Activated protein C resistance testing for factor V Leiden.

Activated protein C resistance assays can detect factor V Leiden with high accuracy, depending on the method used. Factor Xa inhibitors such as rivaroxaban and direct thrombin inhibitors including dabigatran, argatroban, and bivalirudin can cause falsely normal results. Lupus anticoagulants can cause incorrect results in most current assays. Assays that include dilution into factor V-deficient plasma are needed to avoid interference from factor deficiencies or elevations, which can arise from a wide variety of conditions such as warfarin, liver dysfunction, or pregnancy. The pros and cons of the currently available assays are discussed.

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

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

Specificity and sensitivity of anti-beta2-glycoprotein I as compared with anticardiolipin antibody and lupus anticoagulant in Thai systemic lupus erythematosus patients with clinical features of antiphospholipid syndrome.

Antibodies to beta(2)-glycoprotein I (anti-beta(2)-GPI) have been reported to have stronger association with clinical antiphospholipid syndrome (APS) than anticardiolipin antibodies (aCL) and lupus anticoagulant (LAC). We investigated the sensitivity and specificity of ELISA for anti-beta(2)-GPI in Thai systemic lupus erythematosus (SLE) patients with clinical features of APS and compared the results with IgG/IgM aCL and LAC to find the test with the best association. The hospital records of 151 Thai SLE patients whose sera had been sent for either IgG/IgM anticardiolipin antibodies or lupus anticoagulant testing were reviewed. Sera of patients either without complete clinical records or those with APS-related manifestations other than vascular thrombosis and pregnancy morbidity (according to the international consensus statement on preliminary classification criteria for definite APS) were excluded. For the remaining subjects (112 patients), their sera were tested for anti-beta(2)-GPI antibody, IgG and IgM anticardiolipin, and lupus anticoagulant. The sensitivity and specificity of each method were compared by using the chi-square test. Among the 112 (74.2%) SLE patients in the study, 35 (31.3%) presented with preliminary clinical criteria for APS (i.e., vascular thrombosis and pregnancy morbidity) whereas 77 (68.7%) did not. The sensitivity and specificity of anti-beta(2)-GPI determination were 57.1 and 79.2%, respectively, whereas those of IgG aCL were 25.7 and 94.8%, of IgM aCL were 5.7 and 98.7%, and of LAC were 44.8 and 77.3%, respectively. The accuracy of the four tests showed similar association with clinical APS (accuracy of test = 72.3, 73.2, 69.6, and 68.3%, respectively). Concerning the sensitivity, specificity, and difficulty of the methods, the combination of anti-beta(2)-GPI and IgG aCL tests was the best for the diagnosis of APS in Thai SLE patients.

Diagnostic value of antibodies to phosphatidylserine/prothrombin complex for antiphospholipid syndrome in Chinese patients.

To evaluate the diagnosis value of antibodies to phosphatidylserine/prothrombin complex (aPS/PT) in patients with antiphospholipid syndrome (APS) and to determine the clinical features of APS patients with avidity of aPS/PT. Serum samples were collected from 108 APS patients. Sixty patients with pregnancy morbidity, 37 patients with thrombosis without a history of autoimmune diseases, and 89 healthy blood donors were included as the control group. The enzyme-linked immunosorbent assay (ELISA) test was performed to detect the concentration of aPS/PT, including IgG/M, IgG, and IgM forms, in the same serum sample. The chi-square (χ2) test was used to examine the difference of frequencies of antibodies in APS patients and patients with other diseases. Spearman correlation analysis was performed to investigate the relationship between aPS/PT and other clinical/laboratory parameters. aPS/PT was detectable in 68 (63.0%) of the 108 APS patients, 12 (13.2%) of the 91 disease control patients and 1 (1.1%) of the healthy controls. It was strongly correlated with the activity of lupus anticoagulant (LA) (OR 15.952, 95% CI 7.132-35.678; P < 0.001). The frequency of aPS/PT was 56.9% in anti-cardiolipid antibody (aCL)-negative, 60.5% anti-ß2 glycoprotein I antibody (aß2GPI)-negative, and 50.0% in both aCL and aß2GPI negative APS patients. The IgG aPS/PT was significantly associated with arterial and venous thrombosis. The aPS/PT antibody could play an important role in the diagnosis of APS, especially in patients with negative aCL and aß2GPI. It was positively related to thrombotic events in APS.

A lupus anti-DNA autoantibody mediates autocatalytic, targeted delivery of nanoparticles to tumors.

Strategies to target nanoparticles to tumors that rely on surface modification with ligands that bind molecules overexpressed on cancer cells or the tumor neovasculature suffer from a major limitation: with delivery of toxic agents the amount of molecules available for targeting decreases with time; consequently, the efficiency of nanoparticle delivery is reduced. To overcome this limitation, here we propose an autocatalytic tumor-targeting mechanism based on targeting extracellular DNA (exDNA). exDNA is enriched in the tumor microenviroment and increases with treatment with cytotoxic agents, such as doxorubicin (DOX), due to release of DNA by dying tumor cells. We tested this approach using poly(lactic-co-glycolic acid) (PLGA) nanoparticles surface-conjugated with fragments of 3E10 (3E10EN), a lupus anti-DNA autoantibody. We demonstrated that 3E10EN-conjugated nanoparticles bound to DNA and preferentially localized to tumors in vivo. The efficiency of tumor localization of 3E10EN-conjugated, DOX-loaded nanoparticles increased with time and subsequent treatments, demonstrating an autocatalytic effect. 3E10EN-conjugated DOX-loaded nanoparticles exhibited a significant anti-tumor effect that was superior to all controls. This work demonstrates the promise of autocatalytic drug delivery mechanisms and establishes proof of concept for a new anti-DNA autoantibody-based approach for enhancing delivery of nanoparticles to tumors.

Edoxaban: Impact on routine and specific coagulation assays. A practical laboratory guide.

Assessment of plasma concentration/effect of edoxaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced. It was our aim to determine coagulation tests useful for the assessment of edoxaban's pharmacodynamics and provide recommendations for the interpretation of haemostasis diagnostic tests. Edoxaban was spiked at concentrations ranging from 0 to 1,000 ng/ml in platelet-poor plasma which covers the on-therapy range (from ± 25 ng/ml at Ctrough to ± 170 ng/ml at Cmax). aPTT, PT, dRVVT, chromogenic anti-Xa assays, TGA and a large panel of haemostasis diagnostic tests were performed using several reagents. A concentration-dependent prolongation of aPTT, PT and dRVVT was observed. The effect was dependent on the reagents. FXa chromogenic assays showed high sensitivity and a linear correlation depending on the methodology. TGA may be useful to assess the pharmacodynamics of edoxaban but its turnaround time and the lack of standardisation are limitations. Edoxaban impairs the assessment of lupus anticoagulant, protein S (clotting method), APC-R, antithrombin (FXa-based assay) and measurement of clotting factor activity. Immunological assays and assays acting below the FXa are not influenced by edoxaban. In conclusion, some PT reagents could be used to estimate edoxaban activity. Chromogenic anti-Xa assays are required to assess the plasma concentration. TGA may be useful but requires standardisation. In case of thrombophilia or in the exploration of a haemorrhagic event, immunological assays should be recommended, when applicable. Standardisation of the time between the last intake and the sampling is mandatory to provide a proper assessment of the result.

Ischemic stroke subtypes and thrombophilia in young and elderly Brazilian stroke patients admitted to a rehabilitation hospital.

BACKGROUND AND PURPOSE: We sought to examine ischemic stroke subtypes and prevalence of thrombophilia in Brazilian stroke patients. METHOD: A total of 130 consecutive young and 200 elderly stroke patients were studied. RESULTS: Prevalence of thrombophilia was, respectively: protein S deficiency (11.5% versus 5.5%), protein C deficiency (0.76% versus 1%), resistance to activated protein C (2.3% versus 3.5%), mutation in V Leiden factor (1.5% versus 2%), antithrombin III deficiency (0% versus 0%), lupus anticoagulant (0% versus 0.5%), anticardiolipin antibodies (3% versus 10%; P=0.01), hyperhomocysteinemia (31.5% versus 53.5%; P=0.0001), mutation of the MTHFR gene in homocigosis (10% versus 5%), and heterocigosis (27.6% versus 41.9%; P=0.01). CONCLUSIONS: Prothrombotic conditions were more frequent in stroke of undetermined cause.

Inhibition of Binding of ß2-Glycoprotein 1 to Phosphatidylserine by Polymyxin B, a Lupus-Like Anticoagulant.

Polymyxin B is a cationic peptide that inhibits phospholipid-dependent coagulation tests including activated partial thromboplastin time and to a lesser degree prothrombin time. Thrombin clotting time is insensitive to polymyxin B. ß2-glycoprotein 1 (ß2GP1) is a cofactor of antiphospholipid antibodies. Antiphospholipid autoantibodies also poses lupus anticoagulant activity through interactions with ß2GP1. Using affinity chromatography, polymyxin B can effectively decrease the binding of ß2GP1 to immobilize phosphatidylserine. Since then, anticoagulant effect of polymyxin B is most likely due to the binding to negatively charged phospholipids, preventing formation of coagulation complexes.

Thrombin generation and factor X assays for the assessment of warfarin anticoagulation in thrombotic antiphospholipid syndrome.

Monitoring warfarin anticoagulation in patients with thrombotic antiphospholipid syndrome (APS) may be complicated by the sensitivity of different thromboplastins to lupus anticoagulant. The aim of this study was to compare the degree of anticoagulation intensity in thrombotic APS and non-APS patients (50 in each group) on long-term warfarin, by measurement of the INR with two widely available thromboplastins with instrument-specific ISI values, and to investigate the potential role of amidolytic FX levels and thrombin generation (TG) testing in the assessment of anticoagulant intensity in thrombotic APS patients. There were no overall differences in INR between reagents or patient groups, but 20% (10/50) of APS patients showed ≥0.5 INR unit difference between reagents, which would have resulted in altered clinical management in some patients. FX levels were useful in assessing anticoagulation intensity for INR 2.0-3.0, but showed poor utility at INR ≥3.5 where the lowest measured FX level was 12IU/dL. In contrast, ETP and peak thrombin showed significant inverse correlations with the INR, suggesting that TG testing may be helpful in the determination of true anticoagulant intensity in APS patients, including those with ≥3.5 INR. TG testing also highlighted a subgroup of APS patients with increased peak thrombin relative to the intensity of anticoagulation as assessed by INR and FX, suggesting that TG testing may be useful in identifying an ongoing prothrombotic state in patients with apparently adequate anticoagulation intensity as assessed by INR.

Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays.

INTRODUCTION: Apixaban is an oral direct factor Xa inhibitor developed for the prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary, but the effects on common coagulation reagents and assays constitute clinically valuable information. OBJECTIVES: To investigate the effects of apixaban on commonly used coagulation methods, and to evaluate anti-FXa assays for specific determination of the drug concentration. MATERIALS AND METHODS: Apixaban was added to plasma from healthy subjects in the concentration range 0-1000 µg L(-1) , and analyses were performed with different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, protein C, and protein S. A lupus anticoagulant assay and an APTT assay with varying phospholipid concentrations were used to study the phospholipid dependence. RESULTS: In general, apixaban showed fewer effects in vitro than have been shown for rivaroxaban, another direct FXa inhibitor. The concentration needed to double the APTT varied between 2200 and 4700 µg L(-1) , and the concentration needed to double the PT varied between 700 and 3900 µg L(-1) . The effects on antithrombin, protein C and protein S assays were dependent on the type of reagent. Apixaban did not cause false-positive lupus anticoagulant results. Chromogenic anti-FXa assays showed linear dose-response curves with apixaban. CONCLUSIONS: Therapeutic concentrations of apixaban variably affect different assay groups, and even different reagents within an assay group. The effects were much smaller than with rivaroxaban. The use of APTT and/or PT assays to screen the anticoagulant activity of apixaban cannot be recommended. A chromogenic anti-FXa assay can be used for reliable measurements of apixaban concentration.

APTT reagent with ellagic acid as activator shows adequate lupus anticoagulant sensitivity in comparison to silica-based reagent.

BACKGROUND: Lupus anticoagulant (LA) is an antibody that interferes with phospholipid-dependent coagulation reactions. Activated partial thromboplastin time (APTT) is widely used as a test for LA screening. APTT reagents are composed of activators, such as silica or ellagic acid, and phospholipids, and APTT reagents with silica are recommended for LA screening because of greater sensitivity. However, the effects of activators on LA activity have not been adequately investigated. OBJECTIVES: In this study, we examined whether an ellagic acid-based reagent was highly sensitive to LA in a low phospholipid condition and useful for LA screening. METHODS: Silica-based (SL) and ellagic acid-based (EA) reagents were prepared in-house with the same composition and concentration of phospholipids, while the commercial APTT reagents APTT-SLA (SLA), Actin FSL (FSL), APTT-SP (SP) and PTT-LA (PTT) were also included in the study. RESULTS: The normal reference ranges for SL and EA were 30.1-47.0 and 28.0-40.2 s, respectively, while the cut-off index values for circulating anticoagulant activity (ICA) calculated from the results obtained with SL, EA, SLA, FSL, SP and PTT were 12.9, 11.5, 13.2, 15.6, 14.3 and 14.0, respectively. The sensitivity of those reagents based on those cut-off values was 91%, 96%, 68%, 46%, 91% and 86%, respectively. CONCLUSIONS: Our results showed that the ellagic acid-based reagent was more sensitive to LA than silica-based reagents in a low phospholipid condition and had adequate sensitivity to detect LA. We concluded that the sensitivity of APTT reagents for LA is dependent on phospholipid concentration and not the activator.

The antiphospholipid antibodies.

Antiphospholipid antibodies (APLAs) are a group of autoantibodies directed against certain phospholipids, or their protein cofactors. Assay of APLAs is important because their interaction with anionic phospholipid-protein cofactors can generate a syndrome of hypercoagulability associated with a wide variety of thromboembolic events. This article presents the characteristics of some APLAs [anticardiolipin antibodies (aCLAs), lupus anticoagulant (LA) and anti-beta2-glycoprotein I antibodies (anti-beta2-GPIAs)], their action, and their interaction with blood and endothelial cells. The presence of APLAs has been reported in many diseases (autoimmune diseases, atherosclerosis, infections, malignancies), being related to pathogenic mechanisms and/or to a more severe evolution of the disease.

Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid.

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 microM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.

Mutational analysis of a sequence-specific ssDNA binding lupus autoantibody.

11F8 is a murine anti-ssDNA monoclonal autoantibody isolated from a lupus prone autoimmune mouse. This mAb binds sequence specifically, and prior studies have defined the thermodynamic and kinetic basis for sequence-specific recognition of ssDNA (Ackroyd, P. C., et al. (2001) Biochemistry 40, 2911-2922; Beckingham, J. A. and Glick, G. D. (2001) Bioorg. Med. Chem. 9, 2243-2252). Here we present experiments designed to identify the residues on 11F8 that mediate sequence-specific, noncognate, and nonspecific recognition of ssDNA and their contribution to the overall binding thermodynamics. Site-directed mutagenesis of an 11F8 single-chain construct reveals that six residues within the complementarity determining regions of 11F8 account for ca. 80% of the binding free energy and that there is little cooperativity between these residues. Germline-encoded aromatic and hydrophobic side chains provides the basis for nonspecific recognition of single-stranded thymine nucleobases. Sequence-specific recognition is controlled by a tyrosine in the heavy chain along with a somatically mutated arginine residue. Our data show that the manner in which 11F8 achieves sequence-specific recognition more closely resembles RNA-binding proteins such as U1A than other types of nucleic acid binding proteins. In addition, comparing the primary sequence of 11F8 with clonally related antibodies that differ by less than five amino acids suggests that somatic mutations which confer sequence specificity may be a feature that distinguishes glomerulotrophic pathogenic anti-DNA from those that are benign.

Immunological and molecular analysis of three monoclonal lupus anticoagulant antibodies from a patient with systemic lupus erythematosus.

Antiphospholipid antibodies (APA), including lupus anticoagulants (LAC; as detected by in vitro blood clotting tests) and anti-cardiolipin antibodies (ACA; as assayed by solid-phase immunoassay), are strongly associated with recurrent thrombosis, thrombocytopenia, and recurrent fetal loss in some patients with systemic lupus erythematosus (SLE). The combined presence of APA and these clinical manifestations is termed antiphospholipid syndrome (APS). LAC and ACA comprise heterogeneous and somewhat overlapping autoantibody subsets. To date, it is unclear what degree of heterogeneity is present in an individual patient and between patients. To begin to address these issues, we generated three monoclonal LAC antibodies from a patient with SLE and APS. These antibodies were studied for their binding specificities and variable (V) region nucleotide sequences. All three LAC were unreactive with DNA, cardiolipin or other phospholipids. Sequence analysis of these antibodies revealed extensive overlap in their Ig V genes with anti-DNA antibodies and other autoantibodies characteristic of lupus. These data provide the first V gene sequence information on a group of SLE-derived LAC without ACA activity, representative of a similar subset of LAC found in patients with APS.