Quantitative ELISA Using N10 Antibody, Targeting on VWF-Y1605 Residue, Indicates That N-Linked Blood Group Type A Carbohydrate on VWF Molecule Functions Against ADAMTS13 Cleavage
Authors: Hayakawa Masaki , Kato Seiji , Matsui Taei , Sakai Kazuya , Yoshii Yumi , Yagi Hideo , Fujimura Yoshihiro , Matsumoto Masanori 
Published: October 2019

Abstract
Background
ADAMTS13 specifically cleaves the peptide bond between Y1605 and M1606 within the von Willebrand factor (VWF)-A2 domain, and down-regulates VWF function, involved in platelet thrombi formation in denuded vascular endothelial cells in vivo.
A 250 kD-mature VWF subunit, consisting of 2050 amino acid residues, contains 13 N-linked carbohydrates within the molecule, on which ABO blood group sugars with the respective blood phenotypes localize (Matsui et al, Blood 1993).
In 2002, Bowen (JTH) showed that the loss of VWF function was greater for VWF of group O compared with non-O VWF, in the rank order O≥B>A>AB, by means of VWF multimer analysis. Recently, Rauch et al (Thromb Haemost 2016) reported the increased VWF proteolysis in type O than in non-type O individuals, by means of a novel ELISA using anti-VWF-A2 monoclonal antibody (mAb), but without addressing its epitope.
Since we succeeded to have a unique mAb N10, recognizing the Y1605 residue exposed by ADAMTS13 cleavage (Kato et al, Transfusion 2006), we here describe a quantitative analysis of VWF proteolysis by ADAMTS13 using a sandwich ELISA with N10, and reveal that the degree of VWF proteolysis in normal individuals occurs in this rank order O≥B>A>AB, clearly indicating that blood group type A carbohydrate on VWF functions against ADAMTS13 cleavage
Materials and Methods
Citrate plasmas obtained from 177 normal healthy volunteers (blood group O, n=55; A, n=63; B, n=32; AB, n=27) were analyzed, and the plasma levels of VWF antigen (VWF:Ag) were measured by a sandwich ELISA using anti-VWF polyclonal Abs.
The quantitative ELISA to estimate the amount of plasma VWF-degradation products (VWF-DP), generated by ADAMTS13, was performed as follows: First, a 96-well microplate was coated with the primary mAb N10 (IgG), binding to the residue VWF-Y1605 after exposed, and was then blocked with casein. Second, the diluted plasma samples were applied to the coated wells, and after following incubation and extensive washing the plates, VWF-DP captured to the plates was detected by HRP-labeled anti-VWF mAb, which binds to N-terminal residue 1-1315 of VWF subunit, generated by V8 protease digestion (Titani et al. Biochemistry 1986).
In both the assays above, a value of 100% was defined as the amounts in pooled plasma from normal healthy volunteers aged 20-40 years. The values were expressed the percentage of control by mean (minimum-maximum). This study was conducted with the approval of the ethics committee of Nara Medical University.
Results and Discussion
Plasma levels of VWF:Ag in normal subjects were significantly lower in blood group O [65.8% (34.6-142.0)] than A [101.2%(49.0-178.6)], B [102.6% (60.3-177.0)], and AB [99.7% (43.9-159.0)] (p<0.01), as reported in previous publications.
Plasma levels of VWF-DP in normal subjects of blood group O [43.2% (17.2-137.0)] were also significantly lower than those in groups B [63.6% (30.9-319.8)] (p<0.01), and AB [41.7% (17.2-333.0)] (p<0.05). Notably, however, a significant relationship between the plasma levels of VWF:Ag and VWF-DP were observed in all normal subjects (r=0.58, p<0.01). Thus, we analyzed a ratio between VWF-DP and VWF:Ag (VWF-DP/VWF:Ag ratio), in which blood group O [0.61(0.33-1.89)] was significantly higher than those of blood group A [0.54 (0.18-1.26)] (p<0.01) and of blood group AB [0.45 (0.27-2.13)] (p<0.05). Interestingly, the ratio of group B [0.63 (0.36-2.05)] did not show any significant difference with those of Group O. Thus, our novel ELISA clearly indicated that the amount of VWF-DP in normal plasmas is increased in the rank order O≥B>A>AB.
It is well conceived that the ABO-bearing carbohydrate moieties on VWF molecule affect on the susceptibility to ADAMTS13 cleavage, and blood group O-VWF is more cleavable than non-O-VWF. In addition, here we have shown that B-VWF is more susceptible to ADAMTS13 cleavage than A-VWF. Discrimination of blood group type A or B is generated by the presence of one terminal sugar, group A with α-1,3-linked N- acetylgalactosamine (GalNAc) and group B with α-1,3-linked galactose. Thus, although the precise mechanism is yet unknown, our results may indicate that GalNAc plays a key role to create the resistance to ADAMTS13 cleavage via steric hindrance or charge effect.

Disclosures
No relevant conflicts of interest to declare.