ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor.

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605-M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13-mediated cleavage of VWF. Recombinant human full-length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1.5 mol/l urea, and analyzed via multimer migration distance. The glutathione-S-transferase (GST) and histidine-tagged construct, E1554-R1668 of VWF (VWF115) was assayed via enzyme-linked immunosorbent assay: VWF115 was bound to anti-GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase-labelled anti-histidine tag antibody. All alterations examined in the Y1605-M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A-domain changes in VWF alter ADAMTS13-mediated proteolysis.

A novel type 2A von Willebrand factor mutation located at the last nucleotide of exon 26 (3538G>A) causes skipping of 2 nonadjacent exons.

In this manuscript, we describe a case of type 2A von Willebrand disease (VWD) caused by the novel heterozygous G>A transition at nucleotide 3538, which should result in the putative, nonconservative substitution of G1180R. This mutation was reproduced by site-directed mutagenesis; however, the recombinant mutant protein was efficiently secreted from cells and assembled correctly into multimers. Because the substitution is located at the last nucleotide of exon 26, the patient's platelet von Willebrand factor (VWF) mRNA was analyzed and 3 transcripts were observed: the normal transcript without the 3538G>A transition, a transcript with the in-frame deletion of exon 26, and a transcript with the in-frame deletions of exons 23 and 26. These deletion VWF cDNA constructs were created and the resulting recombinant proteins were analyzed following transfection into COS-7 cells. Cotransfection results demonstrate that the exon-skipped transcripts led to intracellular retention, and the levels of VWF antigen (VWF:Ag) produced by these constructs were as follows: del23/26A transition at nucleotide (nt) 3538 does not result in the expression of the G1180R missense mutation, but rather leads to exon skipping, which is the pathogenic basis of the patient's phenotype. This is the first report of a coding region mutation resulting in the skipping of 2 nonadjacent exons.

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease.

To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312-19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312-19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD.