Summary
A protease present in plasma cleaves von Willebrand factor (vWF) at the peptide bond
842Tyr-843Met of the mature subunit. To quantify this vWF-cleaving protease activity
in plasma we have developed a simple method based on the estimation by IRMA of the
degradation of a constant amount of wild type recombinant vWF used as substrate, by
serial dilutions of test plasma used as protease provider. vWFAg was estimated by
two-site IRMA using as first coating antibody a monoclonal antibody (MoAb) whose epitope
is localized on the C-terminal side of the cleavage site, and as second labeled antibody
a pool of MoAbs specific for the N-terminal side. Because the proteolytic process
leads to the progressive separation of the C- and N-terminal portions of the vWF subunit
such an IRMA also shows a progressive apparent loss of vWFAg. In contrast, the levels
of vWFAg estimated after proteolysis by regular 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
plasma, 14 normal individuals and 13 patients with various types of vWD had normal
levels of protease activity (44-178%) by both methods. The validity of the method
was confirmed by showing a lack of detectable protease activity in a patient with
chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears
as a useful tool for the quantification of the vWF-cleaving protease activity in plasma.
Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However,
this new IRMA has the major advantages of being much simpler and faster, and open
to most research laboratories in the field.
Keywords
von Willebrand factor - monoclonal antibodies - vWF-cleaving protease - proteolysis
measurement
