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Collagen binding provides a sensitive screen for variant von Willebrand disease.

ABSTRACT: BACKGROUND:von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time-consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the utility of VWF:CB as a diagnostic test. METHODS:Participants were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. VWF:CB was analyzed with type III collagen and multimer distribution by agarose gel electrophoresis. The study population included 146 healthy controls, 351 individuals with type 1 VWD, and 77 with type 2 VWD. Differences between individuals with multimer group results within (controls) and outside the reference intervals were assessed with Mann-Whitney tests. RESULTS:The mean VWF:CB/VWF antigen ratio was 1.10 for individuals with multimer distribution within the reference intervals and 0.51 for those with multimer distribution outside the reference intervals (P < 0.001). Sensitivity of VWF:CB for multimer abnormalities was 100% for healthy controls, 99% for patients with type 1, and 100% for patients with type 2A and type 2B VWD using a VWF:CB/VWF antigen cutoff ratio of 0.6, and decreased to 99% for all patients with a ratio of 0.7. With the exception of individuals with novel or unclassified mutations, the VWF:CB was able to correctly categorize participants with variant VWD. CONCLUSIONS:These findings suggest that VWF:CB may substitute for multimer distribution in initial VWD testing, although further studies are needed to validate the clinical utility of VWF:CB.

SUBMITTER: Flood VH

PROVIDER: S-EPMC3852672 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Collagen binding provides a sensitive screen for variant von Willebrand disease.

Flood Veronica H VH Gill Joan Cox JC Friedman Kenneth D KD Christopherson Pamela A PA Jacobi Paula M PM Hoffmann Raymond G RG Montgomery Robert R RR Haberichter Sandra L SL

Clinical chemistry 20130122 4

<h4>Background</h4>von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time-consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the uti ...[more]

PMID: 23340442

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Project description:Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to type IV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its in vivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagens I and IV was measured. VWF-dependent platelet adhesion to collagen IV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagen IV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagen IV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagen IV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen IV binding.

Project description:Essentials von Willebrand factor (VWF) function is shear stress dependent. Platelet accumulation in a microfluidic assay correlates with VWF levels. The microfluidic assay discriminates type 1 von Willebrand disease from healthy controls. The microfluidic flow assay detects responses to therapeutic intervention (DDAVP).SUMMARYBackground von Willebrand disease (VWD) is a mucocutaneous bleeding disorder with a reported prevalence of 1 in 10 000. von Willebrand factor (VWF) function and platelet adhesion are regulated by hemodynamic forces that are not integrated into most current clinical assays. Objective We evaluated whether a custom microfluidic flow assay (MFA) can screen for deficiencies in VWF in patients presenting with mucocutaneous bleeding. Methods Whole blood from individuals with mucocutaneous bleeding was assayed in a custom MFA. Results Thirty-two patients with type 1 VWD (10/32) or reported mucocutaneous bleeding were enrolled. The platelet adhesion velocity (r = 0.5978 for 750 s-1 and 0.6895 for 1500 s-1 ) and the maximum platelet surface area coverage (r = 0.5719 for 750 s-1 and 0.6633 for 1500 s-1 ) in the MFA correlated with VWF levels. Furthermore, the platelet adhesion velocity at 750 s-1 (type 1 VWD, mean 0.0009761, 95% confidence interval [CI] 0.0003404-0.001612; control, mean 0.003587, 95% CI 0.002455-0.004719) and at 1500 s-1 (type 1 VWD, mean 0.0003585, 95% CI 0.00003914-0.0006778; control, mean 0.003132, 95% CI 0.001565-0.004699) differentiated type 1 VWD from controls. Maximum platelet surface area coverage at 750 s-1 (type 1 VWD, mean 0.1831, 95% CI 0.03816-0.3281; control, mean 0.6755, 95% CI 0.471-0.88) and at 1500 s-1 (type 1 VWD, mean 0.07873, 95% CI 0.01689-0.1406; control, mean 0.6432, 95% CI 0.3607-0.9257) also differentiated type 1 VWD from controls. We also observed an improvement in platelet accumulation after 1-desamino-8-d-arginine vasopressin (DDAVP) treatment at 1500 s-1 (pre-DDAVP, mean 0.4784, 95% CI 0.1777-0.7791; post-DDAVP, mean 0.8444, 95% CI 0.7162-0.9726). Conclusions These data suggest that this approach can be used as a screening tool for VWD.

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Collagen binding provides a sensitive screen for variant von Willebrand disease.

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Collagen binding provides a sensitive screen for variant von Willebrand disease.

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