Arg-Ser-775, 792 and 823 in Spacer Region of ADAMTS-18 Is Critical for Thrombin Cleavage

Cleavage of ADAMTS-18 by thrombin represents a new mechanism of platelet thrombus clearance via the release of active ~45-kDa C-terminal fragments that induces oxidative platelet fragmentation. The exact cleavage sites remain unclear, but Arg (R)775/Ser (S)776 in spacer region of ADAMTS-18 has been shown to be one of the cleavage sites of thrombin. Here, we demonstrate that R792/S793 and R823/S824 are also thrombin cleavage sites by sequence analysis, amino acid mutation and mass spectrometry assay. All these cleavage sites are thrombin-specific and insensitive to other enzymes tested (e.g. cathepsin D or trypsin). Simultaneous mutation of R775, 792, 823 to S775, 792, 823 in ADAMTS-18 completely abrogated the cleavage by thrombin and the generation of active C-terminal 45-kDa fragments. Together with previous study, a total of three thrombin-specific cleavage sites have been identified in spacer region of ADAMTS-18.