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.


OBJECTIVE
In this study, we aimed to characterize the inactivation of ADAMTS-13 by thrombin and to assess its physiological significance.


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 K(m) 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 (K(D) 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.
Lam
J K
JK
Department of Haematology, Imperial College London, London, UK.
Chion
C K N K
CK
Zanardelli
S
S
Lane
D A
DA
Crawley
J T B
JT
D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000097:blood / Q000378:metabolism; D000071120:ADAMTS13 Protein; D002460:Cell Line; D006801:Humans; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013917:Thrombin / Q000494:pharmacology*
D011994:Recombinant Proteins; D013917:Thrombin; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human