Recently, we published the existence of 2 populations of anti-␤ 2 -glycoprotein I (␤ 2 -GPI) IgG antibodies. Type A antibodies recognize epitope G40-R43 in domain I of ␤ 2 -GPI and are strongly associated with thrombosis. Type B antibodies recognize other parts of ␤ 2 -GPI and are not associated with thrombosis. In this study we demonstrate that type A antibodies only recognize plasma-purified ␤ 2 -GPI when coated onto a negatively charged surface and not when coated onto a neutrally charged surface. The affinity of type B antibodies toward plasma-purified ␤ 2 -GPI was independent of the charge of the surface to which ␤ 2 -GPI was coated. Type A antibodies did not recognize plasmapurified ␤ 2 -GPI in solution, whereas they did recognize recombinant ␤ 2 -GPI both in solution and coated onto a neutrally charged plate. When the carbohydrate chains were removed from plasma-purified ␤ 2 -GPI, we found that type A antibodies did recognize the protein in solution. This supports the hypothesis that the difference in recognition of plasma-purified and recombinant ␤ 2 -GPI is caused by the difference in glycosylation and that epitope G40-R43 of plasma-purified ␤ 2 -GPI is covered by a carbohydrate chain. Type A anti-␤ 2 -GPI antibodies can only recognize this epitope when this carbohydrate chain is displaced as a result of a conformational change. This finding has major implications both for the detection of pathogenic anti-␤ 2 -GPI antibodies and the comprehension of the pathophysiology of the antiphospholipid syndrome.

Antiphospholipid (aPL)/anti-␤ 2 glycoprotein I (anti-␤ 2 GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2) on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti-␤ 2 GPI monoclonal antibody (E7) and of a constructed dimeric ␤ 2 GPI I (dimer), which in vitro mimics ␤ 2 GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (؊/؊) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (؊/؊) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies. (Blood. 2011;117(4):1408-1414) IntroductionThe association between persistently present antiphospholipid (aPL) antibodies and the clinical manifestations of thrombosis or pregnancy morbidity is known as the antiphospholipid syndrome (APS). 1 aPL antibodies are heterogeneous and recognize a wide variety of plasma proteins with phospholipid-binding properties, such as prothrombin 2 and ␤ 2 glycoprotein I (␤ 2 GPI). 3,4 aPL antibodies directed against ␤ 2 GPI, a plasma protein without known physiologic function, are considered the most pathologically relevant antibodies.There is strong experimental evidence that anti-␤ 2 GPI antibodies have thrombogenic properties. In studies on endothelial cell activation, 5-8 authors have shown the induction of a prothrombotic and proinflammatory phenotype upon exposure to anti-␤ 2 GPI antibodies, indicated by expression of tissue factor (TF) and increased surface expression of adhesion molecules, such as intercellular adhesion molecule-1, vascular-cell adhesion molecule-1, and E-selectin. Activation of monocytes by anti-␤ 2 GPI antibodies leads to TF expression as well. 9 Furthermore, anti-␤ 2 GPI antibodies, or recombinant dimers of ␤ 2 GPI that mimic ␤ 2 GPI-antibody immune complexes, increase platelet deposition to extracellular matrix components in in vitro flow models. 10 Injection of anti-␤ 2 GPI antibodies in murine 11 or hamster 12 thrombosis models leads to increased thrombus formation.Several receptors were postulated to mediate the prothrombotic cellular effects of anti-␤ 2 GPI antibodies. The interaction between annexin A2 and ␤ 2 GPI-antibody immune complexes has been reported to lead to endothelial cell activation. 13 It seems unlikely, however, that annexin A2 is able to convey activation signals across the cell membrane because this phospholipid-binding protei...

To cite this article: Urbanus RT, Pennings MTT, Derksen RHWM, de Groot PG. Platelet activation by dimeric b 2 -glycoprotein I requires signaling via both glycoprotein Iba and apolipoprotein E receptor 2¢.

Summary. Background: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (b 2 GPI). Dimerized b 2 GPI binds to apolipoprotein E receptor 2¢ (apoER2¢) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized b 2 GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized b 2 GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized b 2 GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized b 2 GPI was completely inhibited by the addition of soluble forms of both apoER2¢ and GPIba, and the addition of receptor-associated protein and the removal of GPIba from the platelet surface. GPIba co-precipitated with apoER2¢, suggesting the presence of complexes between GPIba and apoER2¢ on platelet membranes. The interaction between GPIba and dimeric b 2 GPI was of intermediate affinity (K d ¼ 180 nM) and Zn 2+, but not Ca 2+ -dependent. Deletion of domain V from dimeric b 2 GPI strongly reduced its binding to both GPIba and apoER2¢. Antibodies that inhibit the binding of thrombin to GPIba inhibited platelet adhesion to dimeric b 2 GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIba had no effect. Dimeric b 2 GPI showed reduced binding to lowsulfated GPIba compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric b 2 GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIba and apoER2¢. These receptors are present in a complex on the platelet surface.

See also Pierangeli SS. In search for a receptor for antiphospholipid antibodies on target cells. This issue, pp 1678-9.Summary. The antiphospholipid syndrome (APS) is a noninflammatory autoimmune disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity in the presence of autoantibodies that recognize beta2-glycoprotein I (b 2 GPI) bound to phospholipids. We have previously demonstrated that dimerization of b 2 GPI by autoantibodies induces platelet activation, involving the platelet receptor apolipoprotein E receptor 2Õ (apoER2¢) a receptor belonging to the lowdensity lipoprotein receptor (LDL-R) family. Here, we show that dimeric b 2 GPI, but not monomeric b 2 GPI, interacts with four other LDL-R family members: the LDL-R related protein (LRP), megalin, the LDL-R and the very-low density lipoprotein receptor (VLDL-R). Interaction between dimeric b 2 GPI and LDL-R, apoER2¢ and VLDL-R was best described with a one-site binding model (half-maximal binding; $20 nM for apoER2¢ and VLDL-R and $300 nM for LDL-R), whereas the interaction between dimeric b 2 GPI and LRP or megalin was best described with a two-site binding model, representing a high-($3 nM) and a low-affinity site ($0.2 lM). Binding to all receptors tested was unaffected by a tryptophane to serine (W316S) substitution in domain V of b 2 GPI, which is known to disrupt the phospholipid binding site of b 2 GPI. Also deletion of domain I or II left the interaction with the receptors unaffected. Deletion of domain V, however, significantly decreased the affinity for the receptors. In conclusion, our data show that dimeric b 2 GPI can interact with different LDL-R family members. This interaction is dependent on a binding site within domain V of b 2 GPI, which does not overlap with the phospholipid-binding site within domain V.