Quantitative determination of the binding of β₂- glycoprotein I and prothrombin to phosphatidylserine-exposing blood platelets

The plasma protein β₂GPI (β₂-glycoprotein I) has been proposed to mediate phagocytosis of apoptotic cells and to play a role in the antiphospholipid syndrome. This suggestion is based mainly on the presumption that β₂GPI has an appreciable interaction with PS (phosphatidylserine)-exposing cell membranes. However, quantitative data on the binding of β₂GPI to PS-exposing cells under physiologically relevant conditions are scarce and conflicting. Therefore we evaluated the binding of β₂GPI to PS-expressing blood platelets. Flow cytometry showed that binding of β₂GPI is negligible at physiological ionic strength, in contrast with significant binding occurring at low ionic strength. Binding parameters of β₂GPI and (for comparison) prothrombin were quantified by ellipsometric measurement of protein depletion from the supernatant following incubation with platelets. At low ionic strength (20 mM NaCl, no CaCl₂), a dissociation constant (K _d) of 0.2 μM was found for β₂GPI, with 7.4 × 10⁵ binding sites per platelet. Under physiologically relevant conditions (120 mM NaCl and 3 mM CaCl₂), binding of β₂GPI was not detectable (extrapolated K_d > 80 μM). Prothrombin binding (at 3 mM CaCl₂) was much less affected by ionic strength: K_d values of 0.5 and 1.4 μM were observed at 20 and 120 mM NaCl respectively. The low affinity and the presence of many lipid-binding proteins in plasma that can compete with the binding of β₂GPI suggest that only a small fraction (< 5%) of the binding sites on PS-exposing blood cells are likely to be occupied by β₂GPI. These findings are discussed in relation to the alleged (patho-)physiological functions of β₂GPI.