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The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF⅐FVIIa complex and by FXa in the ternary TF⅐FVIIa⅐FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF⅐FVIIa-Xa complex normally but were severely impaired in binary TF⅐FVIIa⅐PAR2 signaling. The residues identified were located in the model-predicted S2 pocket of FVIIa, and complementary PAR2 P2 Leu-38 replacements demonstrated that the P2 side chain was indeed crucial for PAR2 cleavage by TF⅐FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF⅐FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactorassociated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.Coagulation factor VIIa (FVIIa) 2 in complex with its cellular receptor tissue factor (TF) mediates activation of two pathways of major physiological importance: (i) initiation of blood coagulation by activation of coagulation factors IX and X (FIX and FX), and (ii) induction of cell signaling through proteaseactivated receptors (PARs). TF⅐FVIIa-dependent signaling primarily activates PAR2, which belongs to a family of four G-protein-coupled receptors activated by specific proteolytic cleavage of their N-terminal extracellular domain (1). Cleavage unmasks a new N terminus, which serves as a tethered ligand that induces transmembrane signaling (2). Activation of PAR2 by the TF⅐FVIIa binary complex involves cellular pools of TF with low affinity for FVIIa, whereas high affinity cell surface TF mediates coagulation activation and the associated cell signaling of the ternary complex of TF⅐FVIIa⅐FXa (3). In the latter complex, FXa is the primary activator for PAR2 (4). PAR2 triggers typical G-protein-coupled as well as ␤-arrestin-dependent signaling and thereby induces cytokine, chemokine, and growth factor expression and regulates protein synthesis, cell motility, proliferation, and apoptosis (5).Although the TF pathway also induces thrombin-dependent PAR1 signaling and transcriptional respo...

Staphylococcus aureus is a dangerous human pathogen characterized by alarmingly increasing antibiotic resistance. Accumulating evidence suggests the role of Spl proteases in staphylococcal virulence. Spl proteases have restricted, non-overlapping substrate specificity, suggesting that they may constitute a first example of a proteolytic system in bacteria. SplA, SplB, and SplD were previously characterized in terms of substrate specificity and structural determinants thereof. Here we analyze the substrate specificity of SplE documenting its unique P1 preference among Spl proteases and, in fact, among all chymotrypsin-like (family S1) proteases characterized to date. This is interesting since our understanding of the general aspects of proteolysis is based on seminal studies of S1 family members. To better understand the molecular determinants of the unusual specificity of SplE, the crystal structure of the protein is determined here. Conclusions from structural analysis are evaluated by successful grafting of SplE specificity on the scaffold of SplB protease.

The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.

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1144 Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. In contrast to thrombin that directly binds to and activates PAR1, other coagulation factors are dependent on co-receptors for efficient PAR cleavage. The endothelial protein C receptor (EPCR) is crucial for protein C (PC) activation by thrombomodulin-bound thrombin and supports signaling of activated PC through PAR1. EPCR may play additional roles by interacting with procoagulant proteases. EPCR binds the Gla-domain of human coagulation factor VII and conflicting reports exist about the role of EPCR as a receptor for FX. We studied the interaction of murine soluble EPCR extracellular domain (sEPCR) under physiological concentrations of divalent cations Ca2+/Mg2+. BIAcore measurements showed that murine sEPCR bound both human and mouse complexes of soluble tissue factor (TF) with FVIIa, as well as human FX. In a lipid free system measuring only protein-protein interactions, amidolytic activity of soluble TF-FVIIa was not changed by sEPCR. However, sEPCR dose dependently inhibited FX activation by both human and mouse soluble TF-FVIIa, indicating that sEPCR interacted with the TF-FVIIa-FX extrinsic activation complex. On human cells, TF forms two signaling complexes, the non-coagulant TF-FVIIa binary complex that activates PAR2 and the ternary TF-FVIIa-FXa complex that signals through PAR1 or PAR2. Overexpression of PAR2 in HUVECs resulted in PAR2 activation by activated PC, and in these transduced cells TF-FVIIa-FXa ternary complex signaling was inhibited by antibody blockade of EPCR. Human HaCaT keratinocytes constitutively synthesize TF and represent a well characterized model of TF binary and ternary complex signaling through PAR2. FACS analysis showed that EPCR was expressed on the cell surface and antibody blockade of EPCR prevented TF-FVIIa-Xa ternary, but not TF-FVIIa binary complex signaling. Mutation of FVIIa Gla residues had no effect on ternary complex signaling, indicating a primary interaction of EPCR with FX/FXa. To expand these studies to murine cells that constitutively express EPCR and TF, we isolated lung smooth muscle cells (SMC) from wild-type, PAR1-/-, PAR2-/-, and EPCRlow mice. Stimulation of SMC with thrombin, high concentrations of FXa, or FVIIa/FX, but not FVIIa alone induced PAR1-dependent signaling. While thrombin signaling was unaltered, EPCRlow SMC showed no response to the ternary complex measured by ERK1/2 phosphorylation. In order to exclude indirect effects on SMC phenotypes due to prolonged EPCR-deficiency in vivo, we further deleted EPCR in vitro by adenoviral transduction with cre recombinase from EPCRfloxflox SMC or blocked EPCR function with antibodies to murine EPCR. Both approaches inhibited TF ternary complex, but not thrombin signaling. These results show that EPCR interacts with the TF coagulation initiation complex to enable specifically ternary complex signaling and suggest that EPCR may play a role in regulating the biology of TF expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage. Disclosures: Heibroch Petersen: Novo Nordisk: Employment. Persson:Novo Nordisk: Employment. Petersen:Novo Nordisk: Employment. Ruf:Novo Nordisk: Research Funding.

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