Page 285 - Haematologica - Vol. 105 n. 6 - June 2020

Platelet depletion for effective transfusion
induction and two days or two hours after the first DT or antibody/rabbit anti-mouse thrombocyte serum treat- ment, respectively. Additionally, platelet activation was assayed at time points of stable platelet depletion, i.e. seven days after DT injection or 12 hours after antibody injection, as well as three days afterwards (Figure 4A). Different anti-platelet antibodies were applied and com- pared with the iDTR system: while the antibodies anti-GPIbα (R300) and 6A6-IgG2A rapidly induced stable thrombocytopenia of different degree within two hours, anti-mouse thrombocyte serum gradually reduced platelet counts to 10.8±9.8% within 12 hours (Figure 4B). Depletion with antibodies or serum did not significantly increase the expression of P-selectin on the remaining platelets, but seven days after first DT administration, the remaining 0.2±0.2% platelets of iDTRPlt mice showed
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slightly increased reactivity (Figure 4C). However, in com- parison with a positive control of PAR4-AP stimulated blood, platelet activation in iDTRPlt mice was still minor. As expected, thrombocytopenia resulted in decreased plasma CXCL4 concentrations compared to basal levels when using R300, serum or the iDTR model (Figure 4D), underlining the lack of platelet activation upon induced thrombocytopenia. This is also reflected by normal platelet-leukocyte-aggregate (PLA) formation (Figure 4E).
The iDTR model outperforms antibody-based platelet depletion in transfusion studies
As a next step, we compared the iDTR model with anti- body-based platelet depletion as a potential tool for platelet transfusion. DT treatment started seven days and R300 (4 μg/mL) was administered 12 hours prior to trans-
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Figure 4. Neither antibody-based nor diphtheria toxin-induced thrombocytopenia cause platelet activation. (A) Graphical overview of depletion comparison. Diphtheria toxin (DT) treatment started seven days (d) prior to stable depletion and R300 treatment 12 hours (h) prior to stable depletion. Blood sampling time points are indicated as reaction tubes. (B) Percentage of platelet counts, relative to initial counts. (C) Percentage CD62P+ of CD41+ events in diluted whole blood, with PAR4- AP-activated positive controls. (D) CXCL4 plasma concentrations. (E) Percentage CD41+ of CD11b+, CD45+ events in whole blood, with PAR4-AP activated positive controls (n=4-5).
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