Screening For A Tandem Dimer Of TMP With Potent Thrombopoietic Activity And Its Effect On Treating Radiation-induced Thrombocytopenia In Mice

With the extensive use of nuclear technology, the negative effects generated fromnuclear weapon, reactor accidents and nuclear terrorism are inevitable under certainconditions.Thrombocytopenia is often resulted from radiation exposure and radioactivecontamination, and it is also a major hematopoietic disease caused by various reasons suchas Aplastic Anemia (AA), idiopathic thrombocytopenic purpura (ITP) and the side effects ofchemotheropy/radiotherapy for tumor treatment. How to effectively promote thrombopoiesis isdesirable for the treatment of thrombocytopenia. However, the existing drugs with weakpotency or notable side effects can not meet the need of clinical therapy for thrombocytopenia.Therefore, it is urgent for seeking new agents for treating thrombocytopenia.Thrombopoietin (TPO), which is also termed as megakaryocyte growth and developmentfactor (MGDF), contains332amino acids and is produced constitutively in the liver andmarrow stroma. TPO is the most powerful regulator of megakaryocyte (MK) developmentand subsequent platelet formation. Each TPO molecule binds two TPO receptors (c-Mpl),activating certain signal transduction pathways which boost proliferation and differentiationof MKs. However, clinical applications of the first generation of thrombopoietic growthfactors, including recombinant human TPO (rhTPO) and pegylated recombinant humanMGDF (PEG-rhMGDF), were ultimately discontinued due to the emergence of neutralizingantibodies which could cross-react with endogenous TPO and therefore paralyse theintrinsic cytokine.Thrombopoietin mimetic peptide (TMP), which was firstly discovered by phagy display in1997, is consist of14amino acids and endowed with high affinity to bind c-Mpl, whereas it isnot homologous to TPO, thereby having no risk of generating TPO autoantibody in vivo.Nevertheless, the low bioactivity in promoting megakaryocytopoiesis and thrombopoiesislimits the application of TMP. It was demonstrated that the activation of intracellular signal pathways involving inMK proliferation and differentiation entirely depends on the formation of a dimer oftwo-coupled c-Mpls. As known, each TPO molecular has the ability to bind and activatetwo-coupled c-Mpls, while each TMP can only bind one c-Mpl, which leads to the loweractivity of TMP in promoting megakaryocytopoiesis in comparision with TPO. Therefore,subsequent attempts are aiming to generate TMP dimer (dTMP) to bind and activatetwo-coupled c-Mpls, in order to improve its bioactivity.Erythropoietin (EPO) is the most homologous counterpart of TPO in hematopoieticcytokine family. It was reported that EMP33, a homologous twin of the dimer of EPOmimetic peptide1(EMP1) with different linkers between the mono peptide, can not activatethe downstream signal transduction pathway of EPO receptor (EPOR), although it has thesame property with EMP1to bind EPOR. The underlying mechanism is that the angle ofEMP33-EPOR compound is15degrees different from that of EMP1-EPOR, which suggeststhat appropriate linkers used to join the mono peptide is important for keeping the activityof the dimer of EMP, possibly through influencing the stereo structure of the dimer of thepeptide. Similarly, we presume that the linker used to join the two momo TMP may also actas a key point on modulating the spatial conformation and activity of dTMP.On the basis of our previous studies, here we designed and synthesized a series oflinear peptides containing two momo TMP joined by different peptide linkers. Then, theimpacts of the length and animo acid composing on the activity of dTMPs in promotingmegakaryocytopoiesis and thrombopoiesis were evaluated, along with analysis of theunderlying mechanisms by application of molecular dynamics (MD) simulation. Finally, weobtained a preferable dTMP with an appropriate linker with potent activity to promotethrombopoiesis. In a mice model of radiation-induced thrombocytopenia, the preferabledTMP was demonstrated to has a strong ability to promote platelet revovery. The mainresults and conclusions are summarized below:1. A linker consisting8Glycine (8G) could significantly improve the activity ofdTMP on promoting MKs proliferation in vitro.2. The activity of dTMP could be remarkably boosted by adding a single Proline (P)into the linker.3. A preferable pepdide of dTMP (named No.341peptide) with appropriate linker and potent activity to promote MK proliferation was finally obtained after two rounds ofselection.4. The expression level of CD41/CD61in the cells derived from Sca+-I bone marrowcells treated with dTMP (No.341peptide) was much higher than that of IL-3control group(83.26±6.26%vs15.70±2.31%, P<0.01), demonstrating that dTMP is able to promote thedifferentiation and maturation of MKs.5. Western blot revealed that the phosphorylation levels of ERK1/2and STAT5inM07e cell and mouse bone marrow-derived primary MKs were up-regulated remarkablyafter dTMP treatment,,indicating that dTMP promotes MKs proliferation and differentiationpossibly through stimulating ERK1/2and STAT5signal transduction pathways.6. After calculating and analyzing the stereo conformations including dihedral angle,span of peptide terminals, the exposure of receptor binding sites by MD simulation, it wasrevealed that the activity of different dTMPs is closely related to their structural differencescaused by the linkers.7. After treatment with dTMP (No.341peptide), the recovery of blood platelet inradiation-injured (5Gy Co60γray) mice was about4days earlier than that of saline control.Moreover, the minimum value of platelet in dTMP-treated mice was much higher than thatof saline control (166.8×109/L vs52.8×109/L). These results demonstrate that this preferablepeptide is capable of treating radiation-induced thrombocytopenia.In conclusion, after screening the linkers, we finally obtained a tandem dimer of TMPwith outstanding megakaryocytopoietic and thrombopoietic activity, which provides a newavenue for the treatment of thrombocytopenia induced by radiation.