Objective      To screen the dimmer of thrombopoietin mimetic peptide (dTMP) with potent thrombocytopoietic effect on the promotion of thrombopoiesis and the proliferation and differentiation of megakaryocyte.       Methods      A series of linear peptides containing two TMP

joined by peptide linkers at different lengthes were designed and synthesized. The promoting activity of the dTMP on megakaryocyte proliferation was determined by cell counting kit-8 (CCK-8) assay. The ability of dTMP to phosphorylate ERK1/2 and STAT5 signal pathways was measured by Western blotting. The expression level of CD41/CD61 was analyzed by flow cytometry to reveal the ability of dTMP peptide to promote murine marrow Sca

+

-Ⅰ cells to differentiate into megakaryocytes. Finally

dTMP was administered to BALB/c mice with thrombocytopenia induced by 5 Gy total body irradiation through subcutaneous injection

in order to investigate the effect of dTMP on platelet recovery in vivo.       Results      The length of peptide linkers significantly influenced the promoting activity of dTMP peptides on megakaryocyte proliferation. The dTMP peptide with a linker composed by a proline and eight glycine (TMP-PG

8

-TMP) displayed a remarkable effect on megakaryocyte proliferation. Western blot assay demonstrated that TMP-PG

8

-TMP had the ability to activate the phosphorylation of ERK1/2 and STAT5. The expression level of CD41/CD61 in the cells derived from Sca

+

-Ⅰ marrow cells treated with TMP-PG

8

-TMP was much higher than that of IL-3 control group [(83.26±5.38)% vs (15.70±2.12)%

P0.01

]

. Meanwhile

dTMP-treated mice were about 4 d earlier in the platelet recovery than that of saline control

demonstrating that this peptide was able to be used for the treatment of thrombocytopenia.       Conclusion      A dTMP peptide with potent thrombocytopoietic activity is finally obtained by peptide linker screening.