PROTEOLYTIC CLEAVAGE OF RECOMBINANT TYPE 2A VON-WILLEBRAND-FACTOR MUTANTS R834W AND R834Q - INHIBITION BY DOXYCYCLINE AND BY MONOCLONAL-ANTIBODY VP-1

The susceptibility of recombinant type 2A von Willebrand factor (VWF) to a recently identified plasma metalloproteinase and the potential ap plication of proteolysis inhibition in the treatment of the disease we re investigated. Two recombinant type 2A vWF mutants, R834W and R834Q, were spontaneously cleaved by the partially purified plasma proteinas e to smaller forms. When treated with guanidine HCl, both the wild-typ e and the R834W mutant vWF exhibited a biphasic change in proteolytic susceptibility, reaching the same maximum cleavage at 1.25 mol/L guani dine HCl. Proteolysis of the recombinant vWF generated the same 350-kD and 200-kD species (dimers of the 176-kD and 140-kD fragments, respec tively) as those found in normal plasma. The proteinase activity was i nhibited by doxycycline, with an IC50 of approximately 0.25 mmol/L. Th e inhibitory activity of doxycycline was related to its metallic catio n binding activity, Susceptibility of the recombinant VWF to the prote inase was inhibited by monoclonal antibody VP-1 (directed against resi dues 828-842 of the VWF polypeptide), but not by two other monoclonal antibodies M13 and M31. The spontaneous susceptibility to proteolytic cleavage may account for the lack of large multimers in type 2A von Wi llebrand disease (VWD), and the results with tetracyclines and monoclo nal antibody VP-1 offer new strategies for developing specific treatme nt of type 2A vWD. (C) 1997 by The American Society of Hematology.