Inhibition of platelet aggregation of a mutant proinsulin molecule engineered by introduction of a native Arg-Gly-Asp sequence

A 13 amino acid sequence, CRVARGDWNDNYC, originated from disintegrin eristo statin, was introduced into an inactive human proinsulin molecule between t he B29 and A2 sites to replace proinsulin C-peptide by molecular cloning te chniques. The constructed Arg-Gly-Asp (RGD)-proinsulin-7 gene was cloned in to a temperature-inducible vector pBV220 and expressed in Escherichia coli. The expressed RGD-proinsulin was refolded and purified by Sephadex G50 and DEAE-Sephadex A25 separations. The chemical identity was confirmed by both amino acid composition and mass spectrometry analyses. This RGD-proinsulin showed an inhibitory activity of adenosine 5'-diphosphate-induced human pl atelet aggregation with an IC50 value of 200 nM. Its insulin receptor bindi ng activity remained as low as 0.03% with native insulin as a control, and its insulin immune activity retained 27.6% compared with proinsulin.