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 wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight 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 isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.
Subject(s)
Collagen Type II/metabolism , Collagen Type I/metabolism , Hemorrhage/metabolism , Mutation, Missense , Protein Multimerization/genetics , von Willebrand Diseases/metabolism , von Willebrand Factor/metabolism , Amino Acid Substitution , Binding Sites/genetics , Cell Line , Collagen Type I/genetics , Collagen Type II/genetics , Family , Female , Gene Expression , Hemorrhage/genetics , Humans , Male , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Recombinant Proteins/economics , Recombinant Proteins/metabolism , von Willebrand Diseases/classification , von Willebrand Diseases/genetics , von Willebrand Factor/geneticsABSTRACT
Reduced plasma survival of von Willebrand factor (VWF) may contribute towards the pathogenesis of type 1 von Willebrand disease (VWD). However, little is known about mechanism(s) of VWF clearance and factors that may affect it. The half-life of VWF-related parameters following the administration of DDAVP was measured in 26 patients with type 1 VWD and 10 haemophilia A controls. Binding of lectins Ricinus communis (RCA-I) and Erythina crystagalli (ECA) agglutinins to VWF and VWF susceptibility to ADAMTS-13-mediated proteolysis were investigated. Sequence analysis of targeted regions of the VWF gene was performed to inspect for mutations that have been associated with increased clearance. Post-DDAVP clearance of VWF was increased approximately three-fold in the type 1 VWD cohort overall. However this was not shown to consistently associate with steady-state VWF antigen (VWF:Ag) levels. Furthermore, increased VWF clearance was not consistently associated with increased ratios of VWF propeptide (VWFpp) to VWF:Ag indicating that a normal ratio does not necessarily reflect normal post-DDAVP survival in type 1 VWD patients. RCA-I and ECA binding to VWF were increased in type 1 VWD patients and, although inversely correlated with VWF levels, this was independent of VWF clearance. There was no association between VWF clearance and ADAMTS-13-mediated proteolysis. Three novel candidate mutations with an increased clearance phenotype were identified. The data are consistent with heterogeneity in pathogenic mechanisms in type 1 VWD and are consistent with type 1 VWD representing a complex genetic trait.
Subject(s)
ADAM Proteins/blood , Deamino Arginine Vasopressin/administration & dosage , Hemostatics/administration & dosage , Mutation , Protein Processing, Post-Translational/drug effects , von Willebrand Diseases/drug therapy , von Willebrand Factor/metabolism , ADAMTS13 Protein , Cohort Studies , DNA Mutational Analysis , Female , Genetic Predisposition to Disease , Genotype , Glycosylation , Half-Life , Humans , Infusions, Intravenous , Male , Phenotype , Plant Lectins/metabolism , Protein Binding , Protein Precursors/blood , Treatment Outcome , von Willebrand Diseases/blood , von Willebrand Diseases/genetics , von Willebrand Factor/geneticsABSTRACT
To investigate the effects of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and danaparoid (DPD) added to whole blood in vitro on standard and heparinase-modified thromboelastogram (TEG) parameters compared with conventional assays of coagulation. The effects of UFH, LMWH and DPD on standard TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-activated factor X (anti-FXa) activity, at concentrations of these anticoagulants ranging from 0.025 to 1 U/ml. In the second part of the study, the effects of very low concentrations (0.005-0.05 U/ml) of UFH, LMWH and DPD on the difference between standard and heparinase-modified TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-FXa activity. Standard TEG parameters were outside the reference range at lower concentrations of UFH, LMWH and DPD than most conventional coagulation assays were able to detect. Only anti-FXa activity was more sensitive to the presence of these anticoagulants than the standard TEG alone. The lowest concentration of UFH, LMWH and DPD used in this study (0.005 U/ml) caused significant differences between the standard and heparinase-modified alpha-angles of the TEG. In addition, the difference between standard and heparinase-modified TEG parameters distinguished between low concentrations (0.005-0.05 U/ml) of UFH with greater sensitivity than anti-FXa activity, but were less sensitive to LMWH and DPD. The standard TEG is more sensitive to UFH, LMWH and DPD than most conventional coagulation tests, with the exception of anti-FXa activity. Calculation of the difference between standard and heparinase-modified TEG parameters greatly increases the sensitivity of the assay for the effects of these anticoagulants, and is more sensitive to very low quantities of UFH than anti-FXa activity.
Subject(s)
Chondroitin Sulfates/chemistry , Dermatan Sulfate/chemistry , Heparin Lyase/chemistry , Heparin, Low-Molecular-Weight/chemistry , Heparitin Sulfate/chemistry , Thrombelastography , Adult , Factor Xa/analysis , Humans , Male , Partial Thromboplastin Time/methods , Reference Standards , Sensitivity and Specificity , Thrombelastography/methodsABSTRACT
This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen-binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (< or = 0.7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (> 0.7), but the VWF:RCo/VWF:CBA ratios were discordant (< or = 0.7) (P = 0.001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high-molecular-weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet-binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.
