Variations in glycosylation of von Willebrand factor with O-linked sialylated T antigen are associated with its plasma levels by Carina J. M. van Schooten, Cécile V. Denis, Ton Lisman, Jeroen C. J. Eikenboom, Frank W. Leebeek, Jenny Goudemand, Edith Fressinaud, H. Marijke van den Berg, Philip G. de Groot, and Peter J. Lenting Blood Volume 109(6):2430-2437 March 15, 2007 ©2007 by American Society of Hematology

Specificity of PNA binding to VWF Specificity of PNA binding to VWF. (A) Microtiter wells coated with polyclonal anti-VWF antibodies (3 μg/mL) were incubated with plasma that was diluted in PBS to reach a VWF antigen concentration of 50 mU/mL. Specificity of PNA binding to VWF. (A) Microtiter wells coated with polyclonal anti-VWF antibodies (3 μg/mL) were incubated with plasma that was diluted in PBS to reach a VWF antigen concentration of 50 mU/mL. After catching VWF, wells were incubated with neuraminidase (5 mU/mL) in PBS/1 mM CaCl2 overnight at 37°C. Subsequently, wells were incubated for 1 hour at 37°C with btPNA (5 μg/mL) in the presence or absence of various carbohydrate structures (0-1 mM): □ indicate glucose; ○, galactose; ▵, GalNAc; ⋄, GlcNAc; and •, Gal-(β1-3)-GalNAc. After washing, HRP-conjugated streptavidin was added to the wells, and bound btPNA was detected by measuring HRP activity using OPD as a substrate. Plotted is residual PNA binding versus sugar concentration. One-hundred percent refers to PNA binding in the absence of competitors. (B) Microtiter wells coated with polyclonal anti-VWF antibodies (3 μg/mL) were incubated with equimolar concentrations of pd-VWF, wt-VWF, VWF/delta-pro, or VWF/D′-D3 (7.5 nM based on monomer concentration). Where indicated, pd-VWF was incubated with recombinant O-glycosidase (12.5 mU/mL) for 3 hours at 37°C following neuraminidase incubation. PNA binding was determined as described for panel A. Plotted is relative PNA binding using wt-VWF as a reference (100%). (C) Microtiter wells coated with a recombinant anti-VWF A1 antibody were incubated with equimolar concentrations of A1/1260-1481 or A1/1238-1494 (7.5 nM). PNA binding was determined as described for panel A. Plotted is relative PNA binding using A1/1238-1494 as a reference (100%). Carina J. M. van Schooten et al. Blood 2007;109:2430-2437 ©2007 by American Society of Hematology

Detection of T antigen on VWF in plasma. Detection of T antigen on VWF in plasma. (A) Serial dilutions of plasma were added to microtiter wells coated with anti-VWF antibodies, and PNA binding was determined as described in the legend of Figure 1. Plotted is absorbance at 490 nm versus concentration of VWF in diluted samples for NPP (X) and a representative sample (▪). The slope found for NPP was set at 100%. The slope for this particular sample was calculated to be 177%. Relative PNA binding was determined for plasma samples from healthy volunteers (n = 111; B) and samples that were randomly provided by our diagnostic laboratory (n = 66; C). (C) The dotted lines indicate VWF values of 0.5 and 1.5 U/mL. The drawn solid lines were obtained by fitting the data in a model for linear regression (Pearson rank = −0.43; B) or a model describing a single exponential decay (r2 = 0.57; C). (D) Data shown in panel C were subdivided into 3 groups based on VWF antigen levels that were less than 0.5 U/mL (n = 15), between 0.5 and 1.5 U/mL (n = 32), and higher than 1.5 U/mL (n = 19). Data represent the mean of 2 independent determinations, and variance between measurements was 16% or less. Differences between groups were tested with the unpaired 2-tailed t test, with the Welch correction applied when necessary. Bars indicate mean values. Carina J. M. van Schooten et al. Blood 2007;109:2430-2437 ©2007 by American Society of Hematology

Liver cirrhosis is associated with reduced PNA binding. Liver cirrhosis is associated with reduced PNA binding. (A) PNA binding to VWF was determined in plasma samples of 54 patients diagnosed with liver cirrhosis (“Patients, materials, and methods”). Plotted is relative PNA binding versus VWF antigen levels. The drawn line was obtained by fitting the data in a model describing a single exponential decay (r2 = 0.39). (B) Data shown in panel A were subdivided according to Pugh's18 modification of the Child classification of patients. Data represent the mean of 2 independent determinations, and variance between measurements was 16% or less. Differences between groups were tested with the unpaired 2-tailed t test, with the Welch correction applied when necessary. Bars represent mean values. Carina J. M. van Schooten et al. Blood 2007;109:2430-2437 ©2007 by American Society of Hematology

VWD type 1 is associated with increased PNA binding. VWD type 1 is associated with increased PNA binding. VWF antigen levels (A) and PNA binding (B) were determined in plasma samples of healthy volunteers (n = 111), unaffected family members (n = 15), and patients diagnosed with VWD type 1 (n = 32). Data represent the mean of 2 independent determinations, and variance between measurements was 12% or less (VWF antigen levels) and 16% or less (PNA binding). (C) PNA binding versus VWF antigen levels is depicted: Depicted are healthy volunteers (□), unaffected family members (▴), and VWD type 1 patients (○). Bars represent mean values. Carina J. M. van Schooten et al. Blood 2007;109:2430-2437 ©2007 by American Society of Hematology

PNA binding correlates with propeptide/VWF ratios. PNA binding correlates with propeptide/VWF ratios. (A) VWF propeptide and mature VWF antigen levels were determined for healthy volunteers (n = 24) and VWD type 1 patients (n = 32) depicted in Figure .4, and the molar ratio propeptide over VWF was calculated as described.26 Difference between both groups was tested using the unpaired 2-tailed t test with the Welch correction applied. (B) Values for relative PNA binding obtained for VWD type 1 patients were plotted versus the corresponding propeptide/VWF ratios. The drawn line was obtained after linear regression analysis. Correlation was found to be significant (Spearman rank = 0.50; P = .004). Bars represent mean values. Carina J. M. van Schooten et al. Blood 2007;109:2430-2437 ©2007 by American Society of Hematology

Reduced PNA binding to post-DDAVP VWF Reduced PNA binding to post-DDAVP VWF. For a selection (n = 11) of the VWD type 1 patients (Figure 4) as well as VWD type 2A (n = 2), VWD type 2N (n = 1), hemophilia A (n = 1), and storage-pool disease (n = 3) patients, VWF antigen levels (A) and PNA bindin... Reduced PNA binding to post-DDAVP VWF. For a selection (n = 11) of the VWD type 1 patients (Figure 4) as well as VWD type 2A (n = 2), VWD type 2N (n = 1), hemophilia A (n = 1), and storage-pool disease (n = 3) patients, VWF antigen levels (A) and PNA binding (B) were determined in plasma samples that were taken prior or 60 minutes after DDAVP treatment. Mean VWF levels were 0.62 ± 0.46 U/mL and 1.55 ± 0.74 U/mL before and after DDAVP, respectively (P = .001). Mean PNA binding was 131% ± 45% and 85% ± 33% (P = .001). For 2 patients, changes in VWF antigen levels (C-D: right axes) and PNA binding (C-D: left axes) were followed in time. Carina J. M. van Schooten et al. Blood 2007;109:2430-2437 ©2007 by American Society of Hematology