A novel monoclonal antibody against the von Willebrand Factor A2 domain reduces its cleavage by ADAMTS13
© The Author(s). 2017
Received: 30 December 2016
Accepted: 25 January 2017
Published: 6 February 2017
We developed a novel murine monoclonal antibody (mAb) against the C-terminal α-helix of the human von Willebrand factor A2, designated SZ-179. We showed that SZ-179 inhibited the interactions between VWF and ADAMTS13 and prevented the degradation of high molecular weight VWF multimers. Importantly, SZ-179 reduced the proteolysis of VWF-R1597W mutant by rADAMTS13 dose-dependently under native conditions. Our findings reveal a potential therapeutic target for bleeding disorders.
Keywordsvon Willebrand factor Monoclonal antibody ADAMTS13
ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain (VWFA2) . This remarkable cleavage specificity depends largely on the binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal α-helix of VWFA2 . A 73 amino acid residue from D1596 to R1668 in VWF A2 domain, designated VWF73, serves as a minimal substrate for ADAMTS13 . In concert, deletion of the VWFA2 C-terminal α-helix (E1660-R1668) from this minimal substrate leads to nearly complete loss of cleavage by ADAMTS13, indicating that this structure is essential to the binding and cleavage of VWF by ADAMTS13 [4, 5].
Moreover, we found that pre-incubation of plasma with SZ-179 rather than with the isotype control resulted in a dose-dependent decrease in the proteolysis of high molecular weight (HMW) VWF multimers under static/denaturing conditions, with an IC50 of 0.66 μg/ml (Additional file 3: Figure S2). These findings suggested that SZ-179 can bind to native VWF and provided further evidence that SZ-179 may attenuate the susceptibility of VWF to proteolytic cleavage by ADAMTS13 under physiological conditions.
We next determined whether SZ-179 could inhibit rADAMTS13-mediated proteolysis of the VWF-R1597W mutant, which can be cleaved by ADAMTS13 under static conditions and in the absence of denaturants including urea and guanidine [6, 7]. The R1597W mutation is commonly associated with von Willebrand disease (VWD) type 2A and located within VWFA2, close to the ADAMTS13 cleavage site. We found that the proteolysis of HMW VWF-R1597W multimers by rADAMTS13 was dramatically reduced by SZ-179 rather than by IgG1 isotype control in a concentration-dependent manner under native conditions (Fig. 2c, d). The IC50 of SZ-179 for this reaction was 13.54 μg/ml (Fig. 2e). Nevertheless, wild-type VWF treated with rADAMTS13 remained intact, as expected in the absence of chemical denaturation or fluid shear stress (Fig. 2f). These findings suggest that SZ-179 inhibits the rADAMTS13-mediated proteolysis of VWF-R1597W multimers under native conditions.
Mechanistically, SZ-179 may interact with E1660-L1666 residues in the VWF, blocking the binding of the spacer domain of ADAMTS13 to the substrate, thereby inhibiting proteolysis of VWF by ADAMTS13. Several recent reports support this possibility. For example, human neutrophil peptides inhibit ADAMTS13-dependent VWF proteolysis by binding to the central A2 domain of VWF to block interactions between ADAMTS13 and VWF . Antibody mAb508 is specific to the D4 domain of VWF, and has been observed to interfere with ADAMTS13-mediated degradation of VWF in a vortex-based degradation assay . mAb508 is bound to VWF with moderate affinity, and its binding to VWF partially inhibits the interaction between VWF and ADAMTS13. We discovered that SZ-179 has high affinity (50 ng/ml) with native VWF and prevents excessive degradation of HMW-VWF-multimers under denaturing conditions dose-dependently. SZ-179 may provide a promising therapeutic approach for a subset of VWD patients.
A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13
Enzyme-linked immunosorbent assay
High molecular weight
Half maximal inhibitory concentration
Murine monoclonal antibody
von Willebrand disease
von Willebrand factor
We acknowledge Blood Transfusion Hybridoma Laboratory Center for the gift of SP2/0-Ag14 myeloma cell line. Furthermore, we thank Dr. J. Evan Sadler for providing the pSVHVWF1 vector and Dr. Jingfei Dong for providing the pSecTag-ADAMTS13 vector.
This work was supported by Jiangsu Provincial Special Program of Medical Science (BL2012005), Jiangsu Province’s Key Medical Center, National Natural Science Foundation of China (81600105) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article.
LZ and CR were the principal investigators and took primary responsibility for the paper. LZ, JS, FS, and ZM performed the experiments. YZ contributed the research material. LZ and LX wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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