Contacts between Several ADAMTS13 Structural Domains and von Willebrand Factor Domain A2 Contribute to Substrate Cleavage.

ADAMTS13 reduces the size of plasma von Willebrand factor (VWF) multimers by cleaving the Y¹⁶⁰⁵-M¹⁶⁰⁶ bond within the central A2 domain of the VWF subunit. The C-terminus of VWF domain A2 is known to bind an exosite in the Spacer domain of ADAMTS13. However, ADAMTS13 cleaves smaller substrates that lack this C-terminal binding segment of the A2 domain, which suggests that additional sites in domain A2 interact with additional exosites on ADAMTS13. To identify these recognition sites, we constructed a series of recombinant substrates and analyzed their cleavage by ADAMTS13 variants (Table 1). Products were characterized by Western blotting and with a quantitative MALDI-MS assay. The progressive deletion of C-terminal amino acids of VWF domain A2 progressively reduced the rate of cleavage by ADAMTS13. In addition, the removal of 9 amino acids at the N-terminus of VWF73 (VWF64n) abolished cleavage. Comparison of the specificity constants (k_cat/K_m) shows that ADAMTS13 truncated after the first TSP1 domain (construct MDT) cleaved VWF64 ∼9-fold slower than did ADAMTS13 truncated after the Cys-rich domain (construct MDTC). Removal of 18 amino acids from the C-terminus of VWF64 (VWF46) also decreased the rate of cleavage by MDTC ∼9-fold, suggesting that the Cys-rich domain interacts with the segment I¹⁶⁴²-R¹⁶⁵⁹. Further C-terminal deletions of the substrate (VWF35, VWF28) had little effect on the rate of cleavage by enzymes MDTC or MDT. Comparing substrates VWF73 and VWF73d3, internal deletion of the region missing from VWF35 and VWF28 (Q¹⁶²⁴ to R¹⁶⁴¹) had only a small effect (<3-fold) on substrate cleavage by ADAMTS13, MDTC, or MDT. Therefore the corresponding segment of VWF domain A2 appears dispensable for substrate recognition by ADAMTS13, and cleavage is not very sensitive to the spacing between the scissile bond and more C-terminal enzyme binding sites. In contrast, substrate VWF73d2 with internal deletion of the more C-terminal segment I¹⁶⁴²-Q¹⁶⁵², had properties similar to VWF64: it was cleaved much faster by full-length enzyme than by MDT, indicating dependence of the cleavage rate on the Spacer and Cys-rich domains. Substrate VWF73d1 lacking I¹⁶⁴²-R¹⁶⁵⁹ had properties similar to VWF46: the cleavage rate was independent of the Spacer and Cys-rich domains. Finally, a substrate with deletion of a segment just C-terminal to the scissile Y¹⁶⁰⁵-M¹⁶⁰⁶ bond (VWF73d4, G¹⁶⁰⁹-I¹⁶²³) was resistant to cleavage by ADAMTS13. These data support extensive contacts between several domains of ADAMTS13 and discrete segments of VWF domain A2. Segments D¹⁵⁹⁶-V¹⁶⁰⁴ and G¹⁶⁰⁹-I¹⁶²³ flanking the cleaved Y-M bond interact at least with the Metalloprotease domain. Segment I¹⁶⁴²-Q¹⁶⁵² interacts with the Cysteine-rich domain. Segment D¹⁶⁵³-R¹⁶⁶⁸ interacts with the Spacer domain. All of these regions contribute to the recognition and cleavage of VWF by ADAMTS13.