期刊
BLOOD
卷 123, 期 16, 页码 2585-2592出版社
AMER SOC HEMATOLOGY
DOI: 10.1182/blood-2013-11-538173
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资金
- British Heart Foundation Basic Science Research Fellowship [FS/11/51/28920]
- National Institute for Health Research, Biomedical Research Centre Funding Scheme
- British Heart Foundation [FS/11/51/28920, FS/14/21/30733] Funding Source: researchfish
Rheological shear forces in the blood trigger von Willebrand factor (VWF) unfolding which exposes the Y1605-M1606 scissile bond within the VWF A2 domain for cleavage by ADAMTS13. The VWF A2 domain contains 2 structural features that provide it with stability: a vicinal disulphide bond and a Ca2+-binding site (CBS). We investigated how these 2 structural features interplay to determine stability and regulate the exposure of the scissile bond in full-length VWF. We have used differential scanning fluorimetry together with site-directed mutagenesis of residues involved in both the vicinal disulphide bond and the CBS to demonstrate that both of these sites contribute to stability against thermal unfolding of the isolated VWF A2 domain. Moreover, we show that the combination of site mutations can result in increased susceptibility of FL-VWF to proteolysis by ADAMTS13, even in the absence of an agent (such as urea) required to induce unfolding. These studies demonstrate that VWF A2 domain stability provided by its 2 structural elements (vicinal disulphide bond and CBS) is a key protective determinant against FL-VWF cleavage by ADAMTS13. They suggest a 2-step mechanism for VWF A2 domain unfolding.
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