A novel nanobody that detects the gain-of-function phenotype of von Willebrand factor in ADAMTS13 deficiency and von Willebrand disease type 2B

Abstract

Von Willebrand factor (VWF) is unable to interact spontaneously with platelets because this interaction requires a conversion of the VWF A1 domain into a glycoprotein Ib␣ (GpIb␣) binding conformation. Here, we discuss a llama-derived antibody fragment (AU/VWFa-11) that specifically recognizes the GpIb␣-binding conformation. AU/VWFa-11 is unable to bind VWF in solution, but efficiently interacts with ristocetin-or botrocetin-activated VWF, VWF comprising type 2B mutation R1306Q, or immobilized VWF. These unique properties allowed us to use AU/ VWFa-11 for the detection of activated VWF in plasma of patients characterized by spontaneous VWF-platelet interactions: von Willebrand disease (VWD) type 2B and thrombotic thrombocytopenic purpura (TTP). For VWD type 2B, levels of activated VWF were increased 12-fold (P < .001) compared to levels in healthy volunteers. An inverse correlation between activated VWF levels and platelet count was observed (R 2 ‫؍‬ 0.74; P < .003). With regard to TTP, a 2-fold (P < .001) increase in activated VWF levels was found in plasma of patients with acquired TTP, whereas an 8-fold increase (P < .003) was found in congenital TTP. No overlap in levels of activated VWF could be detected between acquired and congenital TTP, suggesting that AU/VWFa-11 could be used to distinguish between both disorders. Furthermore, it could provide a tool to investigate the role of VWF in the development of thrombocytopenia in various diseases. (Blood. 2005;106: 3035-3042)

Platelet reactive conformation and multimeric pattern of von Willebrand factor in acquired thrombotic thrombocytopenic purpura during acute disease and remission

“…Densitometric analysis was performed on the blotted membrane using the graph line obtained by the single lane of the gel and plasma samples with similar VWF concentrations in order to obtain comparable area values. According to Budde and Scheneppenheim [11], the reference plasma lane was divided into small (1-5), intermediate (6)(7)(8)(9)(10) and larger (> 10) multimers of regular size. The proportion of larger multimers in the sample was calculated by dividing the area corresponding to larger multimers by the total area of the lane.…”

“…VWF-GPIb-a/BC measurement VWF-GPIb-a/BC was measured using an immunoadsorbent assay [7], based upon the nanobody AU/VWF-a11 that specifically recognizes the VWF-GPIb-a/BC that spontaneously binds to platelet glycoprotein Ib-a. Antibodies were provided by P. G. de Groot and P. J. Lenting.…”

“…a single domain antibody) with specificity towards the platelet glycoprotein Ib-a binding conformation of VWF (GPIb-a/BC) enables us to measure by immunoassay the proportion of plasma VWF that is highly reactive with this platelet glycoprotein [7]. In TTP, VWF-GPIb-a/BC (measured as the ratio of VWF in its platelet-binding conformation to the whole circulating mass of the protein) was 2-fold (acquired TTP) to 8-fold (congenital TTP) higher than normal in 17 patients with acute disease [7], but only 1.5-fold higher in 22 patients in clinical remission [8]. The higher amounts of VWFGPIb-a/BC suggest that a change in the conformation of circulating VWF is responsible for the heightened platelet reactivity that leads to disseminated microvascular thrombosis.…”

Active platelet‐binding conformation of plasma von Willebrand factor in young women with acute myocardial infarction

“…There is little information on the behaviour of the highly platelet-reactive forms of VWF in myocardial infarction (MI). Therefore, we chose to investigate the role of VWF in its platelet-binding conformation (VWF-GPIb:BC) in young women during the acute phase of MI, by using an immunoassay that distinguishes the plateletreactive from the latent conformations of VWF [4]. We also measured VWF antigen, VWF propeptide (an index of acute endothelial cell perturbation) and the VWF-cleaving protease ADAMTS13.…”

“…The plasma pool contained 40.3 nmol L )1 of VWF antigen and 6.3 nmol L )1 of VWF propeptide, and values in cases and controls were expressed in the same way. VWF-GpIb:BC was measured in Utrecht using the immunoassay based upon the AU/VWF-a11 lIama-derived nanobody that recognizes a VWF epitope, the accessibility of which coincides with the exposure of the GPIb binding site, even though both sites do not overlap [4]. The VWF-GpIb:BC of pooled standard plasma (see above) was arbitrarily given a ratio of 1.0.…”