The binding site in β2-glycoprotein I for ApoER2′ on platelets is located is in domain V

Abstract

The antiphospholipid syndrome is caused by autoantibodies directed against β2-glycoprotein I (β2GPI). Dimerization of β2GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2′ (apoER2′), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric β2GPI. To identify which domain of dimeric β2GPI interacts with apoER2′, we have constructed domain deletion mutants of dimeric β2GPI, lacking domain I (ΔI), II (ΔII), or V (ΔV), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. ΔI and ΔII prolonged the clotting time, as did full-length dimeric β2GPI; ΔV had no effect on the clotting time. Second, ΔI and ΔII bound to anionic PL, comparable with full-length dimeric β2GPI. ΔV and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric β2GPI, ΔI, or ΔII (mean increase 150%) were added to whole blood. No increase was found with plasma β2GPI, AV, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric β2GPI, ΔI, ΔII, and the W316S mutant can interact with apoER2′ on platelets. ΔV did not associate with apoER2′. We conclude that domain V is involved in both binding β2GPI to anionic PL and in interaction with apoER2′ and subsequent activation of platelets. The binding site in β2GPI for interaction with apoER2′ does not overlap with the hydrophobic insertion loop in domain V. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.