tailieunhanh - Báo cáo khoa học: Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIa

The initiation of blood coagulation involves tissue factor (TF)-induced allosteric activation of factor VIIa (FVIIa), which circulates in a zymogen-like state. In addition, the (most) active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372(223) in FVIIa | ỊFEBS Journal Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor Vila Egon Persson and Ole H. Olsen Haemostasis Biochemistry Novo Nordisk A S Novo Nordisk Park Malov Denmark Keywords activation loop allosteric activation factor Vila initiation of coagulation tissue factor Correspondence E. Persson Haemostasis Biochemistry Novo Nordisk A S Novo Nordisk Park DK-2760 Malov Denmark Fax 45 4466 3450 Tel 45 4443 4351 E-mail egpe@ Received 3 December 2008 revised 20 March 2009 accepted 30 March 2009 doi The initiation of blood coagulation involves tissue factor TF -induced allosteric activation of factor VIIa FVIIa which circulates in a zymogenlike state. In addition the most active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372 223 in FVIIa which is the sole residue in activation loop 3 that is capable of forming backbone hydrogen bonds with the unusually long 170 loop and with activation loop 2 by studying the effects of replacement with Ala G372 223 A . G372A-FVIIa both in the free and TF-bound form exhibited reduced cleavage of factor X FX and of pept-idyl substrates and had increased Km values compared with wild-type FVIIa. Inhibition of G372A-FVIIa-sTF by p-aminobenzamidine was characterized by a seven-fold higher Ki than obtained with FVIIa-sTF. Crystallographic and modelling data suggest that the most active conformation of FVIIa depends on the backbone hydrogen bond between Gly372 223 and Arg315 170C in the 170 loop. Despite the reduced activity and inhibitor susceptibility native and active site-inhibited G372A-FVIIa bound sTF with the same affinity as the corresponding forms of FVIIa and burial of the N-terminus of the protease domain increased similarly upon sTF binding to G372A-FVIIa and FVIIa. Thus Gly372 223 in FVIIa appears to play a critical role in maturation of the S1 pocket and .