Normal Cleavage of Von Willebrand Factor by ADAMTS13 In the Absence of Factor VIII In Patients with Severe Hemophilia A

Abstract 2114

The size of von Willebrand factor (VWF), a carrier protein for factor VIII (FVIII), is regulated by plasma metalloprotease ADAMTS13 proteolytic activity. Recently studies by Cao et al. (PNAS, 2008; 105: 7416–7421) found that under shear stress, exogenous FVIII enhanced ADAMTS13 cleavage of VWF, especially the high molecular weight multimers, in a system using purified proteins. Based on this result, the authors suggested that in the absence of FVIII, such as in patients with severe hemophilia A, VWF will have ultra-large multimers due to defects in ADAMTS13 proteolytic process, which can be corrected by infusion of FVIII. Here, we assessed VWF multimers, antigen, and ADAMTS13 activity in citrated plasma from seven patients with severe hemophilia A. The FVIII levels in six patients were less than 1% and in one was 4%. Plasma from two patients was available both pre- and post-FVIII replacement therapy (recombinant FVIII). All patients displayed VWF multimer patterns similar to those in pooled normal plasma (PNP), and the two patients receiving FVIII infusions displayed no change in VWF multimer size or pattern between their pre- and post-infusion samples. In all patients, the VWF antigen level (0.32–0.76) was below the PNP value (designated as 1), and all had increased ADAMTS13 activity (measured by the ability of plasma to cleave a small A2 peptide substrate) (1.09–1.79, PNP designated as 1), yielding an increased ratio of ADAMTS13 activity to VWF antigen in these patients (1.4–5.2 compared to PNP). We also examined cleavage of endogenous VWF by ADAMTS13 in the plasmas of the two patients studied pre- and post-infusion, yielding different FVIII levels. In this assay, we first diluted plasma 10-fold with a buffer containing 10 mM HEPES, 6.5 mM BaCl₂, and 1.5 M urea, incubated at 37°C, and ADAMTS13 cleavage was stopped at different time points with EDTA. VWF multimer patterns were examined on a 1.5% agarose gel. We found that ADAMTS13 cleaved VWF efficiently in patient plasma deficient in FVIII, similar to that in PNP. The extent of cleavage was correlated with the ratio of ADAMTS13 activity to VWF antigen, rather than with the FVIII levels. In conclusion, patients with severe hemophilia A appear to have normal ADAMTS13 processing of VWF multimers in vivo and ex vivo. Further studies of the effect of FVIII and VWF levels on ADAMTS13 cleavage of VWF and clinical correlation are needed.