GLYCOPROTEIN Ib PEPTIDE PRESENTED BY MHC CLASS I ON PLATELETS FROM INDIVIDUALS WITH ITP

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TITLE: GLYCOPROTEIN Ib PEPTIDE PRESENTED BY MHC CLASS I ON PLATELETS FROM INDIVIDUALS WITH ITP

Leann M Hopkins,¹ John M. Davis,² Kenneth A Schwartz,² John A. Gerlach.³

¹Pathobiology and Diagnostic Investigation, Michigan State University; ²Medicine, Michigan State University; ³Medical Technology Program and Human Pathology, Michigan State University, East Lansing, MI

Idiopathic thrombocytopenia purpura (ITP) is a disease characterized in most patients by the premature immune destruction of platelets. ITP can be associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane glycoprotein (GP)IIb/IIIa and/or GPIb/IX. As an autoimmune disease, there is T cell involvement. The purpose of this research was to characterize the most abundant MHC class I associated peptides presented on platelets from an ITP population. With appropriate consent, whole blood samples from five ITP patients and six controls were obtained. Samples were MHC typed. From these samples, platelets were isolated. Using a mild acid wash with citric acid buffer pH 3.1, MHC associated peptides were released from the intact platelets. The peptides released from the MHC were purified by step gradient RP–HPLC on a peptide trap and dried down. The obtained peptides were analyzed by a LC–MS system consisting of a self–packed 75um ID reverse phase column connected to an ion trap mass spectrometer via micro–electrospray device. MS data were collected using a data dependent mode with dynamic exclusion. From the MS/MS data, peptide sequence was assigned by de novo sequencing with the aid of computer analysis. The peptide GPRGA(L/I)S(L/I)(L/I) was identified in four of the five ITP patients and none of the random controls. A BLAST search identified the peptide, GPRGALSLL, as GPIb (4–12). The anchor residues of the peptide (2nd and 9th position) that hold the peptide into the cleft of the MHC are P and L respectively. The four ITP patients from whom the peptide GPRGALSLL was identified coincidently all have the allele HLA–B7. HLA–B7 molecules have been found to bind peptides with the anchor residues P in the 2nd position and L or F in the last. The GPIb peptide (4–12), which fits the binding motif of HLA–B7, was not identified as an abundant peptide in the fifth ITP patient who does not have the HLA–B7 allele. These findings support the notion that GPIb peptide (4–12) presented to T cells by MHC platelet surface molecules may contribute to the etiology of ITP.