EPITOPE MAPPING BY CDNA EXPRESSION OF A MONOCLONAL-ANTIBODY WHICH INHIBITS THE BINDING OF VONWILLEBRAND-FACTOR TO PLATELET GLYCOPROTEIN-IIB/IIIA

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-20501, which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIlb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda-gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library With I-125-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with I-125-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to I-125-MAb 9 by 50 %, and this inhibition was dose-dependent between 3.5 and 120 nm. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.