Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

The activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified.

To develop an assay that can differentiate the platelet-binding activity of VWF multimers.

A novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers.

Of the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab_6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab_6C11 to that of HMWMs, which is significantly higher than all other activity assays tested.

The VWF:GPIbNab_6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.