Abstract
von Willebrand factor (vWF), a macromolecular glycoprotein synthesized in endothelial cells and megakayocytes ciculates in blood as heterogenous multimers and plays a critical role in the formation of platelet plugs. vWF is composed of an identical subunit with a molecular weight (MW) of 270 kDa, which is held together by disulfide bonds. Functional domains of vWF for factor VIII, heparin, platelet glycoprotein (GP) Ib, collagen, and GP IIb/IIIa locate in this order from the N-to C-terminus of subunit. The GP Ib binding domain is cyptic in normal circulation and only becomes expressed when vWF is associated with the subendothelial matrix or with fibrin. In vitro, an antibiotic ristocetin or the snake venom botrocetin isolated from Bothrops jararaca mimics the active component of subendothelial matrix and causes the binding of vWF to GP Ib.
To elucidate the mechanism of in vitro process of vWF-GPIb binding, we describe here the isolation and characterization of two distinct forms of botrocetin. Since ristocetin is a heavily glycosylated proteoid, it is hard to characterize the structure on the basis of amino acid analysis. The apparent MW 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 that 60 Asx and Glx residues/molecules) but significant differences in the content of Cys and Phe residues. The N-terminal sequence of the one-chain was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different N-terminal sequences, distinct from that of the one-chain species: (α) Asp-Cys-Pro-Ser-Gly-Try-Ser-Ser-Tyr-Glu and (β) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of the both species was less than 2% of the total mass. Both the one- and two-chain botrocetins bound to purified vWF and formed an activated complex, which then bound to GPIb. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets. Furthermore, when the concentration of botrocetin was not limiting, the parameters of vWF binding to platelets were identical with either species. These results provide an initial chemical and functional characterization of two-distinct forms of botrocetin.
