Platelt receptors involved in the antiphospholipid syndrome

Abstract

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease clinically characterized by the occurrence of either venous or arterial thrombosis or the presence of specific pregnancy complications. Serological criteria are the persistent presence of antibodies directed against cardiolipin or β2-glycoprotein I (β2-GPI) or a prolonged clotting time due to the presence of lupus anticoagulant in plasma. The precise mechanism behind the pathology of APS is largely unknown. Previous work demonstrated that blood platelets incubated with β2-GPI, dimerized by auto-antibodies or monoclonal antibodies become pro-thrombotic. A recombinant dimeric β2-GPI in which the dimerization domain of clotting factor XI (apple4) was used for dimerization, mimics the pro-thrombotic properties of β2-GPI-anti-β2-GPI complexes. In perfusion studies we found that upon dimerization, β2-GPI binds to the platelet receptors Apolipoprotein receptor E2' and Glycoprotein Ibα (ApoER2' and GPIbα). Platelets express 3 different splice variants of ApoER2 which can all participate in signaling events. The splice variant of ApoER2 on platelets expressed the most abundant and participating the strongest in ApoER2 signaling lacks LDL-binding domains 3, 4, 5 and 6 (ApoER2-750 or ApoER2'). Domain deletion studies showed that the binding site for dimeric β2-GPI resides in the LDL-binding domain I of ApoER2'. LDL-binding domain I was expressed in all three ApoER2 splice variants present on platelets. Solid phase binding assays showed the binding site for dimeric β2-GPI on GPIbα in close proximity of the thrombin biding site. The interaction between dimeric β2-GPI and GPIbα was depending on the presence of Zn2+. Dimeric β2-GPI bound to both ApoER2' and GPIbα via its domain V and the binding site for negatively charged phospholipids and ApoER2' did not overlap. The binding site on dimeric β2-GPI for GPIbα and ApoER2 did not overlap either. We furthermore showed that ApoER2' and GPIbα are present in a receptor complex on platelets independent of the presence of dimeric β2-GPI or cytoskeletal association. To test physiological relevance of the interactions of dimeric β2-GPI with ApoER2' and GPIbα, perfusion studies were performed. The interaction of dimeric β2-GPI with both ApoER2' and GPIbα and subsequent downstream receptor specific signaling events were required for platelet activation and adhesion to dimeric β2-GPI and fibronectin under conditions of flow. The transient dissociation of ApoER2 from its adaptor protein Dab-1 however, was not inhibited when dimeric β2-GPI binding to GPIbα binding was blocked. Also, GPIbα translocation towards the cytoskeleton via its adaptor protein 14-3-3-ζ could not be inhibited by blocking dimeric β2-GPI binding to ApoER2'. These observations suggest the presence of a common pathway downstream of the initial signaling events. This common pathway is currently under investigation. The work presented in this thesis opens new therapeutic windows in the treatment of APS. Molecular understanding of protein-protein interactions and subsequent downstream events will help understanding the mechanisms involved in the pathology of APS. In-vitro, blocking the binding of dimerized β2-GPI to either ApoER2' or GPIbα was sufficient to block the pro-thrombotic phenotype of blood platelets induced by binding of dimeric β2-GPI. Animal studies are currently being used to test efficacy of blocking the interaction of dimeric β2-GPI with either GPIbα or ApoER2'.