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

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 (K_d) 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.