Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor

Abstract

The diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of "VWF activity" by this assay and may not reflect a functional defect or true hemorrhagic risk.

Figure 1

Scatterplot of VWF:Ag (Ag), VWF:RCo (RCo), and VWF:RCo/VWF:Ag ratio (ratio) for all African American (AA; ●) and Caucasian controls (CA; ○) enrolled in the ZPMCB-VWD. The mean value for each assay is shown at the top of the graph for 59 African American and 113 Caucasian healthy controls. Error bars represent ± 1 SD.

Figure 2

Common exon 28 SNPs found in the VWF A1 loop. Circles represent the approximate location of the 3 common SNPs, I1380V, N1435S, and D1472H, which were seen most frequently in the African American controls; gray boxes, known regions implicated in VWF-ristocetin interactions.

Figure 3

VWF activity to antigen ratios by race and D1472H status. (A) VWF:RCo/VWF:Ag ratios for African American (AA) and Caucasian (CA) subjects with or without the D1472H polymorphism. (B) VWF:CB/VWF:Ag ratios for African American and Caucasian subjects with or without the D1472H polymorphism. The mean value for each group is listed at the top of the graph. Error bars represent ± 1 SD.

Figure 4

Botrocetin-induced binding of VWF to platelets. African American subjects homozygous for 1472H (●) are compared with African American subjects homozygous for 1472D (○). Error bars represent ± SEM. 1472H points have been moved slightly to the left to allow better visualization of the data points (n = 6 for each group).

Figure 5

VWF:RCo/VWF:Ag ratio in African American (AA) controls by D1472H allele status. VWF:RCo/VWF:Ag ratios are shown for African American subjects homozygous for the 1472H allele (1472 HH), heterozygotes (1472 DH), and those homozygous for the 1472D allele (1472DD). The mean value for each group is listed at the top of the graph. Error bars represent ± 1 SD.

Figure 6

Ristocetin-induced platelet aggregation. Various concentrations of ristocetin were added to platelet-rich plasma from a control subject (A) and a subject heterozygous for the D1472H polymorphism (B).

Figure 7

VWF:RCo assay compared with VWF GPIb complex-binding assay. The first 2 columns show VWF:RCo/VWF:Ag ratio (●) for subjects with and without the D1472H polymorphism. The second 2 columns show the GPIb complex-binding assay/VWF:Ag ratio (○) for subjects with and without the D1472H polymorphism. The mean value for each group is listed at the top of the graph. Error bars represent ± 1 SD.