ABSTRACT
The antiphospholipid syndrome is caused by autoantibodies directed against beta(2)-glycoprotein I (beta(2)GPI). Dimerization of beta(2)GPI 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 beta(2)GPI. To identify which domain of dimeric beta(2)GPI interacts with apoER2', we have constructed domain deletion mutants of dimeric beta(2)GPI, lacking domain I (DeltaI), II (DeltaII), or V (DeltaV), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. DeltaI and DeltaII prolonged the clotting time, as did full-length dimeric beta(2)GPI; DeltaV had no effect on the clotting time. Second, DeltaI and DeltaII bound to anionic PL, comparable with full-length dimeric beta(2)GPI. DeltaV and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric beta(2)GPI, DeltaI, or DeltaII (mean increase 150%) were added to whole blood. No increase was found with plasma beta(2)GPI, DeltaV, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric beta(2)GPI, DeltaI, DeltaII, and the W316S mutant can interact with apoER2' on platelets. DeltaV did not associate with apoER2'. We conclude that domain V is involved in both binding beta(2)GPI to anionic PL and in interaction with apoER2' and subsequent activation of platelets. The binding site in beta(2)GPI for interaction with apoER2' does not overlap with the hydrophobic insertion loop in domain V.
Subject(s)
Blood Coagulation , Blood Platelets/metabolism , Glycoproteins/metabolism , Phospholipids/metabolism , Receptors, Lipoprotein/metabolism , Amino Acid Substitution , Animals , Binding Sites , Cells, Cultured , Collagen/metabolism , Cricetinae , Dimerization , Glycoproteins/genetics , Glycoproteins/isolation & purification , Humans , Immunoprecipitation , Kidney/cytology , Kidney/metabolism , LDL-Receptor Related Proteins , Megakaryocytes/cytology , Megakaryocytes/metabolism , Peptide Fragments/pharmacology , Plasmids , Platelet Activation , Protein Structure, Tertiary , Receptors, Lipoprotein/genetics , Receptors, Lipoprotein/isolation & purification , Sequence Deletion , Umbilical Cord/cytology , Umbilical Cord/metabolism , beta 2-Glycoprotein IABSTRACT
Following a human leucocyte antigen (HLA)-identical allogeneic stem cell transplantation (allo-SCT), minor histocompatibility antigens (mHags) play an important role in the induction of graft-versus-leukaemia (GvL) and graft-versus-myeloma (GvM). Many mHags show ubiquitous tissue expression and are associated with GvL and graft-versus-host disease. Here we describe a cytotoxic CD4(+) T lymphocyte line and a cytotoxic, CD4(+) T cell clone (CTC), 3AB11, which recognized a tissue-restricted mHag. This CTC was isolated from a multiple myeloma patient with clinical GvM following an HLA-matched allo-SCT. CTC 3AB11 was activated in a HLA-DP*0401 restricted fashion and the antigen was expressed by 27% of HLA-DP*0401 positive Epstein-Barr virus (EBV)-transformed B-cell lines (EBV-B). Tissue distribution analysis of antigen 3AB11 showed it to be expressed by patient-derived EBV-transformed B cell lines (EBVp), the myeloma plasma cell-line UM9 and monocytes. It was weakly expressed by peripheral blood-derived phytohaemagglutinin-induced T-cell blasts and absent on CD40L stimulated peripheral B (CD40L B) cells and stromal cells. The relatively high prevalence of the HLA class II-restricted 3AB11 antigen, together with its apparent haematopoietic-restricted expression, makes it an antigen of interest for cellular immunotherapy.
