Isolation and Chemical Characterization of Two Structurally and Functionally Distinct Forms of Botrocetin, the Platelet Coagglutinin Isolated from the Venom of Bothrops jararaca

TWO distinct forms of botrocetin, the von Willebrand factor (vWF)-dependent platelet coagglutinin isolated from the venom of the snake Bothrops jararaca, were purified and characterized structurally and functionally. The apparent molecular mass of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater than 60 Asx and Glx residues/molecule) but significant differences in the content of Cys and Phe residues. The NH₂-terminal sequence of the one-chain botrocetin was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different NH₂-terminal sequences, distinct from that of the one-chain species: (α) Asp-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Glu-and (β) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of both species was less than 2% of the total mass, and the pI was 4.0–4.1 for the one-chain species, and 4.6, 5.3–5.4, and 7.7–7.8 for the two-chain species. No free sulfhydryl group was detected in each species. Both types of botrocetin were resistant to proteolysis at neutral pH. Incubation of ¹²⁵I-labeled one-chain botrocetin with the crude venom solution resulted in no detectable structural change. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets. A new experimental approach revealed that vWF and botrocetin form a soluble complex, and the binding of ¹²⁵I-labeled two-chain botrocetin to vWF was clearly inhibited in a dose-response manner by one-chain botrocetin as well as the unlabeled two-chain counterpart. Furthermore, when the concentration of botrocetin was not limiting, the parameters of vWF binding to platelets were identical with either species. These results clearly indicate the existence of two different forms of botrocetin which differ in their molecular structure and affinity for vWF, and provide their initial chemical characterization.