Von Willebrand factor cleaving protease activity in the physiopathology of microangiopathic disorders

The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve

Requirement of a plasma fraction for the loss of large Von Willebrand factor multimers induced by high wall shear rate

The perfusion of serum, citrated whole blood and citrated plasma, through a simple tube system resulted in a significant loss of large von Willebrand factor (vWf) multimers, without decrease in antigen levels. Maximum loss of large multimers was observed at a shear rate of 15,000 s(-1) for 15 min. Heparin, aprotinin, soybean trypsin inhibitor, phenylmethylsulphonylfluoride, N-ethylmaleimide, leupeptin or calpain inhibitor peptide could not prevent the loss of large vWf multimers in citrated plasma. The addition of EDTA calcium salt partially prevented it, and it was totally prevented by EDTA without calcium. Perfusion of purified vWf did not induce the loss of large multimers, but this did happen after the addition of either whole serum or a plasma fraction. The activity of this plasma fraction disappeared at pH < 6.8. Besides, we have found that the binding to subendothelium of purified vWf diluted in dialized serum was lower at pH 7.2 than at pH 6.0. Chromatographic studies demonstrated that the loss of large vWf multimers, induced by high shear rates, involves a plasma substance(s) of molecular weight larger than 200 kD; calpain and granulocyte or cysteine proteases do not seem to be this plasma substance(s).