Figure 5.

N146Q mutant is less effective than WT ADAMTS13 in cleaving VWF multimers but not in cleaving FRETS-VWF73. (A) Kinetic analysis of VWF multimer cleavage by WT ADAMTS13 (concentration 0.87 nmol/L) or N146Q mutant (concentration 1.08 nmol/L). (B) Kinetic analysis of FRETS-VWF cleavage by WT ADAMTS13 (0.2 nmol/L) or N146Q mutant (0.18 nmol/L). The reaction velocity (WT:♦; N146Q: ◇), based on the increase of the dimeric 176-kDa fragment level on immunoblots against a reference curve constructed from serial dilutions of normal human plasma for panel A and on the increase of fluorescence intensity for panel B, was curve-fitted to Michaelis-Menten equation (solid and dashed lines) to obtain the equivalent values of K_m and k_cat. Because neither the reaction velocity nor the unit of VWF multimers was measured in molar concentrations, this analysis yielded kinetic value equivalents that were suitable only for intra-assay comparison. The difference between N146Q and WT ADAMTS13 against VWF multimers was significant (<.05) for K_m but not for k_cat. No significant difference was detected in either K_m or k_cat against FRETS-VWF73. Similar results were obtained in a repeated analysis.