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Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wildtype recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecularweight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that iso- Introductionvon Willebrand factor (VWF) is a large multimeric glycoprotein that has 2 important roles in hemostasis. These are stabilizing factor VIII (FVIII), by acting as its carrier protein in the circulation, and attaching activated platelets to the subendothelium via binding to the GpIb receptor on platelets and to collagen in the subendothelial matrix. Enhancement of the platelet-subendothelium interaction is vital at sites of vascular injury during conditions of high shear stress. VWF binds to collagen via 2 sites: the A3 domain (residues 1683-1874) contains the main site for fibrillar collagen types I and III found within perivascular connective tissue, 1,2 and a second site in the A1 domain (residues 1260-1471) binds nonfibrillar collagen type VI within the subendothelial matrix. 3,4 However, the A1 domain can also bind collagen types I and III, and the relative importance of the 2 domains has been extensively but inconclusively investigated. 5,6 In all cases, it appears that affinity of VWF for collagen is heavily dependent on the presence of VWF multimers of high or ultrahigh molecular weight. 7 Type 2 von Willebrand disease (VWD) is characterized by a qualitative defect in VWF and is diagnosed by demonstration of a discrepancy between circulating plasma levels of VWF and its functional activity. In addition to measuring GpIb␣-dependent function using ristocetin cofactor activity (VWF:RCo), it has been recommended that collagen-binding activity (VWF:CB) is analyzed in the subclassification of type 2 VWD. 8 A single defect in VWF collagen binding in association with normal multimeric structure has previously been reported. 9 In this report, we describe the phenotypic and genotypic characterization of 3 families with bleeding symptoms...

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Type 3 von Willebrand disease (VWD) is a severe autosomal recessive inherited bleeding disorder. In affected individuals the underlying von Willebrand factor gene (VWF) mutations frequently remain uncharacterized. The aim of this study was to investigate the molecular basis of type 3 VWD in patients (11 Caucasians and 9 of Asian origin) attending the haemophilia centres at Central Manchester NHS Trust. A combination of DNA sequencing of VWF genomic and complementary DNA was performed to identify mutations in the patient cohort. Fifteen different VWF mutations were identified at the genomic DNA level: two gene conversion events, three nonsense, three frameshift, one missense, two splice site, one insertion-deletion and three deletion mutations. Homozygosity or compound heterozygosity for mutations was present in 15 of the 20 patients. In the remaining five individuals, heterozygosity for a single VWF mutation was identified in four cases and one patient had no detectable VWF mutation. Analysis of platelet-derived VWF RNA from these five individuals revealed heterozygosity for a deletion of exons 4 and 5 in four cases. The remaining patient was heterozygous for a three base deletion which had already been identified at the DNA level. Overall the observed VWF genotype explained the phenotype in 18 of the 20 patients investigated. In genetic studies in type 3 VWD, if VWF mutations are not detected at the DNA level, RNA analysis should be performed to search for intronic mutations, heterozygous deletions or aberrant splicing/post-transcriptional events. However, this may still not explain all cases of previously phenotypically diagnosed type 3 VWD.

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