Rhodocetin, a novel platelet aggregation inhibitor from the venom of Calloselasma rhodostoma (Malayan pit viper): Synergistic and noncovalent interaction between its subunits

A novel platelet aggregation inhibitor, rhodocetin, was, purified from the crude venom of Calloselasma rhodostoma. It inhibited collagen-induced plate let aggregation in a dose-dependent manner, with an IC50 of 41 nM. Rhodocet in has a heterodimeric structure with alpha and beta subunits, which could be separated on a nonreducing denaturing gel or reverse-phase: HPLC column. Individually neither subunit inhibited platelet aggregation even at 2.0 mu M concentration. Titration and reconstitution experiments showed that, whe n these subunits are mixed to give a 1:1 complex, most of its biological ac tivity was recovered. The reconstituted complex inhibited platelet aggregat ion with an IC50 of 112 nM, about 3-fold less effective than-the native mol ecule. Circular dichroism analysis revealed that the reconstituted complex had a spectrum similar to that of the native protein. By using surface plas mon resonance studies,: we established that the stoichiometry of binding be tween the two subunits is 1:1 and the subunits interact with a K-d Of 0.14 +/- 0.04 mu M. The complete amino acid sequences of the alpha (15956.16 Da, 133 residues) and beta (15185.10 Da, 129 residues) subunits show a high de gree of homology with each other (49%) and with the Ca2+-dependent lectin-r elated proteins (CLPs) (typically 29-48%) isolated from-other Snake venoms. Unlike the other members of the family in which the subunits are held toge ther by an interchain disulfide bond, rhodocetin subunits.:are held togethe r only through noncovalent interactions. The cysteinyl residues forming the intersubunit disulfide bridge in all other known CLPs: are replaced by Ser -79 and Arg-75 in the alpha and beta subunits of rhodocetin, respectively. These studies support the noncovalent and synergistic interactions between the two subunits of rhodocetin. This is the first reported CLP dimer with s uch a novel heterodimeric structure.