Abstract
Signaling of the tissue factor-FVIIa complex regulates angiogenesis, tumor growth, and inflammation. TF-FVIIa triggers cell signaling events by cleavage of protease activated receptor (PAR2) at the Arg36-Ser37 scissile bond. The recognition of PAR2 by the FVIIa protease domain is poorly understood. We perform molecular modeling and dynamics simulations to derive the PAR2-FVIIa interactions. Docking of the PAR2 Arg36-Ser37 scissile bond to the S1 site and subsequent molecular dynamics leads to interactions of the PAR2 ectodomain with P and P' sites of the FVIIa catalytic cleft as well as to electrostatic interactions between a stably folded region of PAR2 and a cluster of basic residues remote from the catalytic cleft of FVIIa. To address the functional significance of this interaction for PAR2 cleavage, we employed two antibodies with epitopes previously mapped to this cluster of basic residues. Although these antibodies do not block the catalytic cleft, both antibodies completely abrogated PAR2 activation by TF-FVIIa. Our simulations indicate a conformation of the PAR2 ectodomain that limits the cleavage site to no more than 33 A from its membrane proximal residue. Since the active site of FVIIa in the TF-FVIIa complex is approximately 75 A above the membrane, cleavage of the folded conformation of PAR2 would require tilting of the TF-FVIIa complex toward the membrane, indicating that additional cellular factors may be required to properly align the scissile bond of PAR2 with TF-FVIIa.