Mechanisms of thrombosis in antiphospholipid syndrome (APS)

Antiphospholipid syndrome (APS) is an autoimmune-mediated acquired thrombophilia characterized by venous, arterial and small-vessel thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies. Other clinical manifestations are cardiac valvular disease, renal thrombotic microangiopathy, thrombocytopenia, livedo reticularis, hemolytic anemia and cognitive impairment.Antiphospholipid antibodies constitute a heterogenous group of autoantibodies directed against phospholipid-binding plasma proteins, mainly β2-glycoprotein I (β2GPI)-a plasma apolipoprotein and prothrombin (PT). A subgroup of these antibodies termed “Lupus Anticoagulant” (LA) elongate the clotting times in vitro, which are not corrected by adding normal plasma in the detection system. Of the different antiphospholipid antibodies, LA is the strongest predictor of features related to APS. In addition, anti-β2GPI antibodies and especially a specific population of these, recognizing a cryptic epitope on domain I of β2GPI, are found to be highly associated with clinical symptoms.The exact pathogenic mechanisms by which these autoantibodies induce thrombosis remain obscure. Resistance to natural anticoagulants such as protein C, disruption of anticoagulant protein annexin A5, impaired fibrinolysis, activation of endothelial cells and monocytes to a pro-coagulant and pro-inflammatory phenotype and activation of platelets, are among the mechanisms partially supported by experimental evidence. Complement activation and contribution of innate immunity might have a central pathogenetic role.In the current study, the mode of interaction between β2GPI/anti-β2GPI complexes and platelet ligands or receptors on membrane surfaces, as well as the subsequent platelet activation was investigated. It is well established that β2GPI does not have specific receptors on cell surfaces and besides it lacks intracellular domains, therefore the initiation of cell signaling is unclear. To reveal potential platelet ligands of β2GPI, a specific affinity chromatography method was performed and platelet factor 4 (PF4)38appeared as the dominant β2GPI binding protein, following mass spectrometry analysis. Evidence for this binding arose from different experimental procedures, including affinity chromatography with a β2GPI affinity column, loaded with enriched platelet membrane protein extracts from healthy donors and APS patients, direct binding assays in solid and soluble phase, coprecipitation of β2GPI and PF4, as well as size exclusion chromatography (SEC).PF4 is a member of the C-X-C chemokine family and is secreted by the a-granules of activated platelets. It also has the ability to bind to the platelet surface via glycosaminoglycans. Interestingly, PF4 has a proven precoagulant role and it contributes prominently in the pathogenesis of another prothrompotic condition, the so called heparin-induced thrombocytopenia and thrombosis syndrome (HIT). Thus PF4 could be considered as a common denominator in the pathogenesis of APS and HIT which share also clinical characteristics such as thrombocytopenia and thrombosis.Our study demonstrates that PF4, assembled in homotetramers, binds two β2GPI molecules simultaneously and the complex is highly recognized by anti-β2GPI antibodies. The formed immunocomplexes exert important thrombogenic influence concerning platelet activation, as confirmed by p38MAPK phosphorylation and thromboxane B2 production. The anti-β2GPI/β2GPI/PF4 immunocomplexes activate platelets by their F(ab)2 fragments.This study is the first to describe a novel interaction between β2GPI and PF4, both in the solid phase and in solution. The main significance of this interaction is the stabilization of β2GPI dimeric structure, attributed to PF4 binding, which facilitates the antibody recognition. This interaction can probably be involved in the procoagulant tendency of AP