Multifunctional specificity of the protein C/activated protein C Gla domain

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.     

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.     

Structural prediction and analysis of endothelial cell protein C/activated protein C receptor.

The endothelial cell receptor (EPCR) for protein C (PC)/activated protein C (APC) is a 221 amino-acid residues long transmembrane glycoprotein with unclear physiological function. To facilitate future studies and to rationalize recently reported experimental data about this protein, we have constructed three-dimensional models of human, bovine and mouse EPCR using threading and comparative model building. EPCR is homologous to CD1/MHC class I molecules. It consists of two domains, which are similar to the alpha1 and alpha2 domains of MHC class I molecules, whereas the alpha3 domain of MHC is replaced in EPCR by a transmembrane region followed by a short cytosolic tail. The alpha1 and alpha2 domains of CD1/MHC proteins form a groove, which binds short peptides. These domains are composed of an eight-stranded antiparallel beta-pleated sheet with two long antiparallel alpha-helices. The distance between the helical segments dictates the width of the groove. The cleft in EPCR appears to be relatively narrow and it is lined with hydrophobic/aromatic and polar residues with a few charged amino acids. Analysis of the human EPCR model predicts that (a) the protein does not contain any calcium binding pockets; (b) C101 and C169 form a buried disulphide bridge, while C97 is free, and buried in the core of the molecule; and (c) four potential glycosylation sites are solvent exposed.     

The endothelial cell protein C receptor (EPCR) functions as a primary receptor for protein C activation on endothelial cells in arteries, veins, and capillaries.

Plasma protein C functions as an anticoagulant when it is converted to the active form of serine protease. Protein C activation has been found to be mediated by the endothelial cell surface thrombin/thrombomodulin (TM) complex. In addition, we recently identified the endothelial cell protein C/activated protein C receptor (EPCR) which is capable of high-affinity binding for protein C. In this study, we established monoclonal antibodies (mAbs) against EPCR including several function blocking antibodies. Immunohistochemical analysis using these mAbs demonstrated that EPCR is widely expressed in the endothelial cells of arteries, veins, and capillaries in the lung, heart, and skin. Function blocking anti-EPCR mAbs strongly inhibited protein C activation mediated by primary cultured arterial endothelial cells which express abundant EPCR. Anti-EPCR mAbs also prevent protein C activation mediated by microvascular endothelial cells. These results indicate that EPCR functions as an important regulator for the protein C pathway in various types of vessels.     

Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium

Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. (125)I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time- and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively (125)I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in (125)I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR.     

Endothelial protein C receptor is expressed in rat cortical and hippocampal neurons and is necessary for protective effect of activated protein C at glutamate excitotoxicity

Activated protein C (APC) is an anticoagulant and anti-inflammatory factor that acts via endothelial protein C receptor (EPCR). Interestingly, APC also exhibits neuroprotective activities. In the present study, we demonstrate for the first time expression of EPCR, the receptor for APC, in rat cortical and hippocampal neurons. Moreover, exposing the neurons to glutamate excitotoxicity we studied the functional consequence of the expression of EPCR. By cytotoxicity assay we showed that EPCR was necessary for the APC-mediated protective effect in both neuronal cell types in culture. The effect of APC was abrogated in the presence of blocking EPCR antibodies. Analysis of neuronal death by cell labelling with dyes which allow distinguishing living and dead cells confirmed that the anti-apoptotic effect of APC was dependent on both EPCR and protease-activated receptor-1. Thus, we suggest that binding of APC to EPCR on neurons and subsequent activation of protease-activated receptor-1 by the complex of APC-EPCR promotes survival mechanisms after exposure of neurons to damaging factors.     

Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus.

Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C. We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs. Eight residues were identified as important for triphosphatase activity. These were Glu-305, Glu-307, and Phe-310 in motif A (IELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-496 in motif C (EVELE). Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p. E492Q retained triphosphatase activity. Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate. Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K(m) for ATP, whereas substitution with lysine increased K(m) 6-fold. Changing Lys-456 to alanine or glutamine increased K(m) an order of magnitude; ATP binding was restored when arginine was introduced. Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function. Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu-495 resulted in thermal instability in vivo and in vitro. A second S. cerevisiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized. Cth1p activity was abolished by E87A and E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo. Yet, fusion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1Delta yeast cells. This finding illustrates that mammalian guanylyltransferase can be used as a vehicle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through its binding to the phosphorylated CTD of RNA polymerase II.     

Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation

Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23 x 10(5) m(-1).s(-1), a dissociation rate constant of 7.61 x 10(-2) s(-1) and equilibrium binding constant (K(D)) of 147 nm. It was activated by thrombin over endothelial cells with a K(m) of 213 nm and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (< twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K(m) (358 nm for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.     

Reconstitution of the human endothelial cell protein C receptor with thrombomodulin in phosphatidylcholine vesicles enhances protein C activation

Blocking protein C binding to the endothelial cell protein C receptor (EPCR) on the endothelium is known to reduce protein C activation rates. Now we isolate human EPCR and thrombomodulin (TM) and reconstitute them into phosphatidylcholine vesicles. The EPCR increases protein C activation rates in a concentration-dependent fashion that does not saturate at 14 EPCR molecules/TM. Without EPCR, the protein C concentration dependence fits a single class of sites (Km = 2.17 +/- 0.13 microM). With EPCR, two classes of sites are apparent (Km = 20 +/- 15 nM and Km = 3.2 +/- 1.7 microM). Increasing the EPCR concentration at a constant TM concentration increases the percentage of high affinity sites. Holding the TM:EPCR ratio constant while decreasing the density of these proteins results in a decrease in the EPCR enhancement of protein C activation, suggesting that there is little affinity of the EPCR for TM. Negatively charged phospholipids also enhance protein C activation. EPCR acceleration of protein C activation is blocked by anti-EPCR antibodies, but not by annexin V, whereas the reverse is true with negatively charged phospholipids. Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates 7-fold over these cells, whereas annexin V is ineffective, indicating that EPCR rather than negatively charged phospholipid provide the surface for protein C activation. EPCR expression varies dramatically among vascular beds. The present results indicate that the EPCR concentration will determine the effectiveness of the protein C activation complex.     

Identification of a specific exosite on activated protein C for interaction with protease-activated receptor 1

Activated protein C (APC) is a vitamin K-dependent plasma serine protease which down-regulates the clotting cascade by inactivating procoagulant factors Va and VIIIa by limited proteolysis. In addition to its anticoagulant effect, APC also exhibits cytoprotective and antiinflammatory activity through the endothelial protein C receptor-dependent cleavage of protease activated receptor 1 (PAR-1) on endothelial cells. Recent mutagenesis data have indicated that the basic residues of two surface loops including those on 39 and the Ca2+-binding 70-80 loops constitute interactive sites for both factors Va and VIIIa, thereby mediating the interaction of APC specifically with these procoagulant cofactors. The basic residues of both loops have been discovered to be dispensable for the interaction of APC with PAR-1. It is not known if a similar exosite-dependent interaction contributes to the specificity of APC recognition of PAR-1 on endothelial cells. In this study, we have identified two acidic residues on helix-162 (Glu-167 and Glu-170) on the protease domain of APC which are required for the protease interaction with PAR-1, but not for its interaction with the procoagulant cofactors. Thus, the substitution of either Glu-167 or Glu-170 with Ala eliminated the cytoprotective signaling properties of APC without affecting its anticoagulant activity. These mutants provide useful tools for initiating in vivo studies to understand the extent to which the anticoagulant versus antiinflammatory activity of APC contributes to its beneficial effect in treating severe sepsis.     

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.     

Plant heterotrimeric G protein beta subunit is associated with membranes via protein interactions involving coiled-coil formation

The new functional role of activated protein C (APC) in the regulation of tissue factor (TF) expression was investigated using the cultured human monoblastic leukemia U937 cell line. A flow cytofluorometric analysis demonstrated that treatment with APC resulted in time- and dose-dependent decrease in TF expression in unstimulated and phorbol ester-stimulated cells. The effect was antagonized by the monoclonal antibody (mAb) to endothelial protein C/APC receptor (EPCR), 252, which strongly inhibited the interaction between APC and EPCR. In contrast, mAbs 49 and 379, which bind to EPCR without blocking APC binding, had no or only a modest effect. It is concluded that culturing U937 cells in the presence of APC caused down-regulation of TF expression through the EPCR-dependent mechanism, independent of whether induction was triggered by phorbol ester.     

Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.     

The occupancy of endothelial protein C receptor by its ligand modulates the par-1 dependent signaling specificity of coagulation proteases

Several recent studies have demonstrated that the activation of protease-activated receptor 1 (PAR-1) by thrombin and activated protein C (APC) on cultured vascular endothelial cells elicits paradoxical proinflammatory and antiinflammatory responses, respectively. Noting that the protective intracellular signaling activity of APC requires the interaction of the protease with its receptor, endothelial protein C receptor (EPCR), we recently hypothesized that the occupancy of EPCR by protein C may also change the PAR-1-dependent signaling specificity of thrombin. In support of this hypothesis, we demonstrated that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that the occupancy of EPCR by the Gla-domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through the coupling of PAR-1 to the pertussis toxin sensitive G(i) -protein. Thus, when EPCR is bound by protein C, a PAR-1-dependent protective signaling response in cultured endothelial cells can be mediated by either thrombin or APC. This article will briefly review the mechanism by which the occupancy of EPCR by its natural ligand modulates the PAR-1-dependent signaling specificity of coagulation proteases.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor

Cytoadhesion of Plasmodium falciparum‐infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C–EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine‐rich interdomain region (CIDRα1) domain from a variety of domain cassette (DC) 8 and DC13 P. falciparum erythrocyte membrane protein 1 proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate and strong inhibition of the activated protein C (APC)–EPCR interaction. Overall, there was a positive correlation between CIDRα1–EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti‐EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.     

Characterization of a factor Xa binding site on factor Va near the Arg-506 activated protein C cleavage site

Prothrombin is proteolytically activated by the prothrombinase complex comprising the serine protease Factor (F) Xa complexed with its cofactor, FVa. Based on inhibition of the prothrombinase complex by synthetic peptides, FVa residues 493-506 were proposed as a FXa binding site. FVa is homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-activating complex, and FVIIIa residues 555-561 (homologous to FVa residues 499-506) are recognized as a FIXa binding sequence. To test the hypothesis that FVa residues 499-505 contribute to FXa binding, we created the FVa loop swap mutant (designated 499-505(VIII) FV) with residues 499-505 replaced by residues 555-561 of FVIIIa, which differ at five of seven positions. Based on kinetic measurements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity for FXa. The fully formed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k(cat) and K(m)) for cleavage of prothrombin at Arg-320. However, small changes in both Arg-320 and Arg-271 cleavage rates result together in a moderate change in the pathway of prothrombin activation. Although residues 499-505 directly precede the Arg-506 cleavage site for activated protein C (APC), the 499-505(VIII) FVa mutant was inactivated entirely normally by APC. These results suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different specificity for binding FXa and FIXa while retaining compatibility as substrate for APC. In an updated three-dimensional model for the FVa structure, residues 499-505, along with Arg-506, Arg-306, and other previously suggested FXa binding sequences, delineate a continuous surface on the A2 domain that is strongly implicated as an extended FXa binding surface in the prothrombinase complex.     

Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.