Quinine-induced thrombocytopenia: drug-dependent GPIb/IX antibodies inhibit megakaryocyte and proplatelet production in vitro by José Perdomo, Feng Yan, Zohra Ahmadi, Xing-Mai Jiang, Roland Stocker, and Beng H. Chong Blood Volume 117(22):5975-5986 June 2, 2011 ©2011 by American Society of Hematology

Detection of megakaryocytic markers on Mks derived from CD34+ cells in culture. Detection of megakaryocytic markers on Mks derived from CD34+ cells in culture. (A) Experimental protocol for the culture conditions used. CD34+ cells were isolated from human mobilized peripheral blood samples cultured in the presence of TPO (50 ng/mL) for 4 days, washed, counted, and cultured for up to 12 days. D indicates days from the start of culture. (B) Cultured cells were stained with anti-human CD41-FITC and CD42a-Alexa Fluor 647 simultaneously and analyzed by flow cytometry. Shown is the dot-plot profile of GPIIb and GPIX double-positive Mks on day 8 of culture. (C) Kinetic expression of GPIIb (light gray) and GPIX (dark gray) on Mks on days 4, 7, 8, 9, 11, and 12 of culture. The graph shows the means ± SD. José Perdomo et al. Blood 2011;117:5975-5986 ©2011 by American Society of Hematology

Binding of QITP antibodies to platelets, CHO-GPIb/IX cells, CHO-GPIIb/IIIa cells, and Mks. Binding of QITP antibodies to platelets, CHO-GPIb/IX cells, CHO-GPIIb/IIIa cells, and Mks. QITP antibodies (WJ) were bound in the presence (+Qn, red) and absence (−Qn, green) to the platelets, CHO cells expressing human GPIb/IX (CHOαβIX) or GPIIb/IIIa, and cultured Mks derived from CD34+ cells. Control NS plus Qn is shown in blue. José Perdomo et al. Blood 2011;117:5975-5986 ©2011 by American Society of Hematology

QITP antibodies bind mature Mks. QITP antibodies bind mature Mks. Human platelets (A) and Mks (B) were incubated with QITP sera or NS without (left panels) and with Qn (right panels) and the anti-GPIX monoclonal antibody FMC25, followed by anti-human IgG–Alexa Fluor 488 (green) and anti-mouse IgG (H+L)–Alexa Fluor 594 (red). Binding of human IgG (green) was considerably enhanced only in the presence of the drug (right panels, middle image). The microphotographs were acquired by confocal laser scanning microscopy (Olympus FV300, 60× oil objective, Fluoview Version 4.3 software). Scale bars indicate 20 μm. Images were cropped and revised using Adobe Photoshop CS5. José Perdomo et al. Blood 2011;117:5975-5986 ©2011 by American Society of Hematology

Reduction of viable Mks and induction of apoptosis on QITP antibody treatment. Reduction of viable Mks and induction of apoptosis on QITP antibody treatment. Cultured CD34+ cells were treated on day 4 with QITP serum or purified IgG plus Qn or with controls (QITP serum or QITP IgG − Qn or NS or NS IgG + Qn). The total viable cell number was determined after 4 days of treatment by Trypan blue exclusion. Cells were treated with serum (A) or with purified IgG or WJ serum preabsorbed on platelets to remove auto-antibodies (WJpa) (B). (C) Treated Mks stained with anti-GPIIb and PI. The percentage of GPIIb+/PI+ cells was determined by flow cytometry. (D) Viable cells enumerated by Trypan blue exclusion and stained with anti-GPIIb, anti-GPIX, and anti-GPIbα antibodies. The percentage of GPIIb+ (empty bars), GPIX+ (gray bars), and GPIbα+ (solid bars) Mks present in the culture was determined by flow cytometry. The total cell number was calculated by multiplying the number of cells (direct counting) by the percentage of Mks positive for each marker. (E) Cells treated for 20 hours with the indicated sera or with 25μM paclitaxel followed by staining with Annexin V–FITC. The percentage of Annexin V+ cells in the control culture (NS + Qn) was normalized to 1 and the -fold increase in Annexin V+ cells on treatment plotted (n = 4). (F) Cells were treated as in panel E, stained with anti activated caspase 3-FITC antibody, and the -fold increase in activated caspase 3+ cells was plotted (n = 3). The mean ± SD is shown in all cases. José Perdomo et al. Blood 2011;117:5975-5986 ©2011 by American Society of Hematology

Inhibition of GPIX and GPIbα expression on treatment of CD34+ cells with QITP sera or QITP IgG. Inhibition of GPIX and GPIbα expression on treatment of CD34+ cells with QITP sera or QITP IgG. Cultured CD34+ cells were treated on day 4 with QITP serum or purified IgG plus Qn or with controls (QITP serum or QITP IgG − Qn or NS or NS IgG + Qn) and analyzed after 4 days in culture. (A) Left panels: flow cytometric data for GPIIb+, GPIX+, and GPIbα+ Mks treated with NS + Qn (blue), QITP serum − Qn (green), and QITP serum + Qn (red). Right panels: flow cytometric data for GPIIb+, GPIX+, and GPIbα+ Mks treated with NS IgG + Qn (blue), QITP IgG − Qn (green), and QITP IgG + Qn (red). Treatment with QITP serum or QITP IgG + Qn greatly decreased the expression of GPIX and GPIbα. (B) Mature Mks were stained with QITP serum + Qn or with controls (QITP serum − Qn or NS + Qn) for 30 minutes, followed by staining with PE-conjugated anti-human IgG antibody and anti-GPIX antibody conjugated with Alexa Fluor 647. The flow cytometric data show that the binding of the anti-GPIX antibody was not affected by prior incubation with QITP serum + Qn. (C) Number of receptors present on Mks determined as described in the supplemental “Methods.” The number of GPIX and GPIbα molecules on the cell surface was significantly reduced after QITS + Qn treatment. The mean ± SD is shown. José Perdomo et al. Blood 2011;117:5975-5986 ©2011 by American Society of Hematology

Treatment of Mks with QITP serum plus drug does not affect endomitosis. Treatment of Mks with QITP serum plus drug does not affect endomitosis. Cultured CD34+ cells were treated on day 4 with QITP serum + Qn or with controls (QITP serum − QN or NS + Qn) and analyzed after 5 or 6 days in culture. (A) Mks stained with anti-GPIIb antibody and PI in hypotonic citrate buffer. The DNA content of the GPIIb+ population was analyzed by flow cytometry. No differences were observed among the groups analyzed. (B) Mks stained with anti-GPIX antibody conjugated to Alexa Fluor 488 and imaged by confocal microscopy. Cells were chosen randomly (n = 94) and their area calculated using ImageJ software. The graph shows the mean ± SD. No significant differences were observed. (C) Cultured Mks centrifuged onto slides, stained for morphology with Wright/Giemsa staining, and imaged with a Zeiss Axioskop microscope and AxioVision 3.1 software (10× objective using an Axiocam camera; Zeiss). Scale bar indicates 50 μm. (D) Mks at different stages of development (20× objective). Bar indicates 20 μm. Images were cropped and revised using Adobe Photoshop CS5. (E) Percentage of monolobed, bilobed, or multilobed Mks calculated by randomly selecting 750 cells from each treatment group. The graph shows the percentage ± SD. José Perdomo et al. Blood 2011;117:5975-5986 ©2011 by American Society of Hematology

Reduced proplatelet production by QITP antibodies in the presence of quinine. Reduced proplatelet production by QITP antibodies in the presence of quinine. CD34+ cells were cultured for 5-6 days, and then seeded for treatment with QITP sera plus Qn or with controls (QITP sera − Qn or NS + Qn) and analyzed after 5 or 6 days in culture. (A) Representative image of typical morphologic features of proplatelets taken with a Leica DMIRB inverted microscope (20× objective using a DC200 camera; Leica). Areas containing proplatelets are circled, with proplatelet-bearing Mks indicated by arrows and proplatelet extensions indicated by white arrowheads (see inset for representative features of proplatelets). Bar indicates 50 μm. The images were cropped and revised using Adobe Photoshop CS5. (B) Proplatelets were enumerated and are presented as the number of proplatelets ± SD per 104 cells. Treatment with QITP sera + Qn significantly reduced proplatelet production. José Perdomo et al. Blood 2011;117:5975-5986 ©2011 by American Society of Hematology