Circulating β2 glycoprotein I-IgG anti-β2 glycoprotein I immunocomplexes in patients with definite Antiphospholipid Syndrome

Abstract

Beta2-glycoprotein I (β2GPI), a relevant antigen in Antiphospholipid Syndrome (APS), binds anionic macromolecules including heparin (Hep). A possible formation of ternary complexes between β2GPI, antibodies and Hep in APS is thus possible. The aim of this study was to evaluate Hep-β2GPI 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 β2GPI was estimated by fluorescence spectroscopy, yielding dissociation constant (Kd) values of 1.1, 24.0 and 89.4 µM, demonstrating that the longer UFH binds to β2GPI 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 β2GPI and anti-β2GPI IgG. The results of Hep-affinity chromatography and ELISAs concurrently indicate that either β2GPI and anti-β2GPI IgG elute from the Hep column in the same chromatographic peak, at a retention time identical to that of the purified, isolated β2GPI, thus suggesting that circulating immunocomplexes containing β2GPI are present in patients with APS.