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 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.
Tsai
H M
HM
Division of Hematology, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY 10467, USA.
Sussman
I I
II
Ginsburg
D
D
Lankhof
H
H
Sixma
J J
JJ
Nagel
R L
RL
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001120:Arginine / Q000235:genetics; D002413:Cations, Divalent; D003720:Densitometry; D004318:Doxycycline / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000235:genetics; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D005973:Glutamine; D014364:Tryptophan; D001120:Arginine; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D004318:Doxycycline