Proteolysis of ADAMTS-13 by thrombin does not influence the cleavage of, or binding to, VWF115

(A) 5 n ADAMTS-13 or thrombin-cleaved ADAMTS-13 (ADAMTS-13/FIIa) was incubated at 37 °C with 5 μ VWF115, 150 m NaCl, 5 m CaCl, 20 m Tris (pH7.8). Subsamples were stopped with EDTA (0–2 h) and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and Coomassie staining. Proteolysis of VWF115 was visualized by the disappearance of a 16.9 kDa band and the appearance of 10 kDa and 6.9 kDa cleavage products. (B) Kinetic analysis of VWF115 proteolysis, as in (A) except using 700 n VWF115. Proteolysis of VWF115 was quantified by high-performance liquid chromatography, and represented graphically. The catalytic efficiency for VWF115 proteolysis was derived from fitted data for both ADAMTS-13 and ADAMTS-13/FIIa (8 × 10 s and 11 × 10 s, respectively). (C) The affinity of ADAMTS-13 and thrombin-cleaved ADAMTS-13 for VWF115 was measured by plate assay. 140 n VWF115 was immobilized to microtiter wells, and incubated with varying concentrations of ADAMTS-13 or thrombin-cleaved ADAMTS-13 (ADAMTS-13/FIIa), as in Materials and methods. A similar high-affinity interaction was determined (∼20 n) for both ADAMTS-13 and ADAMTS-13/FIIa with VWF115.<p><b>Copyright information:</b></p><p>Taken from "Further characterization of ADAMTS-13 inactivation by thrombin"</p><p></p><p>Journal of Thrombosis and Haemostasis 2007;5(5):1010-1018.</p><p>Published online Jan 2007</p><p>PMCID:PMC2408652.</p><p>© 2007 The Authors. Journal Compilation © 2007 International Society on Thrombosis and Haemostasis