Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor

Abstract

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...

Current issues in diagnosis and treatment of von Willebrand disease

“…The VWF:CB assay can be used to determine if there are any binding defects between VWF and collagen I or III. Several mutations in the A3 domain have been shown to disrupt the VWF collagen interaction and lead to bleeding symptoms including p.Ser1731Thr,15 p.Trp1745Cys,16 p.Met1761Lys,42 p.Ser1783Ala,16 and p.His1786Asp 17…”

How I treat type 2 variant forms of von Willebrand disease

“…Only a few mutations in the A3 domain leading to selective VWF:CBA defects without loss of HMWM have been reported. 12 Hence, a reduced VWF:CBA/VWF:Ag ratio (,0.6) helps discriminating between type 2A and type 2M VWD. 13 Type 2B is characterized by mutations in the A2 domain causing an increased affinity of VWF for platelet GpIb, resulting in enhanced ristocetin-induced platelet agglutination (RIPA).…”

“…41,49 In patients with isolated collagen-binding defects, desmopressin improves all VWF/FVIII measurements including VWF:CBA, albeit with a persistent discrepancy of VWF:CBA/VWF:Ag ratio. 12 Minor side-effects include tachycardia, headache, and flushing; hyponatremia and fluid overload with seizures may be a risk, particularly in small children receiving repeated infusions. In these situations, serum electrolytes should be monitored and fluid intake should be limited.…”

“…However, VWF:CBA would still be required to differentiate the reported rare mutations in the collagen-binding A3 domain from type 1 VWD. 12,37 Confirmatory multimeric and/or genetic analysis are useful complements to the definitive classification, but they are often of little clinical value, apart from differentiating type 2N from mild hemophilia A for genetic counseling, and not readily available to all laboratories.…”

Molecular diagnosis of von Willebrand disease

“…(VWF:CB/VWF:Ag <0.6) [32,33]. In this case, the majority of mutations are located in the A3 domain, and exons 29-32 should be investigated.…”