Influences of Site-directed Mutagenesis of a Recombinant Didintegrin from Gloydius blomhoffi brevicaudus on Its Biological Activities / 中国生物化学与分子生物学报

ABSTRACT

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence found within several glycoprotein ligands and also in snake-venom disintegrins. Adinbitor, a protein with 73 amino acid residues including 12 cysteins and an RGD motif, was cloned from Gloydius blomhoffi brevicaudus in my laboratory. As a new member of disintegrin family, adinbitor can inhibit both human platelet aggregation induced by ADP and angiogenensis in vivo and in vitro, the typical characters of disintegrin family. To separate the effect of inhibiting platelet aggregation from that of inhibiting angiogenensis, the motif KGD was introduced into adinbitor cDNA to replace RGD by site-directed and PCR-based mutagenesis. The recombinant protein (recombinant adinbitor (KGD)) was expressed in E. coli BL21 and purified through the His· Bind affinity chromatography. Recombinant adinbitor (KGD) could inhibit ADP-induced platelet aggregation with IC50 value of 85 nmol/L. Considerably, it was a more effective inhibitor on platelet aggregation than recombinant adinbitor (RGD), which has an IC50 of 150 nmol/L. Interestingly, recombinant adinbitor (KGD) has no potency in inhibiting angiogenesis in vivo compared with recombinant adinbitor (RGD). These findings showed that KGD containing adinbitor was more suitable for inhibiting ADP-induced human platelet aggregation as a potential and specific inhibitor of human platelet aggregation, which might have promising therapeutic potential as an antithrombotic agent.