Altered Von Willebrand Factor Subunit Proteolysis and Multimer Processing Associated with the Cys1599(2362)Phe Mutation in the B2 Domain.

Cysteine residues clustered in carboxyl- and amino-terminal domains mediate the assembly of von Willebrand factor (VWF) subunits into dimers and multimers, respectively. We have identified a patient with bleeding symptoms and very low plasma VWF antigen and ristocetin cofactor activity, compatible with clinically severe von Willebrand disease (VWD), whose abnormal plasma VWF multimers show a distinctly altered proteolytic processing. Analysis of plasma VWF multimers in low resolution agarose gels demonstrated a smearing extending to the origin of the separating gel in both the proband and her parents, suggestive of the presence of unusually large species with low electrophoretic mobility. In high resolution gels, these large multimers were stacked at the origin of the gel, clearly seen in a sample of proband’s plasma obtained after DDAVP infusion even though the treatment caused only a modest increase in VWF:Ag and VWF:RCo (from 4.7 to 7.5 U/dL and from 5.2 to 8 U/dL, respectively). Individual VWF multimers in the proband’s plasma comprised only one clearly visible band with different mobility as compared to the main band of normal multimers, and lacked the satellite bands that form the normal triplet structure. Analysis of the proband’s plasma VWF subunit composition using anti M7 antibodies (which probe the aminoterminal region of VWF) revealed the apparent 225 kDa mature form and a single 205 kDa fragment, but notably absent was the 140 kDa aminoterminal fragment generated by ADAMTS-13 cleavage. Subunit analysis using anti-M31 antibodies (which probe the carboxyl-terminal region of VWF) revealed the 225 kDa intact subunit and the 205 kDa fragment, but notably absent were the fragments generated by ADAMTS-13 cleavage. Analysis with anti-22K antibodies, which react with one or more epitope(s) located in the extreme carboxyl terminal region of VWF downstream of Val1927, revealed the presence of only the intact subunit of 225kDa. Concentration of ADAMTS-13 antigen and VWF-cleaving activity in the patient plasma were within normal limits.The patient carried a homozygous Cys to Phe mutation at position 1599 in the mature VWF subunit B2 domain (Cys2362 of pre-pro-VWF). In her heterozygous parents, the 205 kDa fragment was distinctly visible along with the normal VWF cleavage products. Our finding indicate that a proper conformation of the B2 domain, dependent on critical Cys residues, may be required for normal proteolytic processing of VWF multimers. The association of these structural and functional abnormalities with a missense mutation in the B2 domain provides insights into the mechanism that may regulate the post-secretion processing of VWF multimers, and, thus, their prothrombotic potential.