Study Of The Effect Of Anti-platelet GP?a49-66 Monoclonal Antibodies On Endothelial Cell Function

Impairment of angiogenesis within the tumor microenvironment may provide a clinically useful approach for tumor therapy.We previously showed that antibody(Ab)against platelet integrin p3(GP?a)49-66 inhibits pulmonary metastasis by fragmenting activated platelets in the tumor microenvironment.Platelet integrin??b?3 and endothelial integrin ?v?3 share the common ?3 chain.Nevertheless,it remains largely unknown the effect of anti-GP?a49-66 Ab on endothelial cell functions and angiogenesis.Here,we explored the biological effects of multiple murine anti-GPIIIa49-66 monoclonal Ab(mAb)1C1,1E7 and 5A10 on angiogenesis using human umbilical vein endothelial cells(HUVECs).The results were as follows:1.Flow cytometry assay showed that anti-platelet GPIIIa49-66 mAb 1C1,1E7 and 5A10 could specifically bind to HUVECs in a dose-dependent manner.2.MTT assay showed that 5A10 demonstrated the best inhibition rate on HUVECs proliferation in a time-and dose-dependent manner.3.Flow cytometry analysis of apoptosis and cell cycle distribution showed that this inhibitory effect of 5A10 on HUVECs proliferation is mainly due to 5A10-induced HUVECs apoptosis.4.Western blot analysis showed that 5A10 promoted HUVECs apoptosis through suppressing ERK1/2/MAPKs-mediated Bcl-2 signaling pathway in endothelial cells.5.Transwell migration assay showed that 5A10 inhibited cell migration of HUVECs in a dose-dependent manner.6.Matrigel angiogenesis assay in vitro showed that 5A10 inhibited capillary-like structure formation of HUVECs in a dose-dependent manner.In conclusion,anti-platelet GPIIIa49-66 mAb(5A10)could inhibit with varieties of biological functions of vascular endothelial cells including their proliferation,migration and capillary-like structure formation,and promoted endothelial cell apoptosis via suppressing ERK1/2/MAPKs-mediated Bcl-2 signal transduction pathway.These findings indicated that mAb 5A10 was a potent angiogenesis inhibitor and showed a potential clinical application for cancer therapy.The epitope of GPIIIa49-57 for specific target of 5 A10 was potential molecular target for prevention and treatment of tumor.In the future,development of molecules targeting this locus may be of clinical value to combat cancer via dually inhibiting tumor angiogenesis and dissolving platelet-tumor thrombus.