absence of 5microM of fluvastatin, and expression of the leukocyte adhesion molecules, VCAM-1 and E-selectin, analyzed by flow cytometry and by quantitative reverse transcriptase-PCR (QRT-PCR). The expression of tissue factor and the chemokine MCP-1 was analyzed by QRT-PCR alone. Incubation of EC with patient IgG increased the expression of VCAM-1, E-selectin, tissue factor and MCP-1. Prior treatment of the cells with fluvastatin further increased the expression of these proteins. The fluvastatin effect was reversed by co-incubation with mevalonate or geranylgeranylpyrophosphate and mimicked by the geranylgeranyl transferase inhibitor GGTI-286. Our results show that in cultured human EC, statins increase the extent of inflammatory activation induced by APLA. This effect appears to be mediated by an inhibitory effect of statins on one or more geranylgeranylated protein(s).

Topics: Adolescent; Adult; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cell Adhesion Molecules; Cells, Cultured; Chemokine CCL2; Endothelium, Vascular; Fatty Acids, Monounsaturated; Female; Fluvastatin; Gene Expression Regulation; Humans; Immunoglobulin G; Indoles; Inflammation; Male; Middle Aged; Thromboplastin; Umbilical Cord

The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL) in patients with thromboembolic complications. In APS, most aPL are autoantibodies to beta2-glycoprotein I and prothrombin, which play a major role in the APS pathogenesis. Nevertheless, antibodies with the same antigen specificity are also found in aPL patients with leprosy, in whom thromboembolic complications are uncommon. The in vivo upregulation of the tissue factor (TF) pathway and the imbalance of cytokines have been proposed as potential mechanisms of thrombosis in the APS. We measured the circulating levels of TF, interleukin 6 (IL-6), IL-6 receptor (sIL-6R), tumor necrosis factor (TNF-alpha) and interferon gamma (IFN-gamma) in 83 patients with autoimmune aPL (42 with and 41 without clinical features of definite primary APS), 48 leprosy patients (33 with aPL) and 48 normal controls. There was a trend (P = 0.06) to higher median sTF in patients with autoimmune aPL (139 pg/mL) compared with leprosy patients (103.5 pg/mL) and controls (123 pg/mL). In addition, the frequency of raised sTF levels (> 187 pg/mL) was significantly higher in the group with autoimmune aPL [22.9% (APS 21.4%, non-APS 24.4%)] but not in leprosy (10.4%) compared with controls (4.2%). Elevated levels of IL-6 and TNF-alpha and a trend to lower IFN-gamma were found in patients with definite APS. Leprosy patients with aPL, however, had increased TNF-alpha and IFN-gamma but normal IL-6 levels. Levels of sIL-6R did not differ between controls and either patients with autoimmune aPL or leprosy. The different cytokine profiles as well as differences in circulating levels of TF might contribute to the high thrombotic risk found in patients with autoimmune aPL but not in leprosy related aPL patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Child; Female; Humans; Interferon-gamma; Interleukin-6; Leprosy; Male; Middle Aged; Receptors, Interleukin-6; Thromboplastin; Tumor Necrosis Factor-alpha

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Contraindications; Endothelium, Vascular; Fatty Acids, Monounsaturated; Fibrinolytic Agents; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; NF-kappa B; Pyridines; Simvastatin; Thromboplastin; Thrombosis; Up-Regulation

Increasing evidence suggests that autoantibodies directly contribute to hypercoagulability in the antiphospholipid syndrome (APS). One proposed mechanism is the antibody-induced expression of tissue factor (TF) by blood monocytes. Dilazep, an antiplatelet agent, is an adenosine uptake inhibitor known to block induction of monocyte TF expression by bacterial lipopolysaccharide. In the current study we characterized the effects of immunoglobulin G (IgG) from patients with APS on monocyte TF activity and investigated whether dilazep is capable of blocking this effect. IgG from 13 of 16 patients with APS significantly increased monocyte TF activity, whereas normal IgG had no effect. Time-course experiments demonstrated that APS IgG-induced monocyte TF mRNA levels were maximal at 2 hours and TF activity on the cell surface was maximal at 6 hours. Dilazep inhibited antibody-induced monocyte TF activity in a dose-dependent fashion but had no effect on TF mRNA expression. The effect of dilazep was blocked by theophylline, a nonspecific adenosine receptor antagonist. In conclusion, IgG from certain patients with APS induce monocyte TF activity. Dilazep inhibits the increased expression of monocyte TF activity at a posttranscriptional level, probably by way of its effect as an adenosine uptake inhibitor. Pharmacologic agents that block monocyte TF activity may be a novel therapeutic approach in APS.

Topics: Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Blood Specimen Collection; Dilazep; Gene Expression Regulation; Glycoproteins; Humans; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Lipopolysaccharides; Monocytes; RNA, Messenger; Thromboplastin; Time Factors

Topics: Antibodies; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cells, Cultured; Glycoproteins; Humans; Leukocytes, Mononuclear; Thromboplastin

Topics: Animals; Antiphospholipid Syndrome; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Thromboplastin; Thrombosis

To investigate whether antiplatelet agents, dilazep and dipyridamole, inhibit tissue factor (TF) expression on monocytes induced by IgG from patients with antiphospholipid syndrome (APS).. Freshly isolated peripheral blood monocytes were allowed to adhere on plastic and then cultured in media containing patient or control antibodies and/or other agonists with or without dilazep or dipyridamole. The TF activity on monocytes was investigated by measuring factor VIIa-dependent generation of factor Xa, using a chromogenic substrate and the TF mRNA expression was examined by real-time PCR (TaqMan PCR).. The TF activity on monocytes induced by APS IgG (250 mg/L) was inhibited by dilazep (0.15-150 micromol/L) and dipyridamole (0.2-200 micromol/L) in a dose-dependent fashion. But, the TF mRNA expression induced by APS IgG was not inhibited. Theophylline (500 micromol/L), an adenosine receptor antagonist, could counteract the inhibitory effect of dilazep and dipyridamole on TF activity.. Antiplatelet agents, dilazep and dipyridamole, block APS IgG-induced monocytes TF expression at a post-transcriptional level, partly by adenosine receptor pathway. Pharmacological agents that block monocytes TF activity, such as dilazep and dipyridamole, are a novel therapeutic approach in APS.

Topics: Adrenergic Antagonists; Antiphospholipid Syndrome; Cell Separation; Dilazep; Dipyridamole; Gene Expression; Humans; Immunoglobulin G; Monocytes; Platelet Aggregation Inhibitors; RNA, Messenger; Theophylline; Thromboplastin

Topics: Antiphospholipid Syndrome; Blood Coagulation; Endothelium, Vascular; Humans; Thromboplastin; Thrombosis

It is well known that monocytes may play an active role in thrombogenesis, since they may express on their surface tissue factor, the major initiator of the clotting cascade. The results of this investigation demonstrate beta-2-glycoprotein I (beta2-GPI) mRNA expression by human peripheral blood monocytes, indicating that these cells synthesize beta2-GPI. In addition, we show beta2-GPI expression on cell surface of these cells by flow cytometric analysis, and the presence of this protein in cell lysate by Western blot. Interestingly, beta2-GPI expression on monocytes is significantly increased in patients with anti-phospholipid syndrome (APS) or systemic lupus erythematosus (SLE) as against healthy blood donors and correlates with tissue factor expression on monocytes. These findings support the view that monocytes are able to synthesize beta2-GPI and suggest that patients with APS may have increased beta2-GPI exposure on cell surface, which leads to persistently high monocyte tissue factor expression and consequently to a prothrombotic diathesis.

Topics: Adolescent; Adult; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Blotting, Western; Child; DNA Fragmentation; Female; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Gene Expression; Glycoproteins; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; Monocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin

We investigated the mechanism by which anti-prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity-purified anti-prothrombin antibodies from LAC-positive plasma samples (alpha-FII-LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified alpha-FII-LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when alpha-FII-LAC+ were added to prothrombin-deficient plasma or when purified anti-prothrombin antibodies from LAC-negative plasma samples (alpha-FII-LAC-) were added. When a combination of prothrombin and alpha-FII-LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The alpha-FII-LAC+ alone or a combination of prothrombin and alpha-FII-LAC- did not show inhibition. Ellipsometry studies showed that, in the presence of alpha-FII-LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with alpha-FII-LAC-. Our results show that complexes of prothrombin and anti-prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti-beta2-glycoprotein I antibodies.

Topics: Antiphospholipid Syndrome; Autoantibodies; Cysteine Endopeptidases; Enzyme-Linked Immunosorbent Assay; Factor Xa; Female; Humans; Lipid Bilayers; Lupus Coagulation Inhibitor; Lupus Erythematosus, Systemic; Male; Neoplasm Proteins; Phospholipids; Protein Binding; Prothrombin; Thromboplastin

Because of the variable responsiveness of thromboplastins to lupus anticoagulants (LA), concerns have been raised about the validity of the prothrombin time-International Normalized Ratio (PT-INR) in monitoring oral anticoagulant treatment in patients with the antiphospholipid syndrome (APS) and LA. To date, few studies have been performed, numbers of patients investigated are relatively small and results are conflicting. We report on a multicentre study organized to investigate further this clinically relevant issue. Each of nine thrombosis centres was asked to collect plasma samples from patients with APS who were on oral anticoagulants (cases) and patients without APS who were on oral anticoagulants (controls). Nine thromboplastins (three human recombinant, one from human placenta and five from rabbit brain) were calibrated at the co-ordinating centre according to World Health Organization guidelines. Measurements of the INR and factor X amidolytic activity for all frozen plasmas were performed centrally. The numbers of patients investigated were 58 cases and 57 controls. Between-reagent variability of the INR was higher in cases [coefficient of variation (CV) = 12.4%] than in controls (CV = 6.7%), but this was because of one of the thromboplastins only (Thromborel R, human recombinant), which measured considerably higher INR values than the others in cases but not in controls. In conclusion, our data indicate that LA interference on the PT-INR measured with the majority of commercial thromboplastins is not enough to cause concern if insensitive thromboplastins, properly calibrated to assign them an instrument-specific International Sensitivity Index are used. New thromboplastins, especially those made of relipidated tissue factor, should be checked for their responsiveness to LA before they are used to monitor oral anticoagulant treatment in patients with APS.

Topics: Animals; Anticoagulants; Antiphospholipid Syndrome; Case-Control Studies; Humans; International Normalized Ratio; Lupus Coagulation Inhibitor; Prospective Studies; Rabbits; Sensitivity and Specificity; Thromboplastin; Treatment Outcome

Two human monoclonal antiphospholipid antibodies (APA) of the IgG type, HL-5B and RR-7F have been generated from a patient with primary antiphospholipid syndrome and recurrent cerebral microemboli (H.L.) and from a patient with SLE without evidence of recurrent thrombosis (R.R.). Both monoclonal APA have similar characteristics in ELISA tests. To further analyse the prothrombotic potential, their effect on human monocytes and platelets, and bovine aortic endothelial cells (BAEC) was investigated. Monocytes were isolated from buffy coats by standard techniques. They were incubated either with the respective monoclonal APA in different concentrations, or a control monoclonal IgG of the same subtype, or plasma of the patients, from whom the antibodies were isolated. Incubation with LPS served as positive control. BAEC were grown to confluence, and then incubated with the appropriate agonists. Procoagulant activity (PCA) was determined by a single stage clotting assay. PCA expression after incubation is given as the ratio of the coagulation times observed with media only divided by that observed with the agonist. A PCA ratio >1 indicates the induction of PCA by the agonist. At 1 microg/ml HL-5B yielded a PCA ratio of 1.63 +/- 0.16 while RR-7F induced no significant rise to 1.06 +/- 0.18. Dose response curves showed that RR-7F can induce PCA at higher concentrations. However, its effect is approx. 1/50 of HL-5B based on equimolar antibody concentration. Further analysis indicates that the majority of the PCA induced by monoclonal APA can be inhibited by a specific tissue factor antibody. Neither monoclonal antibody induced PCA in BAEC. Sera from both patients were able to induce PCA in monocytes. However, the PCA ratio of serum from H.L. was higher (1.78) than that of R.R. (1.44). Neither monoclonal APA had an effect on platelets as determined by flow cytometric analysis of CD62P, CD41, CD42b expression and fibrinogen binding with and without previous activation with 5 microM ADP or 15 microM TRAP-6. Similarly, there were no differences in platelet aggregation to different stimuli including submaximal activation. In summary, these data provide further evidence that induction of tissue factor in monocytes is one of the procoagulant effects of APA. Furthermore, the binding specificity of APA is perhaps not suited to predict the biological effects of the antibodies.

Topics: Adenosine Diphosphate; Animals; Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antibody Specificity; Antiphospholipid Syndrome; Autoimmune Diseases; Biomarkers; Blood Coagulation Factors; Blood Platelets; Cattle; Cells, Cultured; Dose-Response Relationship, Immunologic; Endothelium, Vascular; Female; Fibrinogen; Humans; Immunoglobulin G; Intracranial Embolism; Lipopolysaccharides; Lupus Erythematosus, Systemic; Male; Middle Aged; Monocytes; Peptide Fragments; Platelet Activation; Recurrence; Thrombophilia; Thromboplastin

Topics: Adsorption; Annexin A5; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoimmune Diseases; Binding, Competitive; Humans; Membrane Lipids; Models, Immunological; Protein Binding; Prothrombin Time; Thrombin; Thromboplastin

Anti-beta 2-glycoprotein I antibodies bind to endothelial cells through beta 2-GPI. The antibodies are present in patients with systemic lupus erythematosus and antiphospholipid syndrome and are associated with the pathogenesis of the disease. Anti-endothelial cell antibodies that react with constitutive antigens on ECs are present in patients with vasculiditis and other diseases. Both types of antibodies can activate ECs. Frequent findings in APLS and vasculitis are fibrin deposits and thromboembolic phenomena. These indicate that the coagulation system is activated. However, the mechanism of activation is not clear. ECs generate tissue factor upon stimulation with various substances. In the present study we report that monoclonal anti-beta 2-GPI antibodies and AECAs, derived from a patient with primary APLS and a patient with Takayasu's arteritis, respectively, induce a potent tissue factor in ECs. The production of TF activity, TF antigen and TF mRNA is dose and time dependent. The TF activity was induced also by F(ab)2 but not by Fc fragments and was abolished completely by pre-incubation with ant-TF antibodies. The TF that is induced in ECs by AECAs with and without beta 2-GPI specificity may activate the coagulation and thereby play a major role in the pathogenesis of fibrin deposition and thrombus formation in diseases that are associated with the presence of these antibodies.

Topics: Antibodies; Antigens; Antiphospholipid Syndrome; Apolipoproteins; beta 2-Glycoprotein I; Binding Sites, Antibody; Cells, Cultured; Endothelium, Vascular; Fibrin; Glycoproteins; Humans; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Lupus Erythematosus, Systemic; Membrane Glycoproteins; RNA, Messenger; Takayasu Arteritis; Thromboembolism; Thromboplastin; Vasculitis

Platelets contribute to hemostasis by forming a platelet plug and by providing a procoagulant surface for the assembly and activation of the coagulation factors. The contribution of platelets to prothrombotic disorders has been difficult to analyze. Recently an assay was reported that measured the procoagulant activity of test platelets by making the platelet lipid surface the limiting factor in the production of thrombin. In this report we describe a novel technique, based on this assay, that we used to study patient serum factors that activate control platelets and in turn initiate measurable procoagulant activity. Using this assay we investigated a group of patients with prothrombotic disorders. The patient test serum was incubated with normal platelets in the presence of activated factor Xa. The resultant thrombin was measured in a chromogenic assay. The rate-limiting step was the presence of any potential platelet-activating factors, such as antibodies in the heat-treated test serum, that would allow the Xa to bind to the platelet phospholipid surface. Serum samples from patients with heparin-induced thrombocytopenia (HIT) and the anti-phospholipid antibody syndrome enhanced platelet procoagulant activity, while samples from patients with idiopathic thrombocytopenic purpura and disseminated intravascular coagulation (DIC) did not. HIT serum samples also induced platelet activation, as measured by platelet microparticle shedding, carbon 14-labeled serotonin release, and platelet aggregation. The measurement of serum-induced platelet procoagulant activity provides a method for the investigation of circulating platelet agonists in prothrombotic disorders.

Topics: Antiphospholipid Syndrome; Blood Coagulation Factors; Blood Platelets; Disseminated Intravascular Coagulation; Factor Xa; Heparin; Humans; Platelet Activation; Platelet Aggregation; Prothrombin; Purpura, Thrombocytopenic, Idiopathic; Serotonin; Thrombin; Thromboplastin

Anti-beta2-glycoprotein I (beta2-GPI) antibodies behave as classical Lupus Anticoagulants (LA), as they inhibit phospholipid-dependent coagulation reactions and their activity disappears in the presence of excess exogenous phospholipids (PLs). We have recently shown that a certain amount of PLs in the dilute Russell Viper Venom Time (dRVVT) test system is required to express LA activity of anti beta2-GPI antibodies. We have now extended this observation to two other tests, i.e., Kaolin Clotting Time (KCT) in which PLs are not added, and Tissue Thromboplastin Inhibition test (TTI) in which PLs are extremely diluted. In fact, affinity-purified antibody preparations from 5 patients with antiphospholipid syndrome did not express or only weakly expressed anticoagulant activity in both tests; the mean ratios of coagulation times obtained with purified antibodies and that of control buffer were 1.11 and 1.0 for KCT and TTI, respectively. On the contrary, the mean ratios in dRVVT were 1.31 and 1.49 at a PLs dilution of 1:8 and 1:64, respectively. Therefore, the presence of LA activity due to autoantibodies to beta2-GPI is characterized by a positive dRVVT and negative or only weakly positive KCT and TTI.

Topics: Adolescent; Adult; Antibodies, Anticardiolipin; Antiphospholipid Syndrome; Autoimmune Diseases; beta 2-Glycoprotein I; Blood Coagulation Tests; Female; Glycoproteins; Humans; Immunoglobulin G; Kaolin; Lupus Coagulation Inhibitor; Male; Phospholipids; Prothrombin Time; Sensitivity and Specificity; Thromboplastin

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Topics: Antiphospholipid Syndrome; Autoimmune Diseases; Cell Adhesion Molecules; Cells, Cultured; Endothelium, Vascular; Factor VII; Flow Cytometry; Humans; Infections; Lupus Coagulation Inhibitor; Lupus Erythematosus, Systemic; Microscopy, Confocal; Neoplasms; Receptors, Vitronectin; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins

Placentas from pregnancies complicated by antiphospholipid syndrome often show thromboses and infarction, which may result from aberrations in placental coagulant pathways. We tested the hypothesis that alterations in tissue factor, thrombomodulin, and annexin V expressions contribute to poor pregnancy outcome associated with antiphospholipid syndrome.. Frozen sections from random biopsy samples of the basal plates of placentas from patients with primary antiphospholipid syndrome (n = 9), patients with secondary antiphospholipid syndrome (n = 3), and gestational age-matched control subjects (n = 10) were immunostained for tissue factor, thrombomodulin, and annexin V. Intensity of immunostaining was assessed by means of quantitative image analysis. Annexin V protein expression was evaluated with Western blotting techniques.. Tissue factor was expressed in the perivascular cells of the villous vasculature. Thrombomodulin and annexin V immunostaining was localized to the syncytiotrophoblast. There were no differences in the intensity of immunostaining for tissue factor, thrombomodulin, and annexin V between placentas from women with antiphospholipid syndrome and those from control subjects. Western blot analysis of annexin V expression showed no differences between study patients and control subjects.. Alterations in placental coagulant pathways involving tissue factor, thrombomodulin, and annexin V do not contribute to poor pregnancy outcome associated with antiphospholipid syndrome.

Topics: Adult; Annexin A5; Antiphospholipid Syndrome; Blotting, Western; Case-Control Studies; Female; Humans; Immunohistochemistry; Placenta; Pregnancy; Pregnancy Complications; Pregnancy Outcome; Prospective Studies; Thrombomodulin; Thromboplastin

Leg ulcres can be seen as manifestations of antiphospholipid syndromes but their pathogenic relationship with vascular thrombotic events secondary to antiphospholipid antibodies remains to be defined with precision. A significant association between anticardiolipin antibodies and venous leg ulcers has been described. We conducted this study to determine whether such an association is found in venous ulcers and if it could also be present in cases involving the arterial and/or arteriolar circulation.. From December 1995 to March 1997, 48 patients with leg ulcers involving venous (27 cases), arteriovenous (9 cases) or arteriolar (12 cases) circulations were admitted. The etiologic diagnosis was based on clinical presentation and duplex Doppler findings examining the superficial and deep venous and arterial circuits. Antiphospholipid antibodies were searched for in all cases: VDRL, ELISA for IgG and IgM antiphospholipid antibodies, antiprothrombinase circulating anticoagulant.. Circulating anticoagulants were found in 22 of the 48 patients (46%): 12/27 (44%) involved venous leg ulcers (anticardiolipin antibodies, 5 cases; circulating anticoagulants, 4 cases; both, 2 cases); 1/9 involved arteriovenous ulcers (anticardiolipin antibodies, 5 cases); 9/12 involved arteriolar ulcers (anticardiolipin antibodies, 3 cases; circulating anticoagulants, 6 cases; both, 3 cases). Seven of the 9 patients also had severe arteritis. A past history of venous thrombosis was found in 3 cases with venous ulcers and antiphospholipid antibodies. One patient among the 5 with arteriolar ulcers had a past history of arterial thrombosis.. Our cohort is too small for a formal conclusion but underlines two points: 1. antiphospholipid antibodies can be associated with venous ulcers independently of thrombosis history. The hypothesis that leukocyte stasis and endothelial cell activation causes an immune reaction implicating antiphospholipid antibodies has been put forward. The usefulness of an antiaggregate treatment or an anticoagulant treatment should be discussed, 2. The possible association between arteriolar ulcers and antiphospholipid antibodies requires further large scale studies.

Topics: Aged; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Immunoglobulin M; Leg Ulcer; Male; Middle Aged; Prospective Studies; Risk Factors; Thromboplastin; Ultrasonography, Doppler, Duplex; Venous Thrombosis

Antiphospholipid antibodies (aPL) may stimulate tissue factor (TF) expression in cultured endothelial cells and monocytes, but there are discrepancies as to the expression of TF in the patients with antiphospholipid syndrome (APS). By using reverse transcription and polymerase chain reaction amplification, we have analysed TF mRNA accumulation in freshly isolated mononuclear blood cells (MBC) of 14 patients with primary APS (PAPS) and six normal controls. TF mRNA accumulation was low or absent in uncultured MBC from all normal controls, but was elevated in uncultured MBC from nine of the patients as well as in normal MBC incubated with 100 ng/ml lipopolysaccharide (LPS). Mean levels of TF mRNA, as measured by densitometry, were higher in MBC from patients (N = 14) than in those from controls (N = 6, P = 0.009), and in MBC from patients with a history of thrombosis (N = 9) than in those from patients without thrombosis (N = 5, P = 0.02). Uncultured MBC of patients with thrombosis accumulated TF mRNA at similar levels to LPS-treated normal MBC. Increased levels of TF mRNA were found in eight of ten patients with conventional aPL (ie, anti-cardiolipin antibodies [aCL] and/or lupus anticoagulant [LA]) and little if any accumulation of TF mRNA was observed in three of four patients without aPL at the time of study. These data strongly suggest that circulating monocytes of many patients with PAPS are subjected to an up-regulated TF expression that may well explain their prothrombotic state. Although the presence or absence of TF mRNA in MBC was associated with, respectively, the presence or absence of conventional aPL in 11 of the 14 patients studied, our study cannot exclude the involvement of factors other than aCL or LA in inducing TF expression.

Topics: Adult; Aged; Antiphospholipid Syndrome; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Polymerase Chain Reaction; RNA, Messenger; Thromboplastin

Topics: Abortion, Habitual; Adult; Animals; Antibodies, Antiphospholipid; Anticoagulants; Antiphospholipid Syndrome; Autoimmune Diseases; Drug Monitoring; Female; Humans; International Normalized Ratio; Intracranial Embolism; Intracranial Thrombosis; Male; Middle Aged; Pregnancy; Recombinant Proteins; Recurrence; Risk; Seizures; Thromboembolism; Thrombophilia; Thrombophlebitis; Thromboplastin; Warfarin

The antiphospholipid syndrome (APS) is characterised by both arterial and venous thrombosis, recurrent pregnancy loss and thrombocytopaenia in association with antiphospholipid antibodies (aPL). To explore further the pathogenesis of thrombosis in APS, we evaluated the behaviour of tissue factor (TF) pathway in patients with APS. Plasma antigen levels of soluble TF and tissue factor pathway inhibitor (TFPI), a physiological regulator of TF dependent coagulation activation, were measured in 57 APS patients (36 primary and 21 secondary to systemic lupus erythematosus). Significantly elevated levels of both TF and TFPI were found in APS patients compared with 25 healthy controls (279 +/- 15 vs. 217 +/- 17 pg/ml, p = 0.01; 56.24 +/- 2.00 vs. 47.92 +/- 2.22 ng/ml, p = 0.01, respectively), suggesting in vivo upregulation of TF pathway in patients with APS. By flow-cytometry, monocytes from a healthy donor displayed higher TF antigen expression when incubated in the presence of APS plasmas than in control plasmas (24.23 +/- 3.11 vs. 12.78 +/- 1.57%, p = 0.002). Peripheral blood mononuclear cells (PBMC) also expressed more procoagulant activity (PCA) when incubated in the presence of APS plasmas than in control plasmas (1.80 +/- 0.12 vs. 1.35 +/- 0.054, p = 0.001) implying that TF up-regulation in APS was reproducible in vitro. Human monoclonal anticardiolipin antibodies induced PCA on PBMC and also TF mRNA on both PBMC and human umbilical vein endothelial cells shown by reverse-transcription polymerase chain reaction. These data strongly suggest that the TF pathway is implicated in the pathogenesis of aPL related thrombosis.

Topics: Adult; Aged; Antiphospholipid Syndrome; Blood Coagulation; Female; Humans; Lipoproteins; Male; Middle Aged; Pregnancy; Thromboplastin; Up-Regulation

Although multiple sclerosis (MS) and antiphospholipid syndrome (AS) are usually defined by specific criteria that make them distinguishable, in some cases, transition between the two diseases based on clinical and brain imaging findings is not clear.. Our study included 62 patients (sex ratio F/M = 1.48; mean age 43.4 +/- 23.6 years) with diagnosis of MS according to Poser criteria and 31 control subjects (sex ratio F/M = 9.3, mean age 37 +/- 17 years). We examined the level of antibodies against phospholipids (anticardiolipid, anti beta 2-glycoprotein 1 and antiphosphatidylethanolamine antibodies), antinuclear, anti native DNA, antiprothrombinase antibodies and rheumatoid factor.. Antiphospholipid antibodies were found with a significant level (anticardiolipid > 30 UI, anti beta 2-glycoprotein 1 positive) in only five patients (8%) with MS; two others showed an increase in antinuclear antibodies (1/320 degrees and 1/1280 degrees).. In contrast with data recently reported, this study failed to find a significant level of antiphospholipid antibodies in MS. This result argues for the existence of different pathogenic mechanisms in MS and AS.

Topics: Adult; Antibodies, Antinuclear; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoantibodies; Cardiolipins; Diagnosis, Differential; Female; Humans; Male; Multiple Sclerosis; Phosphatidylethanolamines; Rheumatoid Factor; Thromboplastin

Topics: Adolescent; Adult; Aged; Antiphospholipid Syndrome; Female; Humans; Male; Middle Aged; Thromboplastin

Topics: Antiphospholipid Syndrome; Autoimmune Diseases; Female; Humans; Leukocytes, Mononuclear; Male; Models, Biological; Thrombophilia; Thromboplastin

Topics: Antiphospholipid Syndrome; Humans; Thromboplastin; Up-Regulation

The antiphospholipid syndrome (APS) is a disorder of recurrent thrombosis, pregnancy loss, and thrombocytopenia associated with the production of anticardiolipin antibodies (aCL) and lupus anticoagulant (LAC). The mechanisms of thrombus formation remain unknown. Tissue factor (TF), an inducible cell glycoprotein, is a major initiator of coagulation in vivo. The present study was therefore undertaken to investigate TF expression and procoagulant activity on monocytes from patients with primary APS and its correlation with thrombotic events.. Three groups of patients were studied: group 1 comprised 23 primary APS patients with thrombosis, group 2 consisted of 10 primary APS patients without thrombosis, and group 3 contained 20 patients with thrombosis but without antiphospholipid antibodies. Twenty age- and sex-matched healthy blood donors were used as controls (group 4). Anticardiolipin antibodies were measured by enzyme-linked immunosorbent assay (ELISA) and LAC by standard methodology. Cell surface expression of TF on monocytes was assessed by flow cytometry. The amount of TF in cell lysates (TF(Ag)) and soluble TF(Ag) plasma levels were analyzed by ELISA, and the TF-related procoagulant activity (PCA-TF) on intact cells and cell lysates by a chromogenic assay. Levels of the cytokines that influence TF production, i.e., tumor necrosis factor alpha (TNF alpha) and interleukin-1beta (IL-1beta), were determined by ELISA.. Cell surface expression of TF was increased in group 1 (mean +/- SEM 50.2 +/- 4% positive cells) compared with group 2 (14.6 +/- 1.6%), group 3 (16.8 +/- 3.7%), and group 4 (14.1 +/- 1.6%). TF(Ag) levels were also elevated in group 1 (215.8 +/- 11.2 pg/10(6)) compared with group 2 (150.8 +/- 15.2), group 3 (101.4 +/- 14.8), and group 4 (80.32 +/- 5.5). PCA-TF on intact cells and cell lysates was significantly increased in group 1 (148.8 +/- 16.3 units/10(5) lysate cells, compared with 54.5 +/- 11.5, 38.6 +/- 9.7, and 22.5 +/- 3.1 in groups 2, 3, and 4, respectively). Among group 1 patients, there was a significant increase in the degree of TF expression in those positive for IgG aCL, but not in those positive for IgM aCL or LAC. TNF alpha and IL-1beta plasma levels did not differ significantly between any of the groups.. These results suggest that monocyte TF expression is directly involved in the pathogenesis of thrombotic complications in patients with the primary APS.

Topics: Adult; Antiphospholipid Syndrome; Female; Humans; Interleukin-1; Male; Middle Aged; Monocytes; Thromboplastin; Thrombosis; Tumor Necrosis Factor-alpha

We determined the prevalence and relationship with clinical manifestations of antibodies to thromboplastin (aTP) in 92 patients with systemic lupus erythematosus (SLE). Thirty-two (35%) patients had aTP: 13 (14%) were positive for IgG aTP, 13 (14%) for IgM aTP, and 6 (7%) for both. Patients with aTP had an increased incidence of thrombosis (p = 0.01), thrombocytopenia (p < 0.001), hemolytic anemia (p < 0.001), and fetal losses (p = 0.03). When the IgG and IgM aTP isotypes were analysed separately, the IgG aTP were found to be associated with thrombosis (p < 0.001), thrombocytopenia (p < 0.001), and fetal losses (p = 0.02). The IgM aTP were associated with hemolytic anemia (p < 0.001). A correlation was found between the titers of aTP and those of anticardiolipin antibodies, in both IgG (p < 0.01, r = 0.6) and IgM (p < 0.01, r = 0.64) isotypes, and between the titers of IgG aTP and the diluted Russell's viper venom time used to detect the lupus anticoagulant (p < 0.001, r = 0.42). This test is a reliable, reproducible and sensitive assay for the detection of antiphospholipid antibodies, specially in those patients under anticoagulant therapy.

Topics: Adolescent; Adult; Aged; Anemia, Hemolytic, Autoimmune; Antibodies, Anticardiolipin; Antiphospholipid Syndrome; Autoantibodies; Case-Control Studies; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Female; Fetal Death; Humans; Immunoglobulin G; Immunoglobulin Isotypes; Immunoglobulin M; Lupus Coagulation Inhibitor; Lupus Erythematosus, Systemic; Male; Middle Aged; Pregnancy; Reproducibility of Results; Sensitivity and Specificity; Thrombocytopenia; Thromboplastin; Thrombosis

The identification of the presence of antiphospholipid in plasma is recognised to be of diagnostic and prognostic importance in subjects with thrombotic disease, recurrent miscarriage or collagen vascular disorders. A number of coagulation assays are currently employed for the detection of lupus anticoagulant (LA), many of which are influenced by reagent dependent and methodological variables. In the present study lyophilised plasma samples from three subjects with "strong", "weak" and "absent" LA were tested in 220 centres. The most commonly used tests for LA were Activated Partial Thromboplastin Time (APTT), Dilute Russell Viper Venom Time (DRVVT) and Kaolin CLotting Time (KCT). Median DRVVT ratios were 1.75, 1.17 and 1.10 for the three samples. The presence of a strong LA was not detected by 4% of laboratories. The correct diagnosis was made by 94% of users of DRVVT and 85% of users of KCT. A weak LA was not detected by over half of centres. Correction was observed on addition of plasma and also in platelet neutralisation. The correct diagnosis was made by 37% of users of DRVVT and 27% of users of KCT. Lupus Anticoagulant was falsely considered to be present in a Factor IX deficient plasma by approximately one quarter of laboratories. Amongst users of DRVVT and KCT absence of LA in this sample was correctly reported by 73% and 69% of centres respectively. The accuracy of testing for LA in the present study is suboptimal and this is likely to have important clinical consequences. There is clearly a need for greater conformity in the selection and performance of LA tests to facilitate accurate diagnosis of this important group of disorders.

Topics: Antiphospholipid Syndrome; Blood Coagulation; Blood Coagulation Tests; Diagnosis, Differential; Embolism; Female; Hemophilia B; Heterozygote; Humans; Lupus Coagulation Inhibitor; Lupus Erythematosus, Systemic; Male; Partial Thromboplastin Time; Quality Control; Reproducibility of Results; Retinal Vessels; Thromboplastin; United Kingdom

Antiphospholipid antibodies, including anticardiolipin antibodies (ACA), are strongly associated with recurrent thrombosis in patients with the antiphospholipid syndrome (APS). To date, reports about the binding specificities of ACA and their role(s) in causing and/or sustaining thrombosis in APS are conflicting and controversial. The plasmas of patients with APS, usually containing a mixture of autoantibodies, vary in binding specificity for different phospholipids/cofactors and vary in in vitro lupus anticoagulant activity. Although in vivo assays that allow assessment of the pathogenic procoagulant activity of patient autoantibodies have recently been developed, the complex nature of the mixed species prevented determination of the particular species responsible for in vivo thrombosis. We have generated two human IgG monoclonal ACA from an APS patient with recurrent thrombosis. Both bound to cardiolipin in the presence of 10% bovine serum, but not in its absence, and both were reactive against phosphatidic acid, but were nonreactive against purified human beta-2 glycoprotein 1, DNA, heparan sulfate, or four other test antigens. Both monoclonal autoantibodies lacked lupus anticoagulant activity and did not inhibit prothrombinase activity. Remarkably, one of the monoclonal antibodies has thrombogenic properties when tested in an in vivo mouse model. This finding provides the first direct evidence that a particular antiphospholipid antibody specificity may contribute to in vivo thrombosis.

Topics: Adult; Animals; Antibodies, Anticardiolipin; Antibodies, Monoclonal; Antibody Specificity; Antiphospholipid Syndrome; Cardiolipins; Female; Humans; Mice; Thromboplastin; Thrombosis

Antiphospholipid antibodies (aPLs) are associated with thrombosis, but the mechanisms of this thrombotic tendency are unknown. We studied 56 patients 612 with systemic lupus erythematosus [SLE] and aPLs and previous thrombosis, 12 with SLE and aPLs but no thrombosis, 15 with SLE without aPLs or thrombosis, 11 with primary antiphospholipid syndrome with thrombosis, and 6 asymptomatic subjects with aPLs) to investigate the ability of aPLs to induce tissue factor (TF) expression on human normal monocytes. A double direct immunofluorescence technique (anti-CD14 and anti-TF) was used, and procoagulant activity in viable and disrupted cells was measured after plasma incubation for 6 hours at 37 degrees C with normal mononuclear cells. Hemostasis regulatory proteins, prothrombin fragment 1 + 2, and thrombin-antithrombin III complex levels were determined. Increased TF expression and procoagulant activity were observed using plasma samples from SLE patients with aPLs and thrombosis (P < .01) and from primary antiphospholipid syndrome patients (P < .01) but not from patients with SLE and aPLs but no thrombosis, patients with SLE without aPLs, or asymptomatic patients with aPLs. Purified aPL immunoglobulins from one primary antiphospholipid syndrome and two SLE patients added to normal plasma showed a significant increase in both TF expression and procoagulant activity (P < .05) compared with purified aPL from two SLE patients without thrombosis. The addition of nonspecific IgG from three SLE patients without aPLs and from three control subjects did not increase TF expression. Low free protein S was seen in eight patients. Increased TF expression and low free protein S correlated with thrombosis (P < .01) and with higher prothrombin fragment 1 + 2 and thrombin-antithrombin III values (P < .01). These observations may contribute to a further understanding of the thrombotic risk in aPL patients.

Topics: Antiphospholipid Syndrome; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Monocytes; Protein S; Thromboplastin; Thrombosis

Antiphospholipid (aPL) antibodies are associated with thrombosis, recurrent abortions and thrombocytopenia. Studies to determine the mechanisms of action of these antibodies have been hindered by their heterogeneity and limited availability of techniques to isolate and characterize subgroups of the antibodies. We report a new phospholipid affinity chromatography method which enables separation of antiphospholipid positive sera into more than one antibody subpopulation. Sera from five patients with complications of the antiphospholipid syndrome (APS) were studied. Each serum was applied to chromatography columns prepared by coating polystyrene beads (diameter 100 A) with phosphatidylserine (PS) or cardiolipin (CL). A linear salt gradient (0.03-1.0 M NaCl) was used for elution. Eluates were analyzed for phospholipid binding and for inhibition of the prothrombin-thrombin conversion reaction. Each sample yielded two to three peaks for CL and PS affinity columns. Molarities at which peaks were eluted differed between samples. For individual samples, molarities at which peaks were eluted differed between CL and PS columns. These data suggest that aPL antibodies are heterogenous, with differences existing between patients and even within single serum samples. Subpopulations differed in their avidities for CL and PS but generally all had prothrombinase inhibitory activity.

Topics: Adult; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Chromatography, Affinity; Female; Humans; Lupus Coagulation Inhibitor; Male; Thromboplastin

The dilute prothrombin time (dPT) is a widely accepted sensitive screening test for the lupus anticoagulant (LA). In general, Simplastin (Organon Teknika), a rabbit brain thromboplastin, diluted 1/500, is used in this test. Recently, human tissue thromboplastin obtained by recombinant DNA technology has become available and we have evaluated the usefulness of one such preparation, Innovin (Dade), for the detection of the LA. dPTs, using several dilutions of Innovin were determined on plasmas from 18 normal individuals and 15 patients with a well-documented LA. The dPT ratios, calculated as the individual result divided by the mean normal, were statistically compared. Innovin in dilutions from 1/100 onwards was found to be significantly more responsive to the LA than Simplastin (P < 0.05). Intra-assay coefficients of variation (CVs) ranged from 1.05% to 14.8% with Innovin, versus 2.7%-24.3% with Simplastin (P < 0.05); inter-assay CVs ranged from 5.6%-11.8% with Innovin, versus 4.2%-33.8% with Simplastin (P < 0.001). Analysis of 316 consecutive plasma samples from non-anticoagulated patients, on which a LA determination was requested, showed a 100% sensitivity and a 96% specificity of the dPT performed with Innovin, as compared to 81% and 93% respectively for the dPT using Simplastin.

Topics: Antiphospholipid Syndrome; Female; Humans; Immunization, Passive; Lupus Coagulation Inhibitor; Predictive Value of Tests; Pregnancy; Pregnancy Complications, Hematologic; Prothrombin Time; Recombinant Proteins; Reference Values; Reproducibility of Results; Sensitivity and Specificity; Thromboplastin

Anti-cardiolipin Abs (ACLA) are present in the sera of patients with antiphospholipid syndrome (APLS) and are associated with high incidence of thromboembolic phenomena, fetal loss, thrombocytopenia, and prolongation of the phospholipid-dependent coagulation assays (lupus anticoagulant). Recently, it has been shown that APLS can be induced experimentally by using ACLA. However, the pathophysiology of thrombus formation in this syndrome is unknown. Monocytes generate a potent procoagulant activity (PCA) after stimulation with various substances. Increased PCA has been found in monocytes from patients with diseases that are associated with high incidence of thromboembolic phenomena. In the present study, we report that the monoclonal ACLA that were shown previously by us to induce APLS stimulate mononuclear cells to generate a potent PCA. The PCA resembled tissue factor (TF) in that it accelerated clotting through the extrinsic coagulation pathway, was abolished by phospholipase C, and was inhibited by anti-TF mAbs. The induction of TF-like activity by ACLA in monocytes was dose- and time-dependent. It was induced in monocytes and monocytic cell lines, but not in lymphoid or myeloid cells, and did not require T lymphocytes for expression. The generation of PCA was dependent on protein synthesis inasmuch as it was prevented by adding puromycin to the system and was not affected by cytarabine. The TF-like activity that is induced by ACLA in monocytes may activate coagulation and thereby play a major role in the pathogenesis of thrombus formation in APLS.

Topics: Adult; Antibodies, Anticardiolipin; Antibodies, Monoclonal; Antiphospholipid Syndrome; Blood Coagulation Factors; Cardiolipins; Female; Humans; In Vitro Techniques; Male; Monocytes; Thromboplastin

Primary antiphospholipid syndrome (PAPS) is an autoimmune disorder manifested by recurrent thrombosis in the venous and arterial system. We report a group of seven patients with lower limb ischaemia associated with PAPS. Four were male patients and three were females, with a mean age of 37 years. All had a previous deep vein thrombosis and the majority, five out of seven, had a prior cerebrovascular accident (CVA). Prolonged activated thromboplastin time was demonstrated in all our patients and PAPS was established by positive thromboplastin titration index, circulating anticoagulant index and increased anticardiolipin levels. Symptoms included claudication in three, rest pain in four and gangrene in five patients. Angiography demonstrated thrombosis of various segments of the arterial tree including: aorta, iliac, femoral and popliteal arteries. Two patients were treated conservatively and one by percutaneous transluminal angioplasty (PTA) of the distal aorta. A total of eleven vascular surgical procedures were performed in four patients resulting in early postoperative thrombosis (2h-30 days) in 10 cases. Only one graft remained patent, when full heparinisation (1000 units/h) was used perioperatively. We conclude that PAPS patients are at high risk for graft thrombosis and should only be operated upon on full anticoagulation, starting at operation and proceeding indefinitely.

Topics: Adolescent; Adult; Aged; Antiphospholipid Syndrome; Female; Graft Occlusion, Vascular; Humans; Ischemia; Leg; Male; Middle Aged; Partial Thromboplastin Time; Postoperative Complications; Thrombectomy; Thromboembolism; Thromboplastin; Ultrasonography; Whole Blood Coagulation Time

Twenty-three patients with the 'primary' antiphospholipid syndrome were studied over 2-6 years. Twenty-two (96%) had antiphospholipid antibodies detected by ELISA (87% had antibodies to thromboplastin and 70% to cardiolipin), and 18 out of the 21 tested patients (86%) had lupus anticoagulant activity by coagulative assays. Mean age of the cohort was 29.9 years and the sex ratio (female:male) 4.75:1. Eleven patients presented 18 venous and/or arterial thrombosis and 13 had 25 foetal losses (84% occurred during the second and third trimester). Other clinical features were migraine, livedo reticularis, and epilepsy. Three patients had relatives with systemic lupus erythematosus. Thrombocytopaenia was seen in 33%, antinuclear antibodies in low or moderate titre in 30%, and haemolytic anaemia in 13%. During the follow-up, two patients presented recurrent thrombosis despite anticoagulant therapy, one of them dying because of recurrent pulmonary thromboembolism. Four patients achieved successful term pregnancies after treatment with aspirin and a further patient after treatment with aspirin and low dose prednisolone. No patient developed systemic lupus erythematosus or any other definable connective tissue disease. The 'primary' antiphospholipid syndrome may exist as a distinct clinical entity and all younger patients presenting with thrombotic events, foetal losses and/or thrombocytopaenia, without any evidence of a well defined disease, should be tested for antiphospholipid antibodies in order to rule out this syndrome.

Topics: Adult; Antibodies, Anticardiolipin; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Aspirin; Enzyme-Linked Immunosorbent Assay; Female; Fetal Death; Humans; Lupus Coagulation Inhibitor; Male; Middle Aged; Pregnancy; Pregnancy Complications; Thromboplastin; Thrombosis