III. Instantaneous inhibition by compound 48/80 of tissue factor-initiated extrinsic coagulation is mediated by the downregulation of factor VII activation

Our previous study has demonstrated a unique biological function of compound 48/80 (48/80) in the downregulation of monocytic tissue factor (TF)-initiated hypercoagulation in response to bacterial endotoxin (lipopolysaccharide, LPS) [A. J. Chu et al. (1999) Biochim. Biophys. Acta 1472, 386-395]. The inhibition was not due to the blockade of LPS cell signaling as evidenced by the unaffected LPS-induced TF synthesis. In the present study, we investigate the direct inhibitory action of 48/80 on the extrinsic coagulation cascade. TF-initiated coagulation was assayed by a single-stage clotting assay. Chromogenic assays dissected the extrinsic pathway to measure the activities of FVII, FX, and prothrombin by monitoring the hydrolyses of nitroaniline-conjugated substrates, identifying the inhibitory site(s). We report that 48/80 in vitro instantaneously inhibited rabbit brain thromboplastin (rbTF)-initiated coagulation in a dose-dependent manner. 48/80 preferentially inhibited FVII activation without any detectable effect on FVIIa, FXa, and thrombin activities. Neither FX activation nor prothrombin activation was affected. The significant inhibition on FVII activation was found to be noncompetitive with a fourfold reduction in the apparent Vmax of FVIIa formation from 7.1 to 1.7 nM/min, while the apparent Km (approximately 365 nM) remained unaffected. Western blotting analysis further confirmed that FVIIa formation derived from FVII was significantly diminished by 48/80, which was accompanied by blocked FVII binding to rbTF. In conclusion, 48/80 readily blocked FVII binding to rbTF, leading to diminished FVII activation and FVIIa formation. As a result, TF-initiated extrinsic coagulation was downregulated.     

The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII.

The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 0. 6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVII with TF. Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity.     

Tissue factor mediates inflammation

The role of tissue factor (TF) in inflammation is mediated by blood coagulation. TF initiates the extrinsic blood coagulation that proceeds as an extracellular signaling cascade by a series of active serine proteases: FVIIa, FXa, and thrombin (FIIa) for fibrin clot production in the presence of phospholipids and Ca2+. TF upregulation resulting from its enhanced exposure to clotting factor FVII/FVIIa often manifests not only hypercoagulable but also inflammatory state. Coagulant mediators (FVIIa, FXa, and FIIa) are proinflammatory, which are largely transmitted by protease-activated receptors (PAR) to elicit inflammation including the expression of tissue necrosis factor, interleukins, adhesion molecules (MCP-1, ICAM-1, VCAM-1, selectins, etc.), and growth factors (VEGF, PDGF, bFGF, etc.). In addition, fibrin, and its fragments are also able to promote inflammation. In the event of TF hypercoagulability accompanied by the elevations in clotting signals including fibrin overproduction, the inflammatory consequence could be enormous. Antagonism to coagulation-dependent inflammation includes (1) TF downregulation, (2) anti-coagulation, and (3) PAR blockade. TF downregulation and anti-coagulation prevent and limit the proceeding of coagulation cascade in the generation of proinflammatory coagulant signals, while PAR antagonists block the transmission of such signals. These approaches are of significance in interrupting the coagulation-inflammation cycle in contribution to not only anti-inflammation but also anti-thrombosis for cardioprotection.     

Substitution of the Gla domain in factor X with that of protein C impairs its interaction with factor VIIa/tissue factor: lack of comparable effect by similar substitution in factor IX

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.     

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF_1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF_1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.     

Activation and active site occupation alter conformation in the region of the first epidermal growth factor-like domain of human factor VII

The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.     

Aspirin before reperfusion blunts the infarct size limiting effect of atorvastatin

We assessed whether aspirin (acetylsalicylic acid, ASA), administered before reperfusion, abrogates the infarct size (IS)-limiting effect of atorvastatin (ATV). Statins reduce IS. This dose-dependent effect is mediated by upregulation of cycloxygenase-2 (COX2) and PGI(2) production. Administration of selective COX2-inhibitors either with ATV for 3 days or immediately before coronary occlusion blocks the IS-limiting effect of ATV. Sprague-Dawley rats received 3-day ATV (10 mg x kg(-1) x day(-1)) or water alone. Rats underwent 30 min coronary artery occlusion and 4 h reperfusion (IS protocol, n=8 in each group), or rats underwent 30 min coronary artery occlusion and 10 min reperfusion (enzyme expression and activity protocol, n=4 in each group). Immediately before reperfusion rats received intravenous ASA (5, 10, or 20 mg/kg) or saline. Area-at-risk (AR) was assessed by blue dye and IS by triphenyltetrazolium chloride. ATV reduced IS (10.1 +/- 1.4% of the AR) compared with controls (31.0 +/- 2.2%). Intravenous ASA alone did not affect IS (29.0 +/- 2.6%); however, ASA dose dependently (5, 10, and 20 mg/kg) attenuated the protective effect of ATV on IS (15.8 +/- 0.9%, 22.0 +/- 1.6%, and 23.7 +/- 3.8%, respectively). ASA dose dependently blocked the upregulation of COX2 by ATV. COX2 activity was as follows: control, 8.93 +/- 0.90 pg/mg; ATV, 75.85 +/- 1.08 pg/mg; ATV + ASA5, 34.39 +/- 1.48 pg/mg; ATV + ASA10, 19.87 +/- 1.10 pg/mg; and ATV + ASA20, 9.36 +/- 0.94 pg/mg. ASA, administered before reperfusion in doses comparable to those used in the clinical setting, abrogates the IS-limiting effect of ATV in a model with mechanical occlusion of the coronary artery. This potential adverse interaction should be further investigated in the clinical setting of acute coronary syndromes.     

Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.     

Tissue factor upregulation drives a thrombosis-inflammation circuit in relation to cardiovascular complications

The extrinsic coagulation is recognized as an 'inducible' signalling cascade resulting from tissue factor (TF) upregulation by exposure to clotting zymogen FVII upon inflammation or tissue injury. Following the substantial initiation, an array of proteolytic activation generates mediating signals (active serine proteases: FVIIa, FXa and FIIa) that lead to hypercoagulation with fibrin overproduction manifesting thrombosis. In addition, TF upregulation plays a central role in driving a thrombosis-inflammation circuit. Coagulant mediators (FVIIa, FXa and FIIa) and endproduct (fibrin) are proinflammatory, eliciting tissue necrosis factor, interleukins, adhesion molecules and many other intracellular signals in different cell types. Such resulting inflammation could ensure 'fibrin' thrombosis via feedback upregulation of TF. Alternatively, the resulting inflammation triggers platelet/leukocyte/polymononuclear cell activation thus contributing to 'cellular' thrombosis. TF is very vulnerable to upregulation resulting in hypercoagulability and subsequent thrombosis and inflammation, either of which presents cardiovascular risks. The prevention and intervention of TF hypercoagulability are of importance in cardioprotection. Blockade of inflammation reception and its intracellular signalling prevents TF expression from upregulation. Natural (activated protein C, tissue factor pathway inhibitor, or antithrombin III) or pharmacological anticoagulants readily offset the extrinsic hypercoagulation mainly through FVIIa, FXa or FIIa inhibition. Therefore, anticoagulants turn off the thrombosis-inflammation circuit, offering not only antithrombotic but anti-inflammatory significance in the prevention of cardiovascular complications.     

Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF--FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.     

Role of endogenous opioids in ischemic preconditioning but not in short-term hibernation in pigs

Endogenous opioids are involved in ischemic preconditioning (IP) in several species. Whether or not opioids are important for IP and short-term myocardial hibernation (STMH) in pigs is currently unknown. In 34 enflurane-anesthetized pigs, the left anterior descending coronary artery was flow constantly perfused. Subendocardial blood flow (Endo), infarct size (IS; percent area at risk), and the free energy change of ATP hydrolysis (DeltaG) were determined. After 90-min severe ischemia and 120-min reperfusion, IS averaged 28.3 +/- 5.4% (means +/- SE) (n = 8; Endo: 0.047 +/- 0.009 ml. min(-1) x g(-1)). IP by 10-min ischemia and 15-min reperfusion reduced IS to 9.9 +/- 3.8% (P < 0.05, n = 8; Endo: 0.044 +/- 0.009 ml. min(-1) x g(-1)). After naloxone (1 mg/kg iv followed by 2 microg x kg(-1) x min(-1)), IS averaged 25.8 +/- 7.0% (n = 6; Endo: 0.039 +/- 0.008 ml x min(-1) x g(-1)) without and 24.7 +/- 4.7% (n = 6; Endo: 0.044 +/- 0.006 ml x min(-1) x g(-1)) with IP. At 5-min moderate ischemia in the presence of naloxone, Endo decreased from 0.90 +/- 0.07 to 0.28 +/- 0.03 ml x min(-1) x g(-1)and DeltaG decreased from -58.6 +/- 1.0 to -52.6 +/- 0.4 kJ/mol. Prolongation of ischemia to 90 min did not alter Endo, but DeltaG recovered toward control values (57.7 +/- 1.1 kJ/mol), and the myocardium remained viable. These responses are identical to those of nonnaloxone-treated pigs. Endogenous opioids are involved in IP but not in STMH in pigs.     

Expression of recombinant rabbit tissue factor in Pichia pastoris,and its application in a prothrombin time reagent

Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains. It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation. The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies. To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris. Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity. A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.     

Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.     

Intrinsic A(1) adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts

We assessed the role of A(1) adenosine receptor (A(1)AR) activation by endogenous adenosine in the modulation of ischemic contracture and postischemic recovery in Langendorff-perfused mouse hearts subjected to 20 min of total ischemia and 30 min of reperfusion. In control hearts, the rate-pressure product (RPP) and first derivative of pressure development over time (+dP/dt) recovered to 57 +/- 3 and 58 +/- 3% of preischemia, respectively. Diastolic pressure remained elevated at 20 +/- 2 mmHg (compared with 3 +/- 1 mmHg preischemia). Interstitial adenosine, assessed by microdialysis, rose from approximately 0.3 to 1.9 microM during ischemia compared with approximately 15 microM in rat heart. Nonetheless, these levels will near maximally activate A(1)ARs on the basis of effects of exogenous adenosine and 2-chloroadenosine. Neither A(1)AR blockade with 200 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) during the ischemic period alone nor A(1)AR activation with 50 nM N(6)-cyclopentyladenosine altered rapidity or extent of ischemic contracture. However, ischemic DPCPX treatment significantly depressed postischemic recovery of RPP and +dP/dt (44 +/- 3 and 40 +/- 4% of preischemia, respectively). DPCPX treatment during the reperfusion period alone also reduced recovery of RPP and +dP/dt (to 44 +/- 2 and 47 +/- 2% of preischemia, respectively). These data indicate that 1) interstitial adenosine is lower in mouse versus rat myocardium during ischemia, 2) A(1)AR activation by endogenous adenosine or exogenous agonists does not modify ischemic contracture in murine myocardium, 3) A(1)AR activation by endogenous adenosine during ischemia attenuates postischemic stunning, and 4) A(1)AR activation by endogenous adenosine during the reperfusion period also improves postischemic contractile recovery.     

The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor.

Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa*TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa*TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIX(WT)), FIX(Q50P), FIX(PCEGF1) (EGF1 domain replaced with that of protein C), FIX(DeltaEGF1) (EGF1 domain deleted), FX(WT), and FX(PCEGF1). Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa*TF or FVIIa*sTF activated each mutant significantly more slowly than the FIX(WT) or FX(WT). Importantly, in ligand blot assays, FIX(WT) and FX(WT) bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K(i) of approximately 60 microm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.     

Gene Targeting of Tissue Factor, Factor X, and Factor VII in Mice: Their Involvement in Embryonic Development

Inactivation of specific genes in mammals by gene targeting has accelerated our ability to determine gene function. Nearly all genes involved in the blood coagulation system have been knocked out in mice. Tissue factor (TF) is the main initiator of the coagulation system and functions as a cell surface receptor for coagulation factor VII (FVII). Knockout studies have shown that TF deficiency results in lethality around embryonic day (E) 8.5-10.5. The results suggest a role for TF in embryonic blood vessel development and maintenance of vascular integrity in the yolk sac. In addition, TF may be involved in the maintenance of the placental labyrinth. Factor X (FX) deficiency causes partial embryonic lethality between E11.5-12.5.FX^–/– mice that were born died from fatal neonatal bleeding. In contrast, FVII deficiency is not embryonic lethal, but FVII^–/– neonates died from hemorrhage within the first days after birth. The various lethal phenotypes of deficiencies of the different coagulation factors suggest involvement in processes beyond hemostasis. Both TF/FVIIa and FXa can trigger intracellular signaling events in certain cell types. Signaling by coagulation proteases and protease activated receptors (PARs) may have important roles in embryonic development.     

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.