Background: Activated protein C (APC
)
-mediated inactivation of factor (
F
)Va is greatly
enhanced by protein S. For inactivation to occur, a trimolecular complex between FVa,
APC and protein S must form on the phospholipid membrane. However, direct
demonstration of complex formation has proven elusive.
Objectives:
To elucidate the nature of the phospholipid
-dependent interaction
s between
APC, protein S and FVa
.
Methods:
We evaluated binding of active site blocked APC to phospholipid
-coated
magnetic beads in the presence and absence of protein S and/or FVa. The importance
of protein S and FV residues were evaluated functionally.
Results: APC alone bound weakly to phospholipids. Protein S mildly enhanced APC
binding to phospholipid surfaces, whereas FVa did not. However, FVa together with
protein
S enhanced APC binding
(
>14
-fold), demonstrating formation of an APC/protein
S/FVa complex. C4b binding protein
-bound protein S failed to enhance APC binding,
agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A)
with reduced APC cofactor function exhibited essentially normal augmentation of APC
binding to phospholipids, but diminished APC/protein S/FVa complex formation,
suggesting involvement in interactions dependent upon FVa. Similarly, FVaNara
(W1920R), an APC resistant FV variant, also did not efficiently incorporate into the
trimolecular complex as efficiently as wild
-type FVa. FVa inactivation assays suggested
that the mutation impairs its affinity for phospholipid membranes and with protein S
within the complex. Conclusions: FVa plays a central role in the formation of its inactivation complex.
Furthermore, membrane proximal interactions between FVa, APC and protein S are
essential for its cofactor function.