Ristocetin accelerates Von Willebrand Factor (VWF) hydrolysis rate and ameliorates Collagen Binding Assay (CBA) sensitivity

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) in the A2 domain. This cleavage in vitro is stimulated by high fluid shear stress or mild denaturation of VWF with urea or guanidine hydrochloride, suggesting that conformational changes in VWF are necessary to expose the peptide bond cleaved by ADAMTS13. However, Nishio K showed that the molecular recognition of VWF by ADAMTS13 can be also attained under non-denaturing conditions enhanced by a recombinant mutant fragment of platelet GpIba (PNAS, 2004). We hypothesized that ristocetin, which stabilizes a ‘GpIb-bound like’ conformer of VWF, could enhance the interaction with ADAMTS13 without denaturing agents. Accordingly, we developed a method to measure ADAMTS13 activity, modifying the collagen binding activity (CBA) assay reported by Gerritsen et al using ristocetin (CBA-R) instead of urea (CBA-U). For the calibration curve, 100 ml normal human plasma pool aliquots were serially diluted in 1.5 mg/ml de-sulphated ristocetin, 5 mM Tris, pH 8.0 at 37°C. VWF multimers, purified by gel-filtration and heparin-agarose affinity chromatography, were used as substrate. The CBA of the digested VWF substrate was then determined using a CBA ELISA Kit (Gradipore). Plasma samples of normal subjects and patients with congenital TTP were performed. The experimental points of reference curve were fitted to a logistic curve having a sensitivity up to 3% of the ADAMTS13 concentration, compared to 6% of CBA-U. Moreover, CBA-R induced a significantly higher VWF hydrolysis over the time scale of the assay, suggesting that denaturing agents, as urea, may partially impair the protease activity, decreasing the sensitivity of CBA-U. On the whole, these findings showed that the CBA-R is a simple and accurate assay for ADAMTS13 activity. The CBA-R could probably represent a valuable method to simulate in vitro, under non-denaturing conditions, the same flowing conditions occurring in vivo