| With an increasingly wide utilization of ionizing radiation in fields of military,economy, agriculture and medical care, etc, radiation-induced injuries are commonly seennowadays. Radiation injury of hematopoietic tissue is the critical pathogenic factorfor hematopoiesis dysfunction of which the clinical manifestations are dramatic decreasesof WBCs, RBCs and platelets in peripheral blood. The dramatic reduction and dysfunctionof platelets further cause hemorrhage and secondary infection. The level of platelets inradiation-caused injuries is one of the main factors that affect the survival and prognosis.Therefore, rapid recovery of platelets is critical for the treatment of radiation-caused injuries.However, there are limited drugs for clinical treatment of thrombocytopenia at present. Andmost of the drugs hardly meet clinical needs with respect to both efficacy and side effects.Hence, it becomes the pressing concern to develop new high-efficiency and low-toxicitydrugs for promoting platelet production.Platelets are produced by megakaryocytes originated from HSCs. Thus, thedifferentiation potent of megakaryocytes is a key factor for platelet production efficiency.Thrombopoietin (TPO) is not only a growth and differentiation factor formegakaryocyte-lineage cells but also a potent in vivo stimulus of platelet production.Through binding to its receptor c-Mpl, TPO activates the related signal pathways toaccelerate the proliferation and differentiation of megakaryocyte, as well as plateletproduction. However, several studies showed that the peripheral platelet counts in patientswith severe thrombocytopenia did not increase till the5thday, while the peak reached at the12thday after the injection of rhTPO. Thus, rhTPO treatment barely reduced the crisis stageof patients caused by platelet reduction. The delay of platelet recovery may be related to theincapacity of TPO in accelerating terminal differentiation and maturation of megakaryocytes (including pro-platelet formation and platelet release). More importantly,the FDA banned the development of recombined TPO-related genetic engineering drugsbecause of the induction of endogenous neutralizing antibody by the protein. Therefore,people turned to search and develop TPO analogues. TPO mimetic peptide (thrombopoietinmimetic peptide, TMP) of which dimers and diads can bind and activate TPO receptorc-Mpl exerts its activity on accelerating megakaryocyte proliferation and differentiation asTPO does. But, the low molecular weight and short half-life of both TMP dimers and diadslimited their in vivo use.It has been demonstrated that growth hormone (GH) promotes the proliferation and/ordifferentiation of different cells by binding to its receptor GHR. Some reports showed thatDW/J dwarf mice lacking GH had significantly decreased peripheral blood counts(including WBCs, RBCs and platelets), compared with control mice. Several subsequentreports pointed out that large doses of GH significantly promoted bone marrowhematopoietic reconstruction in mice after radiotherapy/chemotherapy or bone marrowtransplantation, largely due to the quick recovery of platelet levels. In addition, a recentclinical research showed that large dose of GH accelerated the recovery of peripheralplatelet level for about3days in patients with hematologic malignancies after intensivetreatment of chemotherapy. These results indicates that GH may play an important role inpromoting platelet production, while which stages it may regulate and the underlyingmechanism is waiting to be revealed.We first analyzed the regulatory role of GH and a TPO mimetic peptide dimer nameddTMP in megakaryocyte proliferation and differentiation, proplatelet formation and plateletrelease, as well as the underlying mechanisms in vitro. Our results showed that dTMPpromoted proliferation and differentiation of megakaryocytes at early stages, while GHexerted its regulatory role at the terminal stages of megakaryocytopoiesis. When GH anddTMP were combined, they act collaborately to promote thrombopoiesis. According to theresults, we generated the GH-dTMP constructs to express their fusion proteins. dTMP-GHfusion protein can accelerate platelet production effectively in vitro, and promote therecovery of platelet level after radiation exposure as well. The main results and conclusionsare summarized as follows:1. Using methods such as immunomagnetic separation, flow cytometry, immunofluorescence staining, scanning electron microscopy, western blot and CCK-8analysis, we founded several platelet-related experimental system, including analyzingproliferation and differentiation of primary megakaryocytes derived from human cordblood-derived CD34+haematopoietic cells, proplatelet formation and platelet production.2. Results from in vitro studies show that GH can not promote proliferation butdifferentiation of megakaryocytic progenitors.3. GH can promote proplatelet formation and platelet production indicating itspositive regulatory role in maturation of megakaryocytes.4. GH can not activate JAK-STAT5signal pathway, but prolong the activation ofERK1/2signal pathway in megakaryocytic progenitors. ERK1/2inhibitor (U0126) canblock the promoting effect of GH on differentiation of megakaryocytes. These resultsindicate that GH promotes differentiation of megakaryocytes through prolonging theactivation of ERK1/2signal pathway.5. GH can induce rapid activation of Akt signal pathway and lead to an evidentincrease in the activation of Rho GTPases, Rac1and Cdc42as well, in maturemegakaryocytes. Inhibitor of Akt (LY294002) significantly blocked the GH-inducedproplatelet formation and platelet production by cultured megakaryocytes.6. dTMP dose-dependently promoted the proliferation of M07e cells. Similarly, thispeptide alone could support and stimulate the differentiation of human cord blood-derivedCD34+cells into megakaryocytes. However, the proplatelet formation and plateletproduction were decreased with increasing concentrations of dTMP, indicating that dTMPmight have an inhibitory effect on the terminal differentiation of megakaryocytes.7. Notably, compared with dTMP alone, co-treatment with rhGH and dTMP resultedin more proplatelet formations and platelet production by megakaryocytes. In addition, anincreased expression of β1-tubulin, typical demarcation membrane (DM) formations andmore pseudopod formations were observed in the cells treated with dTMP plus rhGH. Theseresults indicate GH has a complementary effect on dTMP-induced thrombopoiesis.8. dTMP-GH fusion protein exhibited a more potent promoting effect on thedifferentiation and maturation of megakaryocytes, compared with dTMP alone ordTMP+GH group. This indicates that the proliferation and differentiation-promotingactivity of dTMP fused with GH for megakaryocytes was dramatically enhanced in comparison with equimolecular dTMP. The underlying mechanism is probably that thealtered topology of dTMP when fused with GH increased its bioactivity.9. dTMP-GH significantly increases the expressions of CD41//CD42b, GATA-1,NF-E2, as well as β1-tubulin in megakaryocytes. When compared with equimoleculardTMP, dTMP-GH treatment resulted in an earlier and increased proportion of proplateletformation and platelet production.10. Western blot analyses show that dTMP-GH treatment causes a rapid activation ofSTAT5and a strong and sustained activation of ERK1/2, while in human Meg-01maturemegakaryocytes, dTMP-GH treatment results in a rapid and strong activation of Akt signalpathway. From the perspective of signal pathways, these results further confirm thepromoting effect of dTMP-GH on proliferation and differentiation of megakaryocyticprogenitors and platelet production by mature megakaryocytes.11. Using mice with severe thrombopenia induced by TBI, compared withequimolecular dTMP, we observe that dTMP-GH could more efficiently accelerate plateletrecovery after irradiation and shorten, more significantly shorter the starting time of plateletrecovery and the duration of thrombocytopenia crisis, and increase the30-day survival rateof irradiated mice. This indicates dTMP-GH can accelerate platelet recovery when used forthe treatment of radiation caused injuries.12. Using mice with severe thrombopenia induced by Carboplatin chemotherapy andradiotherapy combined with chemotherapy, we investigated that dTMP-GH could accelerateplatelet recovery and efficiently decrease the mortality of mice.Conclusions: In this study, we show for the first time that GH can promote the differentiationof human primary megakaryocytes and stimulate the proplatelet formation and platelet productionfor cultured megakaryocytes. GH may exert a complementary and synergistic effect withc-Mpl ligands on thrombopoiesis. Accordingly, dTMP-GH fusion protein can promote theproliferation and differentiation of megakaryocytes, and the maturation and terminaldifferentiation of megakaryocytes, lead to a rapid platelet recovery in mice with severethrombocytopenia induced by irradiation and chemotherapy. The dTMP-GH fusion proteinshowed a prospect for the research and development. |
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