Critical von Willebrand factor A1 domain residues influence type VI collagen binding

Abstract

Background: von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen.

Objectives: We report here on several mutations that exclusively alter binding to type VI collagen.

Patients/methods: Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects.

Results: Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen.

Conclusions: VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.

© 2012 International Society on Thrombosis and Haemostasis.

Figures

Figure 1. Family pedigrees for type VI collagen binding mutations

Figure 1A shows family 1, with heterozygous R1399H in the proband and mother. Figure 1B shows family 2, with heterozygous R1399H in the index case and heterozygous exon 18 deletion c.2435delC in the proband, father, and paternal grandmother. Figure 1C shows family 3 with deletion of amino acids 1392-1402 in the proband and siblings. Figure 1D shows family 4 with S1387I in the proband, sibling, and mother. Figure 1E shows family 5 with Q1402P in the proband, daughter, and mother. Arrows denote the proband in each family. VWF:Ag = VWF antigen, VWF:RCo = VWF ristocetin cofactor activity, VWF:CB (VI) = VWF collagen binding with type VI collagen, VWF:CB (I) = VWF collagen binding with type I collagen, VWF:CB (III) = VWF collagen binding with type III collagen.

Figure 1. Family pedigrees for type VI collagen binding mutations

Figure 1A shows family 1, with heterozygous R1399H in the proband and mother. Figure 1B shows family 2, with heterozygous R1399H in the index case and heterozygous exon 18 deletion c.2435delC in the proband, father, and paternal grandmother. Figure 1C shows family 3 with deletion of amino acids 1392-1402 in the proband and siblings. Figure 1D shows family 4 with S1387I in the proband, sibling, and mother. Figure 1E shows family 5 with Q1402P in the proband, daughter, and mother. Arrows denote the proband in each family. VWF:Ag = VWF antigen, VWF:RCo = VWF ristocetin cofactor activity, VWF:CB (VI) = VWF collagen binding with type VI collagen, VWF:CB (I) = VWF collagen binding with type I collagen, VWF:CB (III) = VWF collagen binding with type III collagen.

Figure 1. Family pedigrees for type VI collagen binding mutations

Figure 1A shows family 1, with heterozygous R1399H in the proband and mother. Figure 1B shows family 2, with heterozygous R1399H in the index case and heterozygous exon 18 deletion c.2435delC in the proband, father, and paternal grandmother. Figure 1C shows family 3 with deletion of amino acids 1392-1402 in the proband and siblings. Figure 1D shows family 4 with S1387I in the proband, sibling, and mother. Figure 1E shows family 5 with Q1402P in the proband, daughter, and mother. Arrows denote the proband in each family. VWF:Ag = VWF antigen, VWF:RCo = VWF ristocetin cofactor activity, VWF:CB (VI) = VWF collagen binding with type VI collagen, VWF:CB (I) = VWF collagen binding with type I collagen, VWF:CB (III) = VWF collagen binding with type III collagen.

Figure 1. Family pedigrees for type VI collagen binding mutations

Figure 1A shows family 1, with heterozygous R1399H in the proband and mother. Figure 1B shows family 2, with heterozygous R1399H in the index case and heterozygous exon 18 deletion c.2435delC in the proband, father, and paternal grandmother. Figure 1C shows family 3 with deletion of amino acids 1392-1402 in the proband and siblings. Figure 1D shows family 4 with S1387I in the proband, sibling, and mother. Figure 1E shows family 5 with Q1402P in the proband, daughter, and mother. Arrows denote the proband in each family. VWF:Ag = VWF antigen, VWF:RCo = VWF ristocetin cofactor activity, VWF:CB (VI) = VWF collagen binding with type VI collagen, VWF:CB (I) = VWF collagen binding with type I collagen, VWF:CB (III) = VWF collagen binding with type III collagen.

Figure 1. Family pedigrees for type VI collagen binding mutations

Figure 1A shows family 1, with heterozygous R1399H in the proband and mother. Figure 1B shows family 2, with heterozygous R1399H in the index case and heterozygous exon 18 deletion c.2435delC in the proband, father, and paternal grandmother. Figure 1C shows family 3 with deletion of amino acids 1392-1402 in the proband and siblings. Figure 1D shows family 4 with S1387I in the proband, sibling, and mother. Figure 1E shows family 5 with Q1402P in the proband, daughter, and mother. Arrows denote the proband in each family. VWF:Ag = VWF antigen, VWF:RCo = VWF ristocetin cofactor activity, VWF:CB (VI) = VWF collagen binding with type VI collagen, VWF:CB (I) = VWF collagen binding with type I collagen, VWF:CB (III) = VWF collagen binding with type III collagen.

Figure 2. Comparison of VWF antigen and propeptide for c.2435delC recombinant VWF

VWF:Ag and VWF propeptide (VWFpp) were measured for recombinant full length VWF and a construct containing the c.2435delC mutation. VWF:Ag is shown in black and VWFpp is shown in gray, both in IU/dL. Data represent a minimum of 3 separate transfections, and are given as the mean + 1 SD. VWFpp levels are higher than VWF:Ag due to the use of a plasma standard to determine concentration. Although VWFpp and VWF:Ag are secreted in an equimolar ratio, VWFpp half-life in plasma is much shorter [26]. This graph demonstrates lack of synthesis of either the propeptide or full-length VWF for the c.2435delC construct.

Figure 3. Comparison of 1399R and 1399H rVWF collagen binding

Recombinant full-length VWF was expressed in HEK293T cells using 100% wild-type DNA (1399R, shown in black), 100% variant DNA (1399H, shown in white), or a mixture of 50% of each DNA (1399R/H, shown in gray). Supernatants were analyzed for VWF:Ag and VWF:CB with types I, III, and VI collagen. Graphed here is the ratio of VWF:CB/VWF:Ag for each construct with each type of collagen. Data represent a minimum of 3 separate transfections, and are given as the mean + 1 SD.

Figure 4. Location of type VI collagen binding mutations in the crystal structure of the A1 domain

Shown here is the VWF A1 domain crystal structure (1AUQ [27]) graphed using Pymol. The orange sphere represents the location of S1387, the blue sphere represents the location of R1399, and the yellow sphere represents the location of Q1402. The red spheres represent the 11 amino acid deletion from 1392 to 1402. The pink spheres represent the area between 1387 and 1392, which may also be a part of the type VI collagen binding region. The small grey spheres represent the cysteines forming the disulfide bond (1272-1458).

Figure 5. Type VI collagen binding for recombinant VWF A1 domain mutations

VWF:Ag and VWF:CB with type VI collagen were measured for recombinant full length VWF and constructs containing the del 1392-1402, 1387I ,1402P, and 1399H mutations. VWF:Ag (in IU/dL) is shown in black, graphed on the left y axis, and VWF:CB (VI) (in U/dL) is shown in gray, graphed on the right y axis. Data represent a minimum of 3 separate transfections, and are given as the mean + 1 SD.