Discovery of a Novel Calcium-binding Site of von Willebrand Factor A2 Domain Provides New Insights into the Regulatory Mechanism of A2 Cleavage by ADAMTS13

A team of researchers, led by DING Jianping at the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, CAS, revealed a novel Ca²⁺-binding site at the A2 domain of von Willebrand factor (VWF) and demonstrated interplay of Ca²⁺ and force in the regulation of VWF and primary hemostasis.

 

VWF plays an important role in primary hemostasis through mediating platelet adhesion. The proteolysis of VWF by ADAMST13 is an essential step for regulation of its hemostatic and thrombogenic potential. The cleavage occurs at strand β4 in the structural core of the A2 domain of VWF, thus unfolding of the A2 domain is pre-requisite for the cleavage. ZHOU Minyun, DONG Xianchi and colleagues performed the structural studies of an engineered A2 domain and found a metal ion at a site formed mainly by the C-terminal region of the α3-β4 loop, which was identified to be Ca²⁺ with subsequent biophysical and biochemical studies. Further force probe molecular dynamics simulations revealed that an increase of the force is needed to unfold strand β4 of the A2 domain when Ca²⁺ is bound. Consistently, cleavage assays demonstrated that the Ca²⁺ binding stabilizes the A2 domain and impedes its unfolding, and consequently protects it from cleavage by ADAMTS13. These findings provide new insights into the regulation of VWF cleavage by ADAMTS13 in hemostasis and thrombosis. This work entitled "A novel calcium-binding site of von Willebrand factor A2 domain regulates its cleavage by ADAMTS13" was published in Blood on March 8^th, 2011.

 

This study was conducted in collaboration with the research groups of Professors Frauke Gräter (CAS-MPG Partner Institute for Computational Biology), Timothy Springer (Harvard University Medical School), and LUO Xinping (Fudan University Huashan Hospital), and was supported by grants from the Ministry of Science and Technology of China, the National Natural Science Foundation of China, and the Chinese Academy of Sciences. (SIBCB)

 

AUTHOR CONTACT:

DING Jianping

Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Phone: 86.21.54921619; E-mail: jpding@sibs.ac.cn

 

The protective role of Ca²⁺ binding on the A2 cleavage by ADAMTS13. (A) Overall structure of the engineered VWF A2 domain. The Ca²⁺ bound at α3-β4 loop is shown in gold. (B) Examination of the protective effect of Ca²⁺ on the proteolysis of the wild-type A2 protein with two-step ADAMST13 cleavage assays. (C) Examination of the effect of Ca²⁺ on the proteolysis of the GST-VWF73 protein (Asp¹⁵⁹⁶ to Arg¹⁶⁶⁸ of the A2 domain, the minimal substrate of ADAMTS13) by ADAMTS13. (D) Examination of the effect of Ca²⁺ on the proteolysis of the wild-type A2 protein by ADAMTS13 without adjustment of the calcium concentration in step II. (E) Examination of the effect of Ca²⁺ ion on the proteolysis of the wild-type A2 protein by ADAMTS13 with one-step assays. (Image provided by DING Jianping’s group)