Page 290 - Haematologica - Vol. 105 n. 6 - June 2020

M. Salzmann et al.
with arterial stiffness.22 However, diagnosis of coronary artery disease does not associate with MPV.23 Further, viruses can alter MPV in both directions, depending on the virus species,24,25 and a high MPV can be used to differen- tiate between gastric ulcer and gastric cancer.26
In addition to increased MPV, we also measured increased P-selectin expression of the remaining platelets at day 7 of depletion, an observation also reported by oth- ers.7 Nevertheless, plasma CXCL4 levels, a surrogate marker for platelet degranulation, were not increased, fur- ther underlining that platelets were not activated in circu- lation.
Like the iDTR system, also antibody-based methods, which induce active and rapid clearance of platelets, did not lead to increased plasma CXCL4 levels or platelet acti- vation. In contrast to the iDTR model, antibody-based methods have the advantage that the amount of injected antibody can be fine-tuned to adjust the degree of throm- bocytopenia. The iDTR model completely inhibits platelet production, thus thrombocytopenia cannot be adjusted by titrating DT concentration. This is evident from a rebound effect with rapidly increasing platelet counts after reap- pearance of megakaryocytes. This phenomenon was also observed by others either after short term treatment, where it was used to study young, reticulated platelets.16 In contrast, others were able to keep platelet counts stably low until day 17 using a single injection of 400 ng DT,7 whereas we observed a rebound at day 14. The reason for this discrepancy is currently unclear. Additionally, continu- ous depletion of platelets with the iDTR model has been reported for six weeks.27 However, at day 12-14 we already observed hematomata and impaired wound healing, thus we decided to anesthetize mice for subcutaneous injec- tions to reduce the risk of injuries. Additionally, we observed that female mice tolerated the treatment better than males. This suggests that females are more suitable as recipients during transfusion experiments, whereas untreated males can be readily used as platelet donors.
However, regarding platelet transfusions, the iDTRPlt model shows a significantly better performance than anti- body-based models. In the latter, newly transfused platelets are rapidly depleted by an excess of circulating antibodies, which decreases transfusion efficacy and shortens thrombocytopenia by removing the depleting
antibodies. This became evident, when endogenous platelet counts of R300 antibody-treated mice increased again at day 3 after transfusion although the dosage of antibody used in our transfusion experiment ensured sta- ble platelet depletion over 3.5 days. In contrast to anti- body-based platelet depletion, endogenous platelet counts of iDTRPlt mice remained unaffected by the introduction of new platelets, further demonstrating the superiority of the iDTR system.
Finally, we could show that transfused platelets were fully functional by quantifying macrophage recruitment after induction of sterile peritonitis. Platelets are immunomodulatory cells and play a pivotal role in recruit- ing leukocytes to the site of inflammation.28
In conclusion we were able to delineate advantages and disadvantages of different platelet depletion methods (as summarized in Figure 6). While the iDTR model targets platelets indirectly via megakaryocytes, antibody-based models deplete platelets directly. Hence, stable thrombo- cytopenia is reached with antibodies and anti-mouse thrombocyte serum within 2 or 12 hours, respectively, while the iDTR model takes about five days. Antibody- based methods allow adjustment of platelet counts, depending on the amount of used reagent, whereas the iDTR model results in almost complete thrombocytopenia which may be disadvantageous in certain experimental settings. However, the iDTR model is much more efficient in platelet transfusion compared to antibody based meth- ods, in which freshly transfused platelets and endogenous platelets are scavenged to the same degree.
Altogether, the iDTR model refines platelet transfusion and simplifies its experimental setup, thus reducing the number of required animals. The possibility to transfuse ex vivo-treated platelets or platelets from any global knock- out or knockin mouse strain replaces the need to generate lineage-specific conditional mouse strains, further reduc- ing required animal numbers. Moreover, as transfusion of human megakaryocytes29 and platelets30 into mice has been successfully established, the iDTR model could be potentially useful to evaluate platelet concentrate prepara- tions and storage conditions for clinical applications. Therefore, employing the iDTR-model for platelet deple- tion and transfusion experiments can facilitate multiple angles of platelet research.
References
1. Assinger A, Kral JB, Yaiw KC, et al. Human cytomegalovirus-platelet interaction triggers toll-like receptor 2-dependent proinflamma- tory and proangiogenic responses. Arterioscler Thromb Vasc Biol. 2014;34(4): 801-809.
2. Linden MD, Jackson DE. Platelets: pleiotrop- ic roles in atherogenesis and atherothrombo- sis. Int J Biochem Cell Biol. Vol. 42. Netherlands: 2010 Elsevier Ltd. 2010:1762- 1766.
3. Starlinger P, Haegele S, Offensperger F, et al. The profile of platelet alpha-granule released molecules affects postoperative liver regeneration. Hepatology. 2016;63(5): 1675-1688.
4. Tiedt R, Schomber T, Hao-Shen H, Skoda
RC. Pf4-Cre transgenic mice allow the gen- eration of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood. 2007;109(4):1503- 1506.
5. Nagy Z, Vogtle T, Geer MJ, et al. The Gp1ba-Cre transgenic mouse: a new model to delineate platelet and leukocyte func- tions. Blood. 2019;133(4):331-343.
8. Yamaizumi M, Mekada E, Uchida T, Okada Y. One molecule of diphtheria toxin frag- ment A introduced into a cell can kill the cell. Cell. 1978;15(1):245-250.
9. Chang T, Neville DM, Jr. Demonstration of diphtheria toxin receptors on surface mem- branes from both toxin-sensitive and toxin- resistant species. J Biol Chem. 1978;253(19): 6866-6871.
10. Manning KL, Novinger S, Sullivan PS, McDonald TP. Successful determination of platelet lifespan in C3H mice by in vivo biotinylation. Lab Anim Sci. 1996;46(5):545-
6. Honjo T, Nishizuka Y, Kato I, Hayaishi O.
Adenosine diphosphate ribosylation of
aminoacyl transferase II and inhibition of
protein synthesis by diphtheria toxin. J Biol
Chem. 1971;246(13):4251-4260. 548.
7. Wuescher LM, Takashima A, Worth RG. A novel conditional platelet depletion mouse model reveals the importance of platelets in protection against Staphylococcus aureus bacteremia. J Thromb Haemost. 2015;13(2): 303-313.
11. Harkness JE, Wagner JE. The Biology and Medicine of Rabbits and Rodents (ed 3rd). Philadelphia: Lea & Febiger. 1989.
12. Mitruka BM. Clinical biochemical and hematological reference values in normal experimental animals and normal humans
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