Page 281 - Haematologica - Vol. 105 n. 6 - June 2020

Platelet depletion for effective transfusion
and loxP flanked genes of interest are not available in all cases.
An alternative to overcome these limitations is transfu- sion of genetically or pharmacologically modified platelets into platelet depleted mice. This allows research of altered platelets in a genetically unchanged environment. Additionally, platelets can be treated ex vivo before trans- fusion, eliminating putative side effects of the treatment on other cell types. Platelet depletion prior to platelet transfusion is frequently performed by administration of antibodies targeting platelet-specific epitopes although this harbors certain inherent limitations: Platelets are opsonized by antibodies and actively scavenged from the circulation. This could lead to immune reactions or activa- tion of opsonized and agglomerated platelets. Most importantly though, antibody binding does not differ between endogenous and transfused platelets, which either negates transfusion or demands repeated cycles of depletion and transfusion, to maintain a stable population of exogenous platelets.
A solution to this limitation is provided by a novel model, in which an inducible diphtheria toxin (DT) recep- tor (iDTR) is expressed under the control of a platelet-spe- cific PF4 Cre recombinase, rendering megakaryocytes sus- ceptible to DT-induced termination of protein synthesis.6,7 A single DT molecule in the cytoplasm is sufficient to kill an iDTR-expressing cell,8 whereas wild-type (WT) murine cells are highly insensitive towards DT,9 making megakaryocyte ablation exceptionally specific and effi- cient.
Here, we aimed to validate the platelet iDTR model and compared it with antibody-mediated platelet depletion methods regarding its use in platelet transfusion experi- ments. We show for the first time that the iDTR model can be successfully used in platelet transfusion and that it provides several advantages over antibody-mediated depletion by simplifying the experimental setup and refin- ing platelet transfusion. Additionally, the iDTR model may help to avoid time consuming generation of tissue- specific mice and allows analysis of platelet-specific func- tions, in cases where only a full knockout of the gene of interest is available.
Methods
Detailed methods regarding genotyping, platelet function assays, and in vivo experiments can be found in the Online Supplementary Materials and Methods.
Mice
All experiments and animal studies were conducted according to institutional guidelines and were approved by the Animal Care and Use Committee of the Medical University of Vienna (BMWFM-66.009/0246-WF/V/3b/2016).
All experimental procedures were conducted according to the SYRCLE ’s risk of bias tool for animal studies; e.g. groups were age matched littermates, and after initial scouting experiments, only female mice were used in all studies, to minimize a risk of bias; animals within the same cage were preferably taken into the same experiment; cages of iDTRPlt mice were randomly selected for experiments; blinding was not applicable.
Platelet depletion
Megakaryocyte and consequently platelet depletion were
induced in iDTRPlt mice by subcutaneous injections of 100 ng DT ondays0,2and4,followedby250ngondays7,9and11.Topre- vent hematoma and scarring, mice were anesthetized with isoflu- rane (Abbot Laboratories) for subcutaneous injections after day 7. WT mice were platelet depleted by intravenous administration of 4 μg/g anti-mouse GPIbα (R300, Emfret Analytics), 0.2 μg/g anti- mouse platelet monoclonal antibody 6A6-IgG2A (originating from Dr R. Good, University of South Florida College of Medicine, Tampa, FL, USA) or intraperitoneal injection of 15 μL rabbit anti-mouse thrombocyte serum (AIA31440, Accurate Chemical & Scientific Corporation).
Platelet transfusion
DT treatment was started at day 7 and R300 treatment 12 hours prior to transfusion. Recipients were injected with 4.3x108 washed platelets from WT or naïve male iDTRPlt mice, labeled with anti GPIbβ DyLight649 (X649, Emfret Analytics) and blood samples were collected after 2, 14, 48 and 72 hours. Blood was labeled with anti-CD41-BV421 and analyzed using a CytoflexS flow cytometer with Cytexpert 2.2 software. Exogenous platelets were defined as CD41+ X649+ events, whereas endogenous platelets were CD41+ X649–.
Statistics
Calculations were performed using GraphPad Prism 8.02 soft- ware. Statistical significances are depicted as: *P≤0.05, **P≤ 0.01, ***P≤0.001, ****P≤0.0001.
Comparison of two groups was done by unpaired t-test or Mann-Whitney test if data was not distributed normally. Two or more groups were compared to the respective control group using One-Way ANOVA with Dunnett correction. If all groups were compared with each other, Tukey correction was applied. Two groups with more than one condition were compared by Two- Way ANOVA and Sidak correction. For Figure 1D-E, H-I and Figure 4B-E individual area under the curves (AUC) were calculat- ed and groups compared to the respective control using One-Way ANOVA with Dunnett correction.
Results
Selective killing of megakaryocytes leads to rapid platelet depletion
To selectively ablate megakaryocytes and induce throm- bocytopenia, we crossed mice expressing iDTR down- stream of a loxP-flanked stop cassette with a PF4 iCre strain to generate mice with megakaryocytes expressing iDTR (iDTRPlt) (Figure 1A). Naïve iDTRPlt mice did not show any obvious alterations in bone marrow or megakaryocyte morphology (Online Supplementary Figure S1A), nor was the megakaryocyte count affected (Online Supplementary Figure S1B). For platelet depletion, we administered DT in a weekly routine with 100 ng being injected subcutaneously on days 0, 2 and 4. The dosage was increased at day 7 to 250 ng to avoid rebound effects and maintain efficient thrombocytopenia (Figure 1B), as megakaryocyte count return to basal levels within two days after last 100 ng injection (Online Supplementary Figure S1C). As controls we injected PBS into iDTRPlt mice and DT into WT mice. Megakaryocytes were already remark- ably depleted on day 2 (Online Supplementary Figure S1D) and undetectable from day 4 till day 7 (Figure 1C). Platelet count decreased over time in DT-treated iDTRPlt mice, reaching a nadir with day 4, and remained low until day 12 (Figure 1D). Platelet counts of control groups remained unaffected. In parallel, mean platelet size increased in the
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