Proplatelet Production Is Impaired by Drug-Induced Thrombocytopenia Antibodies

Abstract 384

The development of immune cytopenias is a well recognized side-effect of many drugs. Of a number of causative agents quinine- and quinidine-induced antibodies are classical examples of drugs that cause severe, life threatening thrombocytopenia and their mechanisms of action could be applicable to other drug-induced factors. The antibodies responsible for quinine (Qn)-induced thrombocytopenia (QITP) are immunoglobulins that usually recognize the von Willebrand factor or the fibrinogen receptors, GPIb/IX or GPIIb/IIIa, respectively. While the effect of drug-induced antibodies on platelets has been well documented their effect on megakaryocyte (Mk) biology has not been described in any detail. We analyzed sera from several QITP patients on highly pure Mks (98% CD41⁺; 92% CD42a⁺) derived from human CD34⁺ cells cultured with recombinant human THPO at 50 ng/ml. We demonstrated by confocal microscopy and flow cytometry that immunoglobulins from QITP patients bound Mks efficiently in the presence of Qn (ranging from 38% to 87% positive cells). Incubation of Day 4 Mks with QITP serum (1:5 dilution) for 4 days in the presence of Qn (0.01mM) resulted in a significant decrease in the number of viable cells (trypan blue exclusion, <50% decrease in total cell number, p=0.007) and an increase (p=0.01 to 0.04) in cell death in the CD41⁺ population (propidium iodide staining). Importantly, QITP serum + Qn appeared to preferentially reduce the number of late Mks (CD41, 61%; CD42a, 80% and CD42b, 96%) (Fig. 1A) based on the expression of these markers on surviving cells. Unexpectedly, the ploidy status of CD41⁺ cells remained unchanged after treatment with QITP serum (Fig. 1B). To further evaluate this observation, control and treated Mks were stained with anti CD41 antibody and imaged by confocal microscopy. Cells were randomly selected (n=100) and the cell area determined using ImageJ software. No significant differences in cell area were found between control and treated cells (p=0.18). Furthermore, control and treated Mks were subjected to Wright staining and 250 cells randomly selected and classified by lobularity of the nucleus. No measurable differences were observed between normal serum + Qn, QITP serum –Qn and QITP serum +Qn samples with mono- and bi-lobed cells comprising 90%, 88% and 89% of all cells respectively, while multilobed Mks corresponded to around 10% of the analyzed cells. We then assessed the effect of QITP treatment on the Mk proplatelet production capability. CD34⁺ cells were cultured for 6 days and then seeded at 2×10⁴ cells/well in 24-well plates, incubated for 5 days and the proplatelets enumerated. As shown in Fig. 1C, incubation of Mks with QITP serum +Qn resulted in an 85% reduction (p=0.003) in the number of proplatelets. This suggests that QITP antibodies compromise the ability of mature Mks to form proplatelets. In summary, we show that QITP antibodies considerably reduce the capacity of Mks to produce proplatelets despite undetectable effects on DNA content, morphology and cell size. These observations suggest that the severity of the thrombocytopenia induced by QITP antibodies may be in part due to both a reduction in magakaryocyte numbers and a decrease in their capacity to release newly formed platelets.

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