Factor VIII, a Cofactor for Accelerating Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease.

von Willebrand factor (VWF)-cleaving protease (ADAMTS13), a 195-kDa plasma glycoprotein, preferentially cleaves “unusually large” VWF multimers and generates a range of VWF multimers with sizes from approximately 500,000 Daltons to 20 million Daltons. However, the molecular basis how ADAMTS13 prefers the larger VWF multimers to the smaller ones under physiological condition remains unknown. We hypothesize that coagulation factor VIII, which binds VWF in high affinity, may alter the susceptibility of VWF to ADAMTS13 protease. In the present study, a plasma-derived or recombinant VWF at final concentration of 150 nM was incubated for 3 min with 50 nM of recombinant ADAMTS13 in the absence and the presence of various concentrations of recombinant factor VIII (0–40 nM) in 50 mM HEPES, pH 7.5 containing 150 mM NaCl and 0.5 mg/ml bovine serum albumin under constant vortex-induced mechanic shear stress at rotation rate of 2,500 rpm. The specific cleavage products (the dimer of 176 kDa) were determined by Western blotting with rabbit anti-VWF IgG, followed by a fluorescence (IRDye800)-labeled anti-rabbit IgG and an infrared fluorescent image system. We showed that the rate of proteolytic cleavage of VWF by ADAMTS13 was markedly accelerated by an addition of recombinant factor VIII into the reaction. This augmenting effect was factor VIII concentration-dependent. The maximal augmentation in proteolytic cleavage of VWF by ADAMTS13 in the presence of 20 nM of recombinant factor VIII was approximately 10 fold over the baseline in the absence of factor VIII. The concentration of factor VIII that achieved half of the maximal effect on proteolytic cleavage of both plasma and recombinant VWF was nearly identical (approximately 3.0 nM). The B domain-deleted factor VIII appeared as efficacious as wild type factor VIII in accelerating the proteolytic cleavage of plasma-derived VWF by ADAMTS13, suggesting that the large, central B-domain of factor VIII is not required for its cofactor activity. Moreover, after being activated by thrombin, the activated factor VIII retained its cofactor activity initially, but rapidly lost its ability to enhance the proteolysis of VWF by ADAMTS13 within minutes, which was correlated with the dissociation (or inactivation) kinetics of the activated factor VIII heterotrimer. These data demonstrate that both factor VIII and activated factor VIII may be the cofactors that regulate ADAMTS13 protease function under physiological condition. The findings may provide a novel insight into how the larger VWF multimers are more susceptible to ADAMTS13 protease, likely because the larger VWF multimers are able to bind more factor VIII. The findings may also help design an optimal therapeutic regimen for treatment of thrombotic thrombocytopenic purpura.