Summary
Patients with thrombotic thrombocytopenic purpura (TTP) have a deficiency of von Willebrand
factor (vWF)-cleaving protease, whereas patients with hemolytic-uremic syndrome (HUS)
show normal activity of this protease. Present methods for assaying vWF-cleaving protease
by immunoblotting are time-intensive and cumbersome. We therefore developed a new
functional assay based on the preferential binding of high-molecular-weight forms
of vWF to collagen. In this assay, the diluted plasma sample to be tested is added
to normal human plasma in which protease activity had been abolished. The vWF present
in the protease-depleted plasma is digested by the vWF-cleaving protease in the test
plasma. The proteolytic degradation leads to low-molecular-weight forms of vWF, which
show impaired binding to microtiter plates coated with human collagen type III. The
collagen-bound vWF is quantified using a peroxidase-conjugated rabbit antibody against
human vWF. The values of vWF-cleaving protease activity in tested plasma samples are
read from a calibration curve achieved by incubating the vWF-substrate with dilutions
of a normal human plasma pool (NHP). Testing of plasma from patients with TTP and
HUS showed that the assay can be used to distinguish between these two syndromes.
The presence of an inhibitor can be detected by carrying out the test after incubation
of NHP with the patient plasma sample, thus enabling differentiation of patients with
familial TTP from those with non-familial TTP.
Keywords
von Willebrand factor-cleaving protease - collagen binding assay - TTP - HUS
