MHC class I-associated peptides identified from normal platelets and from individuals with idiopathic thrombocytopenic purpura.

Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8(+) T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I-associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4-12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I.

Characterization of major histocompatibility complex-associated peptides from a small volume of whole blood.

Class I major histocompatibility complex (MHC) presents intracellular-derived peptides on the majority of cells within the human body. Intracellular proteins are degraded into peptides of 8-11 amino acids, allowing them to fit into the groove of an empty MHC class I molecule. Detection of MHC-associated peptides can be challenging with the major difficulty being the ability to obtain peptides in adequate concentration. Published protocols require a large sample size that is unrealistic for a clinically available sample. Based on calculations, it should be possible to characterize MHC-associated peptides from cells obtained from 30 ml of whole blood. A citric acid wash of whole platelets was implemented to release the peptides with sample cleanup by reversed-phase high-performance liquid chromatography on a peptide trap. Peptides were analyzed by liquid chromatography tandem mass spectrometry. Four peptides were identified from an individual's platelets. The binding motifs of the peptides were consistent with the published MHC binding motif of the individual. Since red blood cells do not express MHC, they were used as a negative control. Using citric acid wash of whole cells and a peptide trap, the more abundant MHC-associated peptides can be identified. This report demonstrates the identification of peptides from a sample volume compatible with reasonable clinical availability.