Background: β₂GpI is an abundant plasma protein of yet unknown function and the main autoantigen in the antiphospholipid syndrome (APS). We have recently discovered that β₂GpI may function as a mild anticoagulant, as it binds to α-Thrombin (αT) exosite-2, impairing binding to GpIbα platelet receptor [Pozzi et al., JTH (2013); Acquasaliente et al., Biochem J (2016)]. Earlier studies have shown that β₂GpI Domain-I is the major epitope for APS autoantibodies.

Aims: Check whether β₂GpI interacts with fibrinogen (Fb); identify the binding regions on β₂GpI and Fb involved in β₂GpI-Fb complex formation; study the effect of β₂GpI binding to Fb on fibrin structure.

Methods: Binding measurement were performed by fluorescence and Surface Plasmon Resonance (SPR). Fibrin structure was studied by turbidimetry, Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Fb fragment-X and β₂GpI nicked at Domain-V were both prepared by limited proteolysis with plasmin [Furlan and Beck, 1972; Ohkura et al., Blood (1998)]. Domain-I was produced by solid-phase synthesis [Pozzi et al., Protein Sci (2010)].

Results: Fluorescence and SPR analyses indicate that β₂GpI interacts with Fb at two sites, with K_d¹ = 15±4nM and K_d² > 1µM. Identical results were obtained with Domain-I and Domain-V clipped β₂GpI. Binding of β₂GpI to Fb fragment-X, lacking the Ca-domains, resulted in affinity drop. Turbidimetric, DLS, and SEM measurements indicate that 4mM β₂GpI induces formation of thinner and shorter fibrin fibers.

Conclusions: Given the plasma concentrations of β₂GpI (4mM) and Fb (7mM), and the affinity of β₂GpI for Fb (K_d¹ = 15nM), we conclude that under physiological conditions β₂GpI circulates in the fibrinogen-bound form. β₂GpI-Fb complex formation is driven by β₂GpI Domain-I and Fb Ca-domains. Considered that Domain-I is also the major epitope for APS autoantibodies, our results challenge the current view on the role of β₂GpI in the pathogenesis of APS.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/%ce%b22-glicoprotein-i-%ce%b22gpi-is-a-fibrinogen-binding-protein/