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.

Figure 1. Collagen binding correlates with multimer distribution

This graph shows VWF:CB/VWF:Ag ratios on the y axis for individuals enrolled in the Zimmerman Program with either normal multimer distribution (n=511), abnormal multimer distribution (n=63), or a diagnosis of either type 2A or 2B VWD (n=53). VWF:CB/VWF:Ag was significantly different (P<0.001) for those with normal multimer distribution as compared to either those with abnormal multimer distribution or those with type 2A or 2B VWD. The box extends from the 25% to the 75% percentile and the line represents the median for each group. Whiskers represent the highest and lowest value.

Figure 2. Normal and abnormal multimer distributions seen in Zimmerman program samples

This figure shows characteristic multimer patterns observed in our study. These include type 2B VWD with loss of high molecular weight multimers, type 1 VWD with full spectrum of multimers, type 2A VWD, with loss of high and intermediate molecular weight multimers, type 3 VWD with no visible multimers, a sample with loss of the highest molecular weight multimers, and a normal plasma sample with all multimers present.

Figure 3. Densitometry analysis of multimer distribution

Densitometry was measured for a healthy control and four individuals, one each having type 1C, type 2A, type 2B, and loss of only the highest multimers. Tracings for the individuals with type 2 VWD clearly demonstrate lack of the high molecular weight multimers, whereas the participant only missing the highest molecular weight multimers appears closer to the healthy control.