Characterization of a novel mutation in the von Willebrand factor propeptide in a distinct subtype of recessive von Willebrand disease

Abstract

von Willebrand factor (VWF) is a plasma protein that consists of a series of multimers of which the high-molecular-weight VWF multimers are the most potent in platelet adhesion and aggregation. The propeptide of the VWF (VWFpp) is known to be essential in the process of multimer assembly. Genetic studies were performed in a patient with a phenotype of von Willebrand disease (VWD) characterized by very low plasma factor VIII and VWF levels and a VWF consisting of only a dimeric band and total absence of all multimers in plasma. The patient was found to be homozygous for the novel C570S mutation, caused by a 1709G>C transition in exon 14 of the VWF gene coding for the propeptide. Three asymptomatic relatives were found to be heterozygous. In-vitro mutagenesis and expression in COS-7 cells confirmed the detrimental effect of the mutation on VWF multimerization. Our findings show that the C570S mutation in the VWFpp abolishes multimerization of VWF. The mutation probably disrupts the normal configuration of the VWFpp, which is essential for correct orientation of the protomers and ultimately multimerization. The mutant amino acid is located in a region that is highly conserved across several species which underlines its critical role. This variant constitutes a distinct subtype of recessive 2A VWD with the exclusive presence of the dimeric form of VWF in plasma.

Laboratory and Molecular Characteristics of Recessive von Willebrand Disease Type 2C (2A Subtype IIC) of Variable Severity due to Homozygous or Double Heterozygous Mutations in the D1 and D2 Domains

“…The laboratory phenotype in patients with recessive VWD type 2C (2A subtype IIC) varies widely from severe (with very low levels of FVIII:C and VWF:Ag, unmeasurable VWF:RCo, absent RIPA and strongly prolonged BT as documented in five kindreds [6,7,8, 12, 15]) to rather mild VWD type 2C (with normal values for FVIII:C and VWF:Ag, low levels for VWF:RCo and VWF:CB and prolonged BT as reported in two kindreds [1, 11, 14]). The combination of a null mutation and a missense mutation in the D1 domain may mimic severe VWD type 3 [5].…”

“…In retrospect, the multimeric pattern of the VWD patients homozygous for R273W and C570S clearly showed the absence of high-molecular-weight multimers and a pronounced dimeric band consistent with type 2C (2A subtype IIC) VWD [6, 10]. In addition, expression studies of recombinant R273W, W377C and C570S showed secretion of VWF mainly consisting of dimers which failed to form intermediate- and high-molecular-weight multimers consistent with the clinical diagnosis of VWD 2C (2A subtype IIC) [6,7,8]. These findings indicate that mutations in the D1 and D2 VWFpp region completely abolish multimerization of VWF.…”

“…A few missense mutations related to autosomal recessive severe VWD have been identified in the VWF prosequence D1 and D2 domains [5,6,7,8]. There are two reports on double heterozygous missense/null mutation D141Y/null and C275S/null associated with VWD type 3 with documented hemarthrosis in one of them (table 2) [5].…”

Phenotypic Expression of ADAMTS13 in Glomerular Endothelial Cells

“…After 24 hrs of incubation with serum free medium, cells were stimulated with phorbol-12-myristate-13-acetate (PMA, Sigma–Aldrich, St Louis, MO) at 500 nM for 1 hr or with histamine 100 µM (Sigma-Aldrich) [34] for 15 min, alternatively with estradiol 1 nM (Sigma-Aldrich) [35] for a further 24 hrs. As a positive control for cell stimulation VWF release was measured in cell media by ELISA as described [36]. Of the stimulants used, only histamine exhibited a 2-fold increase in VWF release.…”

Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli – An Anti-thrombotic Mechanism in the Kidney

“…The level of endogenous VWF in the GBM was measured by enzyme-linked immunosorbent assay (ELISA) as previously described (Lanke et al, 2008). Levels of ADAMTS13 (Cloud-Clone Corp, Houston, TX), elastase (Hycult Biotech, Uden, Holland), cathepsin G (Nordic BioSite, Täby, Sweden) and MMP9 (R&D Systems, Minneapolis, MN) were measured by ELISA kits according to the manufacturers' protocols.…”