Further characterization of ADAMTS-13 inactivation by thrombin

Background: The multimeric size and platelet-tethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13). In vitro ADAMTS-13 is susceptible to proteolytic inactivation by thrombin. Objectives: In this study, we aimed to characterize the inactivation of ADAMTS-13 by thrombin and to assess its physiological significance. Methods and results: By N-terminal sequencing of cleavage products, and by mutagenesis, we identified the principal thrombin cleavage sites in ADAMTS-13 as R257 and R1176. Using a library of 76 thrombin mutants, we highlighted the functional importance of exosite I on thrombin in the proteolysis of ADAMTS-13. Proteolysis of ADAMTS-13 by thrombin caused an 8-fold reduction in its affinity for VWF that contributed to its loss of VWF-cleaving function. Intriguingly, thrombin-cleaved ADAMTS-13 both bound and proteolyzed a short recombinant VWF A2 domain substrate (VWF115) normally. Following activation of coagulation in normal plasma, endogenous ADAMTS-13, but not added ADAMTS-13, appeared resistant to coagulation-induced fragmentation. An estimation of the Km for ADAMTS-13 proteolysis by thrombin was appreciably higher than the physiological concentration of ADAMTS-13. This was corroborated by the comparatively low affinity of ADAMTS-13 for thrombin (KD 95 nm). Conclusions: Together, our data suggest that ADAMTS-13 is protected from rapid proteolytic inactivation by thrombin in normal plasma. Whether this remains the case under pathological situations involving elevated/sustained generation of thrombin remains unclear

ADAMTS-13 proteolyzed by thrombin has reduced affinity for von Willebrand factor (VWF)

(A) 6 n ADAMTS-13, 60 n thrombin-cleaved ADAMTS-13 (ADAMTS-13/FIIa) or 60 n thrombin-cleaved ADAMTS-13/6 n ADAMTS-13 (10:1 mix) was incubated with 10 n purified plasma-derived VWF in the presence of 1.5 urea and 5 m BaCl. At time-points (0–4 h) subsamples were stopped with EDTA and VWF function measured using a collagen-binding assay (CBA). Changes in CBA are represented as % original CBA at 0 h. A 10-fold molar excess of proteolyzed ADAMTS-13 partially competed with ADAMTS-13 for VWF. (B) The affinity of ADAMTS-13 and thrombin-cleaved ADAMTS-13 for purified plasma-derived VWF was measured by plate assay. 30 n VWF 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 high-affinity interaction between ADAMTS-13 and VWF was determined (K∼6 n), whereas this was appreciably reduced for ADAMTS-13/FIIa (∼45 n).<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</p

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</p