Circulating ß2 glycoprotein I-IgG anti-ß2 glycoprotein I immunocomplexes in patients with definite antiphospholipid syndrome.

Beta2-glycoprotein I (ß(2)GPI), a relevant antigen in Antiphospholipid Syndrome (APS), binds anionic macromolecules including heparin (Hep). A possible formation of ternary complexes between ß(2)GPI, antibodies and Hep in APS is thus possible. The aim of this study was to evaluate Hep-ß(2)GPI interaction in patients with APS. The affinity of Heps of different length, including unfractionated Hep (UFH), low-molecular weight Hep (enoxaparin) and pentasaccharide (fondaparinux), to human ß(2)GPI was estimated by fluorescence spectroscopy, yielding dissociation constant (K(d)) values of 1.1, 24.0 and 89.4 µM, demonstrating that the longer UFH binds to ß(2)GPI far more tightly than the shorter ones. Plasma and protein G-purified IgGs from eight patients with APS (i.e. five with thromboembolic disease and three with catastrophic APS), were fractionated by affinity chromatography using a Hep (UFH)-bound column, eluted with a linear NaCl gradient. For each chromatographic analysis, fractions were collected in the whole NaCl gradient and tested by ELISA for the presence of ß(2)GPI and anti-ß(2)GPI IgG. The results of Hep-affinity chromatography and ELISAs concurrently indicate that either ß(2)GPI and anti-ß(2)GPI IgG elute from the Hep column in the same chromatographic peak, at a retention time identical to that of the purified, isolated ß(2)GPI, thus suggesting that circulating immunocomplexes containing ß(2)GPI are present in patients with APS.

Discovery and characterization of an antibody directed against exosite I of thrombin.

UNLABELLED: ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. BACKGROUND: We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. OBJECTIVES: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. METHODS: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. RESULTS: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. CONCLUSIONS: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.

ß2 -Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions.

BACKGROUND: This work was aimed at characterizing the interaction of ß(2)-glycoprotein I (ß(2)GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. METHODS: The ß(2)GPI-thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of ß(2)GPI on the procoagulant (fibrin generation and platelet aggregation) and anticoagulant (protein C activation) functions of thrombin were investigated with turbidimetric, immunocytofluorimetric and enzymatic assays. RESULTS: SPR and fluorescence data indicated that ß(2)GPI tightly bound thrombin (K(d) = 34 nM) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the ß(2)GPI-thrombin complex. In particular, blockage of thrombin exosites with binders specific for exosite-1 (hirugen and HD1 aptamer) or exosite-2 (fibrinogen γ'-peptide and HD22 aptamer) impaired the ß2 GPI-thrombin interaction. Identical results were obtained with thrombin mutants having one of the two exosites selectively compromised by mutation (Arg73Ala and Arg101Ala). Fluorescence measurements indicated that ß(2)GPI did not affect the affinity of the enzyme for active site inhibitors, such as p-aminobenzamidine and the hirudin(1-47) domain, in agreement with the structural model. ß(2)GPI dose-dependently prolonged the thrombin clotting time and ecarin clotting time in ß(2)GPI-deficient plasma. ß(2)GPI inhibited thrombin-induced platelet aggregation (IC50 = 0.36 µM) by impairing thrombin cleavage of protease-activated receptor 1 (PAR1) (IC50 = 0.32 µM), both on gel-filtered platelets and in whole blood. Strikingly, ß(2) GPI did not affect thrombin-mediated generation of the anticoagulant protein C. CONCLUSIONS: ß(2) GPI functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function.

Antibodies to Domain I of ß(2)Glycoprotein I are in close relation to patients risk categories in Antiphospholipid Syndrome (APS).

INTRODUCTION: Antiphospholipid Syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies in patients with thrombosis or pregnancy morbidity. Antibodies involved in these disorders are mainly those directed against ß(2)-Glycoprotein I (ß(2)GPI) with the major epitope apparently located on discontinuous antigen with several parts of Domain I (DmI) involved. The relation between anti-DmI antibodies and patients' risk categories is unknown. MATERIALS AND METHODS: The synthetic full-length and correctly-folded DmI (1-64) to set up a competitive inhibition enzyme-linked immunoadsorbent assays (ELISA) was used. Plasma of 22 patients with APS and triple positivity [Lupus Anticoagulant positive (LAC+), IgG anti-cardiolipin positive (aCL+), IgG anti-ß(2)GPI positive (a ß(2)GPI +)], 15 with double positivity (IgG aCL+, IgG aß(2)GPI+), 9 with single positivity (IgG aß(2)GPI+) and 20 controls were evaluated. RESULTS: Median of percentage inhibition was 25.5% [interquartile range (IQR)17.2-33.0] in triple positive patients. Significantly lower inhibition was observed in patients with double positivity, median inhibition 5.0% (IQR 0.0-27.0) and in patients with single positivity median inhibition was 2.0% (IQR 0.5-8.0) (p<0.0001). No inhibition was detected in control subjects or using ß(2)GPI peptides (40-52 and 57-70), or when antithrombin, an insignificant control protein was used. CONCLUSIONS: High risk patients with APS and triple laboratory positivity as compared with double and single positivity patients have significantly higher titre of anti-DmI antibodies as evaluated by an inhibition test.