Absent collagen binding in a VWF A3 domain mutant: utility of the VWF:CB in diagnosis of VWD

摘要: Von Willebrand factor (VWF) tethers platelets to injured subendothelium through binding sites for collagen and platelet glycoprotein Ib (GPIb). The site has been localized the VWF A1 A3 domains [1]. This interaction is measured in vitro by assay, or VWF:CB [2,3]. VWD guidelines recently published NHLBI suggest restricting use of subjects who have abnormal initial screening results with antigen (VWF:Ag) ristocetin cofactor activity (VWF:RCo) [4]. VWF:RCo assay measures VWF-platelet interactions, as induced antibiotic ristocetin, therefore a defect exclusive VWF-collagen axis could potentially be missed omitting assay. We report here on subject type 1 was discovered mutation domain. index case family members were enrolled Zimmerman Program Molecular Clinical Biology after informed consent obtained. VWF:Ag, VWF:RCo, VWF:CB, multimer distribution, blood analysis performed clinical hemostasis laboratory BloodCenter Wisconsin previously described [5]. This had VWF:Ag 41 IU/dL 44 normal but only 22 U/dL (figure 1A). Her history significant epistaxis requiring cautery hemorrhage following tonsillectomy child. She also prolonged bleeding dilation curettage procedure. score, calculated using scoring system from European MCMDM-1 study, elevated at 8 [6]. DNA sequencing full length coding sequence, an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA), showed this heterozygous exon 31 (5356C>G) which led substitution aspartic acid wild-type histidine amino 1786 (H1786D). Figure 1 Panel A shows levels H1786D proband members. listed each along analysis. used III collagen. In addition, ... Recombinant containing 1786D synthesized via site-directed mutagenesis Stratagene QuikChange kit (La Jolla, CA) expressed HEK293T cells. Additional constructs incorporate three reported mutations domain, 1731T [7], 1745C, 1783A [8]. For research assays, ELISA plates coated either I human placental (Sigma, St. Louis, MO) 5 μg/mL (Southern Biotech) μg/mL, diluted carbonate coating buffer (15 mM sodium carbonate, 35 bicarbonate, 3 azide), incubated 4°C overnight. Either plasma recombinant phosphate-buffered saline 1% BSA added well room temperature hour. bound detected biotin-conjugated polyclonal antibody (Dako, Carpinteria, BSA. Both expression compared (WT) VWD. Multimer spectrum multimers, no decrease high molecular weight multimers domain mutants. Collagen studies our WT, 1786D, VWF. Results are ratio VWF:Ag. reduction both collagen, 45% 50% WT. contrast, mutant barely detectable <1% 1B). Similar lack seen 1745C constructs. The profound shown construct supported crystal structure, H1786 one interfaces [9]. Replacement alanine abolished [10]. S1731T Ribba colleagues [7]. recent Riddell details two mutations, W1745C S1783A, Three additional Q1734H, I1741T, Q1762R, associated decreased although affected did not display symptoms [11]. Both capable major thought [12]. assays demonstrate any evidence compensatory binding. It possible that vivo can adequately compensate when activated under flow conditions, may patients defect. Previous work H1786A supports hypothesis, it demonstrated occur [13]. severity phenotype partially alleviated presence some since multimeric would likely contain monomers intact site. The fits best into current classification 2M variant, function despite distribution. Not all however, F1369I I1425F while 11 deletion mutant, Δ1392–1492, appear affect [14]. These patients, presented VWF:RCo/VWF:Ag ratios, prompting their inclusion mutations. different effect than those considered classic 2M. Defects ultimately require alternate scheme distinguish them platelet-binding defects. suggests utility diagnosis variant