Substitution of the Echistatin Amino Acid Motif RGDD with KGDW Enhances Inhibition of Platelet Aggregation and Thrombogenesis

Abstract

Disintegrins are a family of small proteins found in snake venom. These proteins are of great biomedical importance due to their binding affinities with different kinds of integrins, which results in the inhibition of platelet aggregation, the adhesion of cancer cells, and the induction of signal transduction pathways. Disintegrins are multi-functional due to their lower integrin selectivity. To increase their binding specificity with platelets, we modified the gene sequence encoding one kind of disintegrins, echistatin (Ech). DNA recombination technology was used to change the Ech amino acid sequence RGDD to KGDW, which are located at the 24th–27th location site of Ech, thereby creating a mutant protein echistatin (E-KW). The E-KW was expressed, isolated and purified using molecular biological methods. ADP-induced platelet aggregation in human platelet-rich plasma was inhibited by E-KW at 45 nM and by Ech at an IC50 of 140 nM (P < 0.05). E-KW was also more effective than wild-type Ech in inhibiting thrombus formation (12.8 ± 3.2 vs. 27.9 ± 4.1, P < 0.05). Our studies revealed that, compared to wild-type Ech, E-KW had stronger binding specificity to the glucoprotein receptors on platelet membranes and was more effective in inhibiting platelet aggregation and thrombogenesis.