Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to vWF

A protease present in plasma cleaves von Willebrand factor (VWF) at the pep tide bond 842Tyr-843Met of the mature subunit. To quantify this vWF-cleavin g protease activity in plasma we have developed a simple method based on th e estimation by IRMA of the degradation of a constant amount of wild type r ecombinant vWF used as substrate, by serial dilutions of test plasma used a s protease provider. vWFAg was estimated by two-site IRMA using as first co ating antibody a monoclonal antibody (MoAb) whose epitope is localized on t he C-terminal side of the cleavage site, and as second labeled antibody a p ool of MoAbs specific for the N-terminal side. Because the proteolytic proc ess leads to the progressive separation of the C- and N-terminal portions o f the vWF subunit such an IRMA also shows a progressive apparent loss of vW FAg. In contrast, the levels of vWFAg estimated after proteolysis by regula r IRMA remained essentially constant. Results obtained with this new method were compared with the analysis by SDS-agarose gel electrophoresis of the multimeric pattern of proteolyzed WT-rVWF and no significant difference was noted testing a series of 28 plasmas. As compared with normal pooled plasm a. 14 normal individuals and 13 patients with various types of vWD had norm al levels of protease activity (41-178%) by both methods. The validity of t he method was confirmed by showing a lack of detectable protease activity i n a patient with chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears as a useful tool for the quantification of th e vWF-cleaving protease activity in plasma. Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However, this new IRMA ha s the major advantages of being much simpler and faster, and open to most r esearch laboratories in the field.