Studies On The Structure And Activity Of RGD-Ins Proteins And The Gene Therapy Of Thrombosis Thereof

Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motif-containing sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from a venom protein dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS, were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chain and connected with B27 and Al residues of the inactive insulin that lacks the characteristic intramolecular A6-11 disulfide bond within the A chain. The two RGD-containing insulin genes were over-expressed in E. coli, and purified and designated as RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those of expected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC₅₀ of 3μM, while the latter was 3. 5-fold less active. The in vivo assay also indicated that the RGD-Cys-Ins had a higher activity in prolonging bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The result indicates that the introduction of a conformational constraint into the RGD motif is necessary to exhibit a higher anticoagulant activity.A naked plasmid with RGD-Cys-Ins gene was transferred into skeletal muscle of normal BALB/c by electric pulses and gene expression was detected. Bleeding time was prolonged from 90 to 600 seconds. Circulating insulin-like protein was similar to control group injected with the empty vector remained at less than 10mg/ml. The prolonging of bleeding time lasted for more than two months. These studies indicate that electroporational transfer of plasmid with RGD-Cys-Ins gene into skeletal muscle could be a potential...