Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex

Abstract

Interaction of von Willebrand factor (vWF) with its platelet receptor only occurs in vitro in the presence of a modulator such as ristocetin. We have recently confirmed that the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor involved in the ristocetin-dependent binding of vWF by reconstitution with the purified components [Berndt, M.C., Du, X., & Booth, W.J. (1988) Biochemistry 27, 633-640]. We have now developed a similar solid-phase reconstitution assay using an alternate modulator, botrocetin, for the competitive analysis of functional domains in both vWF and the GP Ib-IX complex. Botrocetin was purified from Bothrops jararaca venom by ammonium sulfate fractionation and subsequent DEAE-cellulose and hydroxylapatite chromatography. The purified protein was a 25-kilodalton (kDa) disulfide-linked dimer with apparent subunit molecular weights of 14,000 and 14,500. Binding studies with immobilized botrocetin demonstrated that botrocetin bound to vWF and to a 52/48-kDa region of vWF that contains the GP Ib binding domain, but not to glycocalicin, a proteolytic fragment of GP Ib that contains the vWF binding site. Binding of 125I-labeled vWF to GP Ib-IX complex coated beads and to platelets was strictly botrocetin-dependent with half-maximal binding at a botrocetin concentration of congruent to 0.27 microM. Botrocetin-dependent binding of vWF was specific, saturable, and comparable to that observed with ristocetin. An anti-vWF monoclonal antibody, 3F8, inhibited ristocetin- but not botrocetin-dependent binding of vWF, suggesting the presence of distinct ristocetin and botrocetin modulator sites on vWF. The botrocetin reconstitution assay was at least an order of magnitude more sensitive than the corresponding ristocetin assay for the competitive analysis of functional domains on both vWF and the GP Ib-IX complex and has confirmed the localization of the vWF-binding domain to the 45-kDa N-terminal region of GP Ib.

Identification of the disulphide bonds in human platelet glycocalicin

“…The interaction between von Willebrand factor and glycoprotein Ib can be simulated in vitro by using the antibiotic ristocetin [24] or the snake venom peptide botrocetin [25] or by using animal von Willebrand factor such as bovine or porcine or asialo human von Willebrand factor [26,271. It is nevertheless still not clear how far these approaches really approximate to the natural interaction and, indeed, differences in potential binding sites have been observed [28].…”

Bruton tyrosine kinase is essential for botrocetin/VWF-induced signaling and GPIb-dependent thrombus formation in vivo

“…Botrocetin was prepared as previously described. 26 VCL, a recombinant fragment of VWF, 27,28 was a generous gift from Dr Steven Slack (University of Memphis). 29 …”

Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

“…Human VWF was purified from cryoprecipitates as described previously (33). Botrocetin was also purified as previously described (34). Ristocetin, forskolin, and dimethyl sulfoxide (Me 2 SO) were purchased from Sigma.…”