A Novel Monoclonal Antibody Against A2 Domain of Von Willebrand Factor Reduces Its Proteolysis By ADAMTS13

Introduction:ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. A minimal region as a functional substrate for ADAMTS13 consisted of 73 amino acid residues from D1596 to R1668 of VWF, designated VWF73, and deletion of the E1660-R1668 region (the C-terminal α helix of VWF A2 domain) led to the loss of cleavage by ADAMTS-13. This suggested that the C-terminal α helix of VWF A2 domain contributes to ADAMTS13 binding to substrate. We will explore whether murine monoclonal antibody (mAb) against the C-terminal α helix of human VWF A2 domain affects the susceptibility of VWF to proteolysis by ADAMTS13 in vitro.

Methods:Balb/c mice were immunized with R1659-R1668 of the VWF polypeptide. Monoclonal antibodies (mAbs) were developed by standard hybridoma technology and identified with ELISA. The epitope of mAbs were mapped using recombinant VWF A2 and a series of its truncated mutants with western blot analysis. The effect of mAbs to VWF on its proteolysis by ADAMTS13 was investigated under static/denaturing condition by 1.3% agarose gel electrophoresis and immunologic analysis. The effect of mAbs to GST-VWF73-His on its proteolysis by recombinant ADAMTS13 was measured by 15% SDS-PAGE under reducing condition and western blot analysis.

Results: One mAb, designated SZ-179, was found to have high affinity (50 ng/ml) with both a synthetic peptide encompassing residues 1659-1668 and purified pVWF. SZ-179 has isotype of IgG1. It was shown that SZ-179 reacted specifically with recombinant VWF A2 and GST-VWF73-His, respectively, but not with recombinant VWF A1 nor with recombinant VWF A3. Epitope mapping experiments revealed that SZ-179 bound to the distal portion of the VWF A2 domain (E1660-L1666). SZ-179 inhibited proteolytic cleavage of GST-VWF73-His by recombinant ADAMTS13 in a concentration-dependent manner, with a maximal inhibition at concentrations of 50 ug/ml. SZ-179 also reduced degradation of high molecular-weight (HMW)-VWF-multimers under static/denaturing condition dose-dependently, with a maximal inhibition at concentrations of 1 ug/ml.

Conclusion:SZ-179, a novel mAb to the A2 Domain of VWF, inhibits the interactions between VWF and ADAMTS13, and prevents excessive degradation of HMW-VWF multimers. This mAb might be useful for the development of therapeutic options to treat bleeding episodes among hemorrhagic diseases.