Publications (94) View all
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Article: In vivo von Willebrand factor size heterogeneity in spite of the clinical deficiency of ADAMTS-13.
Journal of Thrombosis and Haemostasis 09/2011; 9(12):2506-8. · 5.73 Impact Factor -
Article: The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo.
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ABSTRACT: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13(-/-) mouse. Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.Journal of Thrombosis and Haemostasis 10/2010; 8(10):2305-12. · 5.73 Impact Factor -
Article: Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura.
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ABSTRACT: ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross-link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti-ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute-phase but not remission-phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute-phase but not remission-phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose-dependently inhibited by addition of exogenous α₂ -antiplasmin. Examination of the endogenous α₂-antiplasmin antigen and activity confirmed deficiency of α₂ -antiplasmin function in acute-phase but not remission-phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase-type plasminogen activator was added to an (unrelated) congenital α₂ -antiplasmin-deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.Journal of Thrombosis and Haemostasis 09/2010; 8(9):2053-62. · 5.73 Impact Factor -
Article: Active platelet-binding conformation of plasma von Willebrand factor in young women with acute myocardial infarction.
Journal of Thrombosis and Haemostasis 04/2010; 8(7):1653-6. · 5.73 Impact Factor -
Article: Multi‐step binding of ADAMTS‐13 to von Willebrand factor
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ABSTRACT: 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.Journal of Thrombosis and Haemostasis 11/2009; 7(12):2088 - 2095. · 5.73 Impact Factor
