Abstract
Abstract 4317
Ristocetin, a peptide antibiotic from the soil bacterium Nocardia lurida, has been used for decades as a tool to diagnose deficiency or dysfunction of von Willebrand factor (VWF) in von Willebrand disease. Ristocetin is able to assess the functional state of VWF because it induces the interaction of VWF with the platelet glycoprotein (GP) Ib-IX-V complex in the absence of shear stress or VWF immobilization, conditions normally required in vivo for their interaction. Presumably, ristocetin is able to do this by inducing an allosteric change in VWF that exposes the binding site for GPIbα. Ristocetin is one of two widely used modulators of the VWF–GPIb α interaction (the other being botrocetin), and the one that induces an interaction that most closely mimics shear-induced platelet adhesion and aggregation. Recently, Shim et al, (Blood, 2008;111(2):651-7) demonstrated that VWF bound to platelets was a better substrate for the plasma metalloprotease ADAMTS13, raising the possibility that exposure of the GPIbα binding site on VWF could be coupled to exposure of the ADAMTS13 cleavage site. Another possibility would be that the tensile force experienced by a VWF strand with multiple bound platelets in a shear field would be sufficient to stretch VWF and expose the ADAMTS13 cleavage site. We therefore evaluated whether ristocetin alone could enhance ADAMTS13 cleavage of VWF in the absence of shear force. We used four VWF sources for these experiments: plasma; purified, multimeric VWF from plasma; a recombinant fragment encompassing the three A domains (A1A2A3); and two recombinant A2 domains, one containing a previously identified ristocetin-binding site between D1459 and P1465, and the other lacking it. Ristocetin at 1.0 mg/ml induced the cleavage of VWF by ADAMTS13 in plasma or of the purified multimeric form as efficiently as did 1.5 M urea, the standard reagent and concentration used for this assay. Similarly, ristocetin accelerated cleavage of the monomeric A1A2A3 fragment. Finally, and somewhat surprisingly, ristocetin accelerated cleavage of the isolated A2 domain, but only when the D1459–P1465 sequence was included in the construct. Vancomycin, a related antibiotic, did not have this effect. Our data suggest that exposure of the ADAMTS13 cleavage site is not only induced by tensile force in vivo, but also by other more subtle biochemical forces. These findings also indicate that exposure of the binding site for GPIbα is coupled to exposure of the ADAMTS13 cleavage site in VWF, perhaps providing part of the explanation for why platelet-bound VWF is a better ADAMTS13 substrate and for why newly released ultralarge VWF is both capable of spontaneously binding platelets and of being cleaved rapidly by ADAMTS13 in the presence of minimal shear stress. Finally, our findings also suggest that ristocetin might be a more specific reagent to evaluate the activity of ADAMTS13 for cleaving multimeric VWF in vitro.
Disclosures:
No relevant conflicts of interest to declare.
Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: targeting adhesion proteins collagen I and von Willebrand factor
