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.