Multi‐step binding of ADAMTS‐13 to von Willebrand factor
| Autor: | Patricia J. Anderson, J E Sadler, Hendrik B. Feys, Karen Vanhoorelbeke, Elaine M. Majerus |
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| Rok vydání: | 2009 |
| Předmět: |
Protein Denaturation
congenital hereditary and neonatal diseases and abnormalities Immunoprecipitation Von Willebrand factor type A domain Blotting Western ADAMTS13 Protein Plasma protein binding Article Von Willebrand factor hemic and lymphatic diseases von Willebrand Factor Humans Binding site Hemostasis Binding Sites biology Chemistry ADAMTS Active site Thrombosis Hematology Molecular biology ADAM Proteins Glycoprotein Ib Multiprotein Complexes Blood Circulation cardiovascular system biology.protein Stress Mechanical Protein Binding circulatory and respiratory physiology |
| Zdroj: | Journal of Thrombosis and Haemostasis. 7:2088-2095 |
| ISSN: | 1538-7836 |
| DOI: | 10.1111/j.1538-7836.2009.03620.x |
| Popis: | Summary. Background: ADAMTS-13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS-13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS-13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS-13. Methods: Immunoprecipiation of VWF–ADAMTS-13 complexes using anti-VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Results: Co-immunoprecipitation demonstrated that ADAMTS-13 binds to native multimeric VWF (Kd of 79 ± 11 nmol L−1) with no measurable proteolysis. Upon shear-induced unfolding of VWF, binding increased 3-fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50–200). Moreover, results with ADAMTS-13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS-13 spacer, probably thrombospondin-1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS-13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). Conclusions: ADAMTS-13 binds to circulating VWF and may therefore be incorporated into a platelet-rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress. |
| Databáze: | OpenAIRE |
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