Perfluorooctanoic acid alters progesterone activity in human endometrial cells and induces reproductive alterations in young women.

Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p < 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of ß-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.

Lupus anticoagulant identifies two distinct groups of patients with different antibody patterns.

BACKGROUND: Whether antibodies directed to ß2-Glycoprotein I (aß2GPI) are responsible for LA activity is not well defined. However, in the absence of such antibodies the molecule responsible for LA phenomenon is unknown. OBJECTIVE: The aim of this study was the biochemical identification of the target antigen epitope of aPL responsible of LA activity in the absence of aß2GPI antibodies together with the biological and clinical characteristics of these patients in comparison with classical triple positive patients. PATIENTS/METHODS: A comparison of patients with LA without (LA+/aß2GPI-) and those with (LA+/aß2GPI+) associated aß2GPI antibodies was performed. Size exclusion chromatography and analytical chromatography were used to identify the molecule with LA activity in patients LA+/aß2GPI-. RESULTS AND CONCLUSIONS: Analytical size-exclusion chromatography revealed a peak of 996Kd with LA activity perfectly overlapping that of IgM anti phosphatidylserine/prothrombin (aPS/PT) antibodies. Similarly, all the 25 LA+/aß2GPI- patients were positive for aPS/PT antibodies. LA+/aß2GPI- compared to 33 LA+/aß2GPI+ patients turned out to be significantly older, with a lower rate of previous thromboembolic events and a weaker LA activity. Search for aPS/PT and aß2GPI antibodies in patients with LA is useful to identify two subgroups of LA at different risk of thromboembolic events.

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.

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.