RGD‐containing trypsin with both platelet aggregation inhibitory activity and proteolytic activity

Abstract

SUMMARY: Arg-Gly-Asp (RGD) motif mediates cell adhesion as a major determinant in interactions of disintegrins with cell surface receptors. In order to obtain a mutant trypsin with both high affinity to integrins and retained proteolytic activity, RGDS and RGD were inserted into a rigid turn region and a flexible loop of trypsin, respectively. Wild type trypsin and substituted mutant trypsins, 37RGDS and 77RGD, were expressed in E. coli and purified. Kinetic properties, autolytic stability as well as platelet aggregation inhibitory activity of both 37RGDS and 77RGD were determined. 37RGDS and 77RGD give retained proteolytic activities of 34% and 87%, respectively, and both become less stable to autolysis. 37RGDS shows an obvious inhibition rate of 29% for platelet aggregation and 77RGD gives a rather weak rate of 14% at the same protein concentration of 3.5pM, while the wild type trypsin shows no inhibitory activity.

Inhibition of Platelet Aggregation of a Mutant Proinsulin Chimera Engineered by Introduction of a Native Lys-Gly-Asp-containing Sequence

“…The KGD-proinsulin chimera is a first reported KGD-containing recombinant mutant proinsulin with glycoprotein IIb/IIIa receptor specificity. The KGD-proinsulin is easy to obtain through large-scale fermentation as compared with other large recombinant anti-thrombosis proteins (Lee et al 1993, Nie & Tang 1998.…”