ACTIVATION LOOP 3 AND THE 170 LOOP INTERACT IN THE MOST ACTIVE CONFORMATION OF FACTOR VIIA

Abstract number: P-M-022

Persson¹ E., Olsen¹ O.H.

¹Haemostasis Biochemistry, Novo Nordisk AS, Måløv, Denmark

How-to-cite Persson E, Olsen OH. ACTIVATION LOOP 3 AND THE 170 LOOP INTERACT IN THE MOST ACTIVE CONFORMATION OF FACTOR VIIA. J Thromb Haemost 2007; 5 Supplement 2: P-M-022

Abstract

Introduction: Tissue factor (TF) stimulates factor VIIa (FVIIa), involving stabilization of the 170 loop (substrate binding) and activation pocket (insertion of N-terminus). In structures of the active conformation of FVIIa, Arg^315{170C} and Ser^333{185} in these distant regions appear to be connected by hydrogen bonds involving Gly^372{223}. Here we study the importance of Gly³⁷² in activation loop 3 for the catalytically most active conformation of FVIIa.

Methods: Mutation G372A was introduced by site-directed mutagenesis. G372A-FVIIa was characterized in terms of soluble TF (sTF) binding, enzymatic activity, inhibitor reactivity and conformation of the protease domain N-terminus.

Results: G372A-FVIIa bound sTF with an affinity identical to that of FVIIa in the native (K_{d ~}5 nM) and active-site inhibited (K_{d ~}1 nM) form. G372A-FVIIa-sTF exhibited a 6-fold decrease in specificity constant (kcat/Km) for factor X activation compared with FVIIa-sTF, and with a peptidyl substrate we saw a 3.5-fold higher Km and 2-fold lower kcat. The same activity difference was seen between free G372A-FVIIa and FVIIa. G372A-FVIIa-sTF was inhibited slower than FVIIa-sTF by antithrombin and had a 6-fold higher K_i for p-aminobenzamidine. The N-terminus of the protease domain in G372A-FVIIa and FVIIa became equally less exposed after sTF binding according to carbamylation and PEG conjugation experiments.

Conclusions: Activity and inhibition data show that Gly³⁷², i.e. a hydrogen bond with Arg³¹⁵, is crucial for the most active conformation of free and TF-bound FVIIa. This bond stabilizes the substrate binding cleft and is weakened/abrogated in G372A-FVIIa. The unaltered conformational distribution of the N-terminus in G372A-FVIIa suggests an intact hydrogen bond to Ser³³³ and all data are consistent with normal binding of sTF and sTF-induced allosteric effects.

To cite this abstract use the following format:

Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number

Session Details

Date:	01/08/2007
Time:	00:00-00:00
Session name:	XXIst ISTH Congress
Subject:
Location:	Oxford, UK
Presentation type:
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