ABNORMALITY OF THE N-TERMINAL PORTION OF VON WILLEBRAND FACTOR (FRAGMENT Sp III ) IN TYPE IIA AND IIC VON WILLEBRAND DISEASE

Abstract

A new analytical method was established, allowing the characterization of von Willebrand Factor (vWF) degradation fragments from minute amounts of plasma without the need for immunopurification of vWF. Ten ul of citrated plasma in 20 ul of 0.1 M Tris-HCl, pH 7.8, 0.1 M NaCl, 10 mM EDTA buffer were treated with S. aureus V-8 protease for 15 min to 3 h at 22°C. Following addition of 1 mM DFP, the cleaved fragments were separated by SDS-polyacrylamide or SDS-agarose gel electrophoresis and identified by 125 I-labeled polyclonal or monoclonal antibodies (MAbs) to vWF andautoradiography. Quantification of each fragmentwas estimated by counting radioactivity in slices of the dry gel. In normal plasma, S. aureus V-8 protease produced two dimeric fragments of vWF, with identical electrophoretic and antigeniccharacteristics to those produced from highly purified vWF, ie a C-terminal fragment of 220 kd (Spll) and a series of N-terminal fragments (Spill) with a major species of Mr 320 kd (SpIIIa) and two minor ones of 265 kd (SpIIIb) and 215 kd (SpIIIc). The method was applied to further characterize the molecular abnormalities of vWF in 15 patient plasmas with variant (type II) von Willebrand disease (vWD). In all cases with type IIA, IIB, IIC and IID tested, fragment Spll had the same mobility as in normal plasma. In 7 patients with type IIA, the amount and distribution ofthe three Spill species were clearly abnormal, with a marked decrease or absence of SpIIIa and an increase of SpIIIb and SpIIIc. Results slightly varied between patients but were consistent within one family. In 4 patients with type IIC, the band SpIIIb was lacking. In 2 patients with type IIB and 2 patients with type IID, there was no significant modification of Spill. In all cases the pattern of Spill was similar whether using polyclonal antibodies to vWF or MAbs to Spill.The pattern of normal or abnormal Spill remainedthe same in fresh or frozen plasma and was not modified by the presence of 5 mM EDTA at the timeof blood collection. Furthermore, the abnormaldistribution of Spill in type IIA or IIC vWD wasalready present after the shortest digestion time(15 min) indicating that the fragment Spill was produced from an abnormal vWF during the primary digestion with S. aureus V-8 protease. In conclusion, the molecular abnormality of vWF in type IIA and IIC vWD appears to reside in Spill, the N-terminal portion of the subunit (residues 1 to 1,365).