The Role Of GP?b Epitope 833-840 In Platelet-tumor Cell Interactions

Part I The effect of GP?b epitope 833-840 on platelet biology functionObjective:Many molecules are involved in the interaction between platelets and tumors,but the role and mechanism of GP?b molecules in the process is still unclear.In this study,we used the monoclonal antibody SZ22,an anti-platelet glycoprotein GP?b,as a target molecule,and used phage display technology to screen a specific binding site that is highly homologous to GP?b.Therefore,we investigated the role of this site in the biological function of platelets.Methods:1.Firstly,phage display screening on SZ22 antibodies was performed using the Ph.D.-12 peptide phage display library kit.After three rounds of screening,bound phages were harvested and twelve isolated plaques were recovered for DNA extraction.The sequenced peptides and the GP?b molecular sequences were aligned,then the candidate specific N2G-peptide was obtained for subsequent studies.2.Enzyme-linked immunosorbent assay(ELISA)was used to identify the specificity of N2G-peptide.Flow cytometry and immunoblotting were used to detect the blocking effect of N2G-peptide on platelet binding to SZ22 antibodies.3.The effect of N2G-peptide on the biological function of platelets was detected by platelet aggregation assay,clot shrinkage assay and flow cytometry.Results:The phage display technology was used to screen a binding site(NPLKVDWG)with high homology to the GP?b molecule,which was located at position 833-840 of the GP?b molecule.The N2G-peptide and its corresponding random sequence NsG-peptide were synthesized.The amino acid fragment containing the N2G epitope of the wild type of the GP?b molecule in the Calf2 domain and the control amino acid fragment were recombinantly expressed for experiments.The results showed that:1.The N2G-peptide and wild-type Calf2-N2G peptide could specifically bind to SZ22 in a concentration-dependent manner,while the NsG-peptide and Calf2-NsG peptide had no binding.2.Compared with NsG-peptide,N2G-peptide could specifically block the binding of SZ22 antibody to denatured or intact platelets in a concentration-dependent manner.3.In presence of 10?M ADP or 0.05 U/ml thrombin,N2G-peptides slightly inhibited platelet activation,and significantly reduced platelet aggregation and clot retraction.Conclusion:These results demonstrated that N2G epitope of GP?b is a specific site for binding to SZ22 antibodies,which was involved in the activation and aggregation of platelets induced by the agonists,and that mimetic N2G-peptides can be utilized to manipulate platelet-platelet aggregation.Therefore,this site can be used as one of the highlights of thrombotic disease research,and then the application of phage technology can provide new directions and clues for the treatment of other diseases mediated by autoantibody-mediated platelet abnormalities and platelet abnormalities.Part II The effect of GP?b epitope 833-840 on tumor biological function and its targeting abilityObjective:Platelets are closely related to cancer,and there are many studies on the molecules that interact between platelets and tumor cells.There are few studies of Glycoprotein GP?b,one of the platelet surface receptors,in the process of forming aggregates between platelets and tumor cells.In the study,we screened a specific epitope(833-840)of GP?b molecule that binds to SZ22,and synthesized mimetic N2G-peptides,which were found to bind to the surface of some tumor cells.Therefore,we investigated whether specific epitope of GP?b molecule was involved in the process of platelet-tumor interaction and whether it has an effect on the biological function of tumor cells.Besides,does it have application value for the treatment of tumor diseases?Methods:1.Mimetic N2G-peptides or the NsG-peptides were synthesized according to the specific epitope(833-840)of GP?b molecule and labeled with FITC.The binding ability of N2G-peptides to various cells was detected by flow cytometry.2.The ligands on the surface of NB4 cells that bound to N2G-peptides were searched by pull down assay and protein profiling,and the binding ligand was also verified using a recombinant GP?b fragment corresponding to the Calf2 domain of GP?b.3.Flow cytometry was used to detect the blocking effect of blocker on N2G-peptides binding to NB4 cells.4.In bioinformatics analysis,a three-dimensional structural model of ATP synthase was used to predict possible sites binding to N2G-peptides.5.ATP synthesis experiments,flow cytometry and western blotting were used to detect the effects of N2G-peptides on ATP synthesis,cell proliferation,differentiation and related signaling pathways.6.The binding ligands on the surface of NB4 cells to the platelets were detected by flow cytometry,and the level of binding of bone marrow cells from leukemia patient and normal human to the platelets were compared.In addition,blocking effects of blockers on the formation of platelet-NB4 cell aggregates were examined.7.N2G-peptides were used as a targeting moiety,coupled with a toxic polypeptide(KLAKLAK)2 to form a complex of N2G-KLA peptides,and the killing effect of N2G-KLA peptides on cells was detected by flow cytometry in vitro.8.Moreover,the mouse leukemia metastasis model and subcutaneously inoculated leukemia solid model were established in vivo.After administration of the N2G-KLA peptides,the survival time and tumor size of the mice were regularly monitored.Results:1.N2G-peptides synthesized according to the specific site of GP?b molecule could bind to leukemia NB4 and HL-60 cells through the ectoATP5B located on the cell surface.And the binding of N2G-peptides to NB4 cells could be blocked by anti-ATP5B antibodies.2.N2G-peptides affected the synthesis of ATP on the cell surface,but hardly affected the proliferation,differentiation and signaling pathway molecules of cells.3.In the three-dimensional structural model of ATP synthase to predict the possible binding sites of N2G-peptides,we found that in the 50 best binding positions,nearly forty-four ways of binding sites were located in the p subunit of ATP synthase,that is,ATP5B molecule.4.rATP5BesT protein was shown to bind to platelets in a dose-dependent manner.5.NB4 cells expressing ectoATP5B could aggregate with platelets,and the binding of bone marrow cells derived from leukemia patients to platelets was higher than that of normal people.6.When rATP5BesT proteins or anti-ATP5B antibodies were added to the co-culture system of platelets and leukemia cells,a slight decrease of PLA was observed.7.Compared with cells that do not express ectoATP5B,N2G-KLA peptides could kill the cells(such as NB4 cells)with high expression of ectoATP5B in vitro.8.In the in vivo leukemia model,we found that the N2G-KLA peptides prolonged the survival time of mice in the leukemia metastasis model and decreased the size of tumor formation in the leukemia solid model.Conclusion:Our studies demonstrated that specific site of GP?b molecule were capable of specifically binding to leukemia cells via ectoATP5B.Although N2G-peptide alone has little effect on the biological function of the cells,its bridge with ectoATP5B could participate in the interaction between platelets and leukemia cells.N2G-peptides may be utilized as a guide to deliver cytotoxic peptides onto ectopic ATP5B-positive leukemia cells,hence the killing effect on tumor cells and inhibition of cancer development will be achieved.