A. Iakhiaev et al., The role of catalytic cleft and exosite residues of factor VIIa for complex formation with tissue factor pathway inhibitor, THROMB HAEM, 85(3), 2001, pp. 458-463
The extrinsic coagulation pathway is initiated by the binding of plasma fac
tor VIIa (VIIa) to the cell surface receptor tissue factor (TF). Formation
of the TF-VIla complex results in allosteric activation of VIIa as well as
the creation of an extended macromolecular substrate binding exosite that g
reatly enhances proteolytic activation of substrate factor X. The catalytic
function of the TF-VIIa complex is regulated by a specific Kunitz-type inh
ibitor, tissue factor pathway inhibitor (TFPI). TFPI inhibition of the TF-V
IIa complex was enhanced by the presence of Xa. This study investigates the
relative contribution of catalytic cleft and exosite residues in Wa for in
hibitory complex formation with TFPI. Wa protease domain residues Q(176), T
-239 and E-296 are involved in the formation of stable inhibitor complex wi
th free TFPI. Kinetic analysis further demonstrated a predominant role of t
he S2' subsite residue Q(176) for the initial complex formation with TFPI.
In contrast, no significant reductions in inhibition by TFPI-Xa were found
for each of the mutants in complex with phospholipid reconstituted TF. Howe
ver, reduced rates of inhibition of the VIIa Gla-domain (R-36) and Q(176) m
utant by TFPI-Xa were evident when TF was solubilized by detergent micelles
. These data demonstrate docking of the TFPI-Xa complex with the macromolec
ular substrate exosite and the catalytic cleft, in particular the S2' subsi
te. The masking of the mutational effect by the presence of phospholipid sh
ows a critical importance of Xa Cia-domain interactions in stabilizing the
quaternary TF-VIIa-Xa-TFPI complex.