Cloning, expression, and hemostatic activities of a disintegrin, r-mojastin 1, from the 3 mohave rattlesnake (Crotalus scutulatus scutulatus)

Abstract

Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, 29 platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic 30 processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause 31 edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in 32 snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of 33 the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical 34 importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of 35 cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216 bp disintegrin 36 cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes 37 for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet 38 adhesion to fibronectin with an IC50 of 58.3 nM and ADP-induced platelet aggregation in whole blood with 39 an IC50 of 46 nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting 40 with an IC50 of 95.6 nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 41 7.9509 kDa.