The Influences Of Site-directed Mutagenesis Of Adinbitor, A Disintegrin From The Snake Venom Of Gloydius Blomhoffi Brevicaudus On Its Biological Activities

Disintegrins are a family of highly homologous proteins with smallmolecular weight or peptides derived from viper venoms that contain manycysteines forming disulfide bonds[1]. Disintegrins contain an Arg-Gly-Asp(RGD) recognition sequence, which appears to mediate the binding of theseantagonists to the integrins, with the exceptions of barbourin and ussuristatin2possessing a KGD and atrolysin E possessing an MVD motif in its inhibitoryloop[2-5]. RGD-containing disintegrins show different binding affinity andselectivity toward integrins that recognize the RGD sequence in their ligands,such as αIIbβ3,α5β1,αvβ3 and α2β1. KGD-containing barbourin inhibits the αIIbβ3integrin with a high degree of selectivity[6]. Adinbitor is cloned from Gloydius blomhoffi brevicaudus andcharacterized as a novel disintegrin, which is composed of 73 amino acidresidues including 12 cysteines and an RGD motif, the signature motif ofdisintegrin. As a new snake venom protein, adinbitor could inhibit ADPinduced human platelet aggregation. Platelet aggregation is a key event in thrombus formation and isdependent upon the binding of RGD sequences in adhesive proteins to theglycoprotein (GP) IIb-IIIa complex (integrin αIIbβ3) on the platelet surface[7-10].However, because the known viper venom GPIIb-IIIa antagonists contain theRGD amino acid sequence, they block the adhesive functions of otherRGD-dependent integrins, such as vitronectin receptor(s)( αvβ3, αvβ5) andfibronectin receptor (α5β1) and are thus not GPIIb-IIIa-specific [11][12] . Becauseof this broad reactivity with other integrins, the therapeutic potential of theseagents for specifically inhibiting platelet-dependent thrombus formation invivo may be limited [13][14] . The single disintegrin, barbourin, contains KGDinstead of RGD. This disintegrin is highly homologous to other GPIIb-IIIaantagonists of the viper venom, but it is the first member that does not containthe Arg-Gly-Asp (RGD) amino acid sequence believed to be required forinhibiting receptor function. The conservative Lys for Arg substitution appearsto be the sole structural feature, which imparts integrin specificity to barbourin,since venom protein analogs with Lys substitutions are also specific forGPIIb-IIIa. Thus, barbourin represents a new structural model useful fordesigning potent and GPIIb- IIIa-specific compounds that may havetherapeutic value as platelet aggregation inhibitors[15]. Using this information in this study, Lys (K) substituted for Arg (R) bysite-directed and PCR-based mutagenesis of cDNA, recombinant protein wasexpressed in E.coli.BL21 and purified by using the His-Bind affinitychromatography. KGD-rAdinbitor could also dose-dependently inhibit ADP-inducedhuman platelet aggregation in human platelet-rich plasma (PRP) system. TheIC50 value of KGD-rAdinbitor for platelet aggregation was 0.08μmol/L. Therewas no obvious difference between the RGD-rAdinbitor and theKGD-rAdinbitor. rAdinbitor significantly inhibited angiogenesis in vivo. Compared to wildrAdinbitor, KGD- rAdinbitor had no potency in angiogenesis in vivo. These results suggested that mutated rAdinbitor containing the KGDsequence was potential and specific inhibitor of human platelet aggregation.Thus, KGD-rAdinbitor represented a new structural model useful fordesigning potential and GPIIb-IIIa-specific protein that might have therapeuticvalue as platelet aggregation inhibitor and could be an excellent candidate forevaluation as antithrombotic agent.