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Proteolytic cleavage of recombinant type 2A von Willebrand factor mutants
R834W and R834Q: inhibition by doxycycline and by monoclonal antibody VP-1
HM Tsai, II Sussman, D Ginsburg, H Lankhof, JJ Sixma and RL Nagel
Division of Hematology, Montefiore Medical Center/Albert Einstein College
of Medicine, Bronx, NY 10467, USA.
The susceptibility of recombinant type 2A von Willebrand factor (vWF) to a
recently identified plasma metalloproteinase and the potential application
of proteolysis inhibition in the treatment of the disease were
investigated. Two recombinant type 2A vWF mutants, R834W and R834Q, were
spontaneously cleaved by the partially purified plasma proteinase to
smaller forms. When treated with guanidine HCI, both the wild-type and the
R834W mutant vWF exhibited a biphasic change in proteolytic susceptibility,
reaching the same maximum cleavage at 1.25 mol/L guanidine HCI. Proteolysis
of the recombinant vWF generated the same 350-kD and 200-kD species (dimers
of the 176-kD and 140-kD fragments, respectively) as those found in normal
plasma. The proteinase activity was inhibited by doxycycline, with an IC50
of approximately 0.25 mmol/L. The inhibitory activity of doxycycline was
related to its metallic cation binding activity. Susceptibility of the
recombinant vWF to the proteinase was inhibited by monoclonal antibody VP-1
(directed against residues 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 Willebrand disease (vWD), and the results with tetracyclines and
monoclonal antibody VP-1 offer new strategies for developing specific
treatment of type 2A vWD.
Volume 89,
Issue 6,
pp. 1954-1962,
03/15/1997
Copyright © 1997 by The American Society of Hematology
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