Anderson Patricia J, Kokame Koichi, Sadler J Evan
Howard Hughes Medical Institute, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
J Biol Chem. 2006 Jan 13;281(2):850-7. doi: 10.1074/jbc.M504540200. Epub 2005 Nov 11.
ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp(1596)-Arg(1668) of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced approximately 2-fold by zinc ions, approximately 3-fold by calcium ions, and approximately 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to approximately 200-fold by zinc ions (K(D) (app) approximately 0.5 microM), calcium ions (K(D) (app) approximately 4.8 microM), and barium ions (K(D) (app) approximately 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a K(m) (app) of 3.7 +/- 1.4 microg/ml (approximately 15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5-10 microg/ml. ADAMTS13 could cleave approximately 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K(m) (app) of 3.2 +/- 1.1 microM, consistent with an approximately 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 microM(-1) min(-1) and 18 microM(-1) min(-1), respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.
ADAMTS13是一种可切割血管性血友病因子（VWF）多聚体的金属蛋白酶。利用多聚体VWF和基于VWF A2结构域Asp(1596)-Arg(1668)的荧光肽底物FRETS-VWF73检测了ADAMTS13活性对金属离子的依赖性。在枸橼酸盐抗凝血浆中，锌离子可使ADAMTS13活性增强约2倍，钙离子增强约3倍，两种离子共同作用时增强约6倍，提示存在协同激活作用。重组ADAMTS13对VWF的切割作用被锌离子（表观解离常数K(D)约为0.5 microM）、钙离子（表观解离常数K(D)约为4.8 microM）和钡离子（表观解离常数K(D)约为1.7 mM）激活达约200倍。钡离子可刺激枸橼酸盐血浆中的ADAMTS13活性，但对无枸橼酸盐血浆无此作用。因此，钡离子对枸橼酸盐血浆中ADAMTS13的刺激作用似乎反映了与枸橼酸盐结合的钙和锌离子的释放。在最佳锌和钙浓度下，ADAMTS13切割VWF的表观米氏常数K(m)为3.7±1.4 microg/ml（VWF亚基约为15 nM），这与血浆中5-10 microg/ml的VWF浓度相当。ADAMTS13可切割约14%经盐酸胍预处理的VWF，提示该底物在蛋白水解敏感性方面具有异质性。ADAMTS13切割FRETS-VWF73的表观米氏常数K(m)为3.2±1.1 microM，与对VWF的亲和力相比降低了约200倍。ADAMTS13切割VWF和FRETS-VWF73的催化效率大致相当，分别为55 microM(-1) min(-1)和18 microM(-1) min(-1)。ADAMTS13对VWF的显著偏好表明，底物识别取决于多聚体VWF上FRETS-VWF73所没有的结构特征或外部位点。
