by Parisa Asvadi, Zohra Ahmadi, and Beng H. Chong Drug-induced thrombocytopenia: localization of the binding site of GPIX-specific quinine-dependent antibodies by Parisa Asvadi, Zohra Ahmadi, and Beng H. Chong Blood Volume 102(5):1670-1677 September 1, 2003 ©2003 by American Society of Hematology

Schematic representation of the chimeric human/mouse GPIXs Schematic representation of the chimeric human/mouse GPIXs. Chimeric human/mouse GPIX constructs were generated to dissect the human GPIX into 3 domains and assess the contribution of each domain to quinine-dependent antibody binding. Schematic representation of the chimeric human/mouse GPIXs. Chimeric human/mouse GPIX constructs were generated to dissect the human GPIX into 3 domains and assess the contribution of each domain to quinine-dependent antibody binding. Figure depicts the leucine-rich motif (LRM) and the regions which precede (N) and proceed (C-ext) it. The portions shown in black are of mouse origin, whereas the white boxes denote human origin. Single-letter amino acid codes are used. Parisa Asvadi et al. Blood 2003;102:1670-1677 ©2003 by American Society of Hematology

Sequence similarity between the human and mouse GPIX Sequence similarity between the human and mouse GPIX. The mature protein sequence of human and mouse GPIX are aligned (single letter amino acid code). Sequence similarity between the human and mouse GPIX. The mature protein sequence of human and mouse GPIX are aligned (single letter amino acid code). Asterisks denote sequence identity. The boxed sequences represent the LRM and the potential transmembrane domain (top to bottom), respectively. The 6 amino acids after the transmembrane domain are the potential cytoplasmic domain. Numbers indicate the position of the last amino acid in the line. Parisa Asvadi et al. Blood 2003;102:1670-1677 ©2003 by American Society of Hematology

The hGPIX C-ext mutants. The hGPIX C-ext mutants. The C-ext region (aa 64-135) of mouse and human GPIX is aligned. The amino acid differences are shown in gray. Six hGPIX mutants were generated by replacing the group of amino acids labeled 1 to 6 in the human GPIX with the corresponding residues from mouse GPIX. Parisa Asvadi et al. Blood 2003;102:1670-1677 ©2003 by American Society of Hematology

Activity of quinine-dependent thrombocytopenia patient sera against mouse platelets. Activity of quinine-dependent thrombocytopenia patient sera against mouse platelets. Patient sera were used in flow cytometry experiments to examine whether the reaction against human and mouse platelets could be distinguished. Figure is representative of the reaction of patient sera against human and mouse platelets. AB type sera, an isotype-matched IgG, and patient sera in the absence of quinine were used as negative controls (solid gray peak). Open histograms: dotted line indicates mouse platelets without quinine; dotted and dashed line, mouse platelets with quinine; thin solid line, human platelets without quinine; and thick solid line, human platelets with quinine. Parisa Asvadi et al. Blood 2003;102:1670-1677 ©2003 by American Society of Hematology

Binding of FMC25 and SZ1 to GPIX chimeras. Binding of FMC25 and SZ1 to GPIX chimeras. The WT GPIX and the 4 human/mouse chimeras were expressed on the surface of CHOαβ cells, and the binding of anti-GPIX moAbs FMC25 and SZ1 was examined. An isotype-matched IgG was used as the negative control (gray solid histogram). Open histograms (black) depict the binding of FMC25 and SZ1 to CHOαβ cells expressing WT GPIX, chimeras 1, 2, 3, and 4. Parisa Asvadi et al. Blood 2003;102:1670-1677 ©2003 by American Society of Hematology

Binding of quinine-induced thrombocytopenia patient sera to WT and chimeric GPIXs. Sera from 6 patients were used against the CHOαβ cells expressing WT and chimeric GPIXs to determine what region of GPIX was the target of antibodies in patient sera. Binding of quinine-induced thrombocytopenia patient sera to WT and chimeric GPIXs. Sera from 6 patients were used against the CHOαβ cells expressing WT and chimeric GPIXs to determine what region of GPIX was the target of antibodies in patient sera. Histograms represent the reaction of one patient serum (patient 1) against the cell lines expressing WT GPIX and the 4 chimeras. The pattern of reaction was representative of the set of 6 sera tested. Solid peaks (gray) denote the binding of negative controls [(–) quinine test samples and isotype-matched IgG]. Open peaks (black) denote patient-derived quinine-dependent antibody binding. Parisa Asvadi et al. Blood 2003;102:1670-1677 ©2003 by American Society of Hematology

Reactivity of FMC25 and SZ1 against the hGPIX C-ext mutants. Reactivity of FMC25 and SZ1 against the hGPIX C-ext mutants. The WT GPIX and the 6 hGPIX C-ext mutants were expressed on the surface of CHOαβ cells, and the binding of anti-GPIX moAbs FMC25 and SZ1 was examined. An isotype-matched IgG was used as the negative control (gray solid histogram). Open histograms (black) depict the binding of FMC21 and SZ1 to CHOαβ cells expressing the mutants. Parisa Asvadi et al. Blood 2003;102:1670-1677 ©2003 by American Society of Hematology