The Comparison Of Antiangiogenic Activity And Anti-platelet Agglutination Among The Lj-RGD3Toxin Protein With Its RGD Mutated Proteins

rLj-RGD3toxin protein with3RGD motifs from the saliva gland of Lampetrajaponica which are rich in homologous with a Histidine-rich glycoprotein (HRG).Both of the RGD toxin proteins and Histidine-rich glycoprotein have antiangiogenicactivities, while with different targets in according to different signal pathways. Ourlaboratory had already finished many researches systematically about RGD motifsproteins with different mutation ways. In this paper, we focused on the comparison ofantiangiogenic activity and anti-platelet agglutination among KGD mutations fromrLj-RGD3with the wild type. KGD motif is capable of specially combination withGPIIb/IIIa which is a kind of platelet cell surface glycoprotein, but never combineswith αVβ3which is highly expressed on vascular endothelial cell surface. It makesprotein with KGD motif can be capable of special anti-platelet agglutination, but don’thave ability of antiangiogenic activity, in order to being good for new antithromboticdrug development. In this study, we derived series KGD motif mutated proteins byreplacing RGD with KGD from rLj-RGD3, including the protein rLj-116, rLj-28w,rLj-26. Used purified proteins, and studied their antiangiogenic activity andanti-platelet agglutination. We made MTT experiment of ECV304cell as angiogenicmodel in vitro for antiangiogenic activity study. The result represented that IC50ofrLj-116and rLj-RGD3were at0.562μmol/l and0.832μmol/l, respectively, whileother mutated proteins had no obviously effect. In cell apoptosis test, rLj-RGD3andrLj-116could induce apoptosis dose-dependent, while rLj-26and rLj-28could not.Transwell assay was used to determine the effect of r rLj-RGD3and the four mutatedproteins on bFGF induced ECV304cells migration, the result suggested rLj-RGD3and rLj-116had obviously effect, while rLj-28was not represented, rLj-26was noteffective. Utilization of Matrigel and Transwell aimed to imitate environment in vivoand research the inhabitation of cell invasion with five forms of proteins. As the result,Matrigel assay revealed that rLj-RGD3and the rLj-116significantly inhibited bFGF induced invasion of ECV304cells, while rLj-28was not represented, rLj-26was noteffective. Then, four forms of proteins were detected by anti-angiogenesis in chickenchorioallantoic membrane (CAM). The result demonstrated that rLj-116, rLj-RGD3inhibited the angiogenesis in CAM, while the rLj-28and rLj-26were not obviously.By the platelet agglutination experiment with the instrument tested and compared PRPto the normal inducer of platelet agglutination factor to receive the inhibition ratio,among the five proteins, only rLj-RGD3and rLj-116could induce obviousagglutination. Above all, based on the tested data, we proposed several possibleconclusions:(1) proteins involved KGD motif had no ability of antiangiogenesis;(2)rLj-RGD3could not represent antiangiogenesis with RGD motif and His-richstructure themselves;(3) KGD motif proteins rLj-116could anti-platelet agglutinationeffectively with its3D-structure provided by His sequence.