The Endothelial Protein C Receptor Supports TF-VIIa-Xa Ternary Complex Signaling through Protease-Activated Receptors.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1144-1144
Author(s):  
Jennifer Disse ◽  
Helle Heibroch Petersen ◽  
Katrine S. Larsen ◽  
Egon Persson ◽  
Naomi Esmon ◽  
...  
Keyword(s):  
Protein C ◽  
Ternary Complex ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Binary Complex ◽  
Endothelial Protein C Receptor ◽  
Free System ◽  
Isolated Lung ◽  
Complex Signaling ◽  
Human And Mouse

Abstract Abstract 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.

The A6936G polymorphism of the endothelial protein C receptor gene is associated with the risk of unexplained foetal loss in Mediterranean European couples

2009 ◽  
Vol 102 (10) ◽  
pp. 656-667 ◽  
Author(s):  
Pascale Fabbro-Peray ◽  
Pierre Marès ◽  
Patrick Mismetti ◽  
Géraldine Lissalde-Lavigne ◽  
Éva Cochery-Nouvellon ◽  
...  
Keyword(s):  
Protein C ◽  
Pregnancy Loss ◽  
Receptor Gene ◽  
Coagulation Factor ◽  
Factor V ◽  
Endothelial Protein C Receptor ◽  
Foetal Loss ◽  
Factor Ii ◽  
Embryonic Loss ◽  
Factor V Gene

SummaryThe endothelial protein C receptor (EPCR) is expressed by trophoblast cells. Mid-gestation pregnancy loss is described in animals with a haemochorial placenta lacking EPCR. The A6936G allele of the EPCR gene (PROCR) may be associated with lower EPCR densities on trophoblasts, but data are lacking for its effect on the risk of pregnancy loss in humans. A 1:2 case-control study on unexplained pregnancy loss was nested in the NOHA First cohort: 3,218 case couples and 6,436 control couples were studied for PROCR A6936G, coagulation factor V gene (F5) G1691A and coagulation factor II gene (F2) G20210A polymorphisms. Ethnicity and time of pregnancy loss defined through biometry-based gestational ages (embryonic loss < 10th week ≥ foetal loss) were analysed. The PROCR A6936G allele, in mothers and fathers, was associated only with foetal loss in both Europeans and non-Europeans. Increasing probability levels of carrying a homozygous child were increasingly associated with the risk of foetal demise. The F5 G1691A and F2 G20210A alleles, only in mothers, were only and independently associated with foetal loss in Europeans. In our population, the PROCR A6936G allele describes women, but also men and thus couples, at risk for first unexplained foetal loss. This risk is independent of the foetal loss risk conferred to our local Mediterranean European women by the F5 G1691A and F2 G20210A alleles. Data confirm that the relationship between thrombophilias and pregnancy loss varies according to ethnicity and loss type.

scholarly journals Sequence Determinants of Mouse Protein C Interaction with Mouse Endothelial Protein C Receptor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4227-4227
Author(s):  
Giulia Pavani ◽  
Katherine A. Stafford ◽  
Paris Margaritis
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Critical Role ◽  
Single Amino Acid ◽  
Factor Vii ◽  
Endothelial Protein C Receptor ◽  
Coagulant Activity ◽  
Single Amino Acid Residue ◽  
Gla Domain ◽  
Single Substitution

Abstract The Endothelial Protein C Receptor (EPCR) is an important component of the Protein C anticoagulant pathway. The interaction of Protein C (PC) through its Gla domain with EPCR enhances PC activation, thus down-regulating thrombin production. EPCR can also bind human activated Factor VII (FVIIa) and modulate its activity and localization. The residues involved in receptor recognition in both PC and FVIIa are Phe4 and Leu8, located in the first portion of the Gla domain. The importance of Phe8 is indicated by the lack of EPCR binding of a PC variant that contains a Val8 from human prothrombin. Remarkably, the similarity between PC and FVIIa for EPCR binding is lacking in the mouse. Others and we have shown that mouse FVIIa (mFVIIa, which contains a Leu4 and a Leu8) interacts with mouse EPCR (mEPCR) poorly, thus failing to model the spectrum of known human FVIIa properties. In previous work, we generated mFVIIa chimeras that contain parts of the mouse PC (mPC) Gla domain and determined that three residues in the mPC Gla domain can confer mEPCR binding to mFVIIa. Specifically, molecule mFVIIa-FMR that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from mPC was functionally similar to mFVIIa and could bind mEPCR as a true gain-of-function. However, little is known on the contribution of any/all of these positions in mPC binding to mEPCR. Here, we wanted to understand the sequence determinants that dictate this interaction. For this, we generated single amino acid mutants of mPC at position 4, 8 or 9 from the corresponding residues of mFVIIa. Using conditioned medium from transiently transfected cells, we tested the ability of each mPC mutant to bind to mEPCR expressed on the surface of CHO-K1 cells. A single substitution of Phe4 with Leu abolished mEPCR binding of mPC, in contrast to modifications at position 8 (Met to Leu) or 9 (Arg to Trp). The importance of Phe4 for the mPC-mEPCR interaction was confirmed in a reverse experiment modifying mFVIIa (that has poor mEPCR affinity) individually at position 4 (Leu to Phe), 8 (Leu to Met) or 9 (Trp to Arg) according to the mPC sequence. We found that Leu4->Phe was the sole modification that could confer mEPCR binding to mFVIIa. We have previously shown that the interaction of mFVIIa-FMR with mEPCR enhances its hemostatic function (vs. mFVIIa) after administration in hemophilic mice that have undergone injury (Pavani G et al, Blood 2014). To further explore the contribution of position 4 (Leu->Phe [L4F]) in these effects, recombinant mFVIIa-L4F was purified. Titration of mFVIIa-L4F on CHO-K1 cells expressing mEPCR showed a specific and dose-dependent receptor binding, in contrast to mFVIIa, confirming our previous data (see above). Moreover, mFVIIa-L4F showed no difference in clotting activity compared to mFVIIa in a prothrombin time-based assay. In order to compare mFVIIa-L4F to mFVIIa in its ability to generate mouse thrombin, we used a thrombin generation assay using hemophilia B plasma spiked with either procoagulant. We found that addition of either mFVIIa or mFVIIa-L4F generated similar amounts of thrombin at all concentrations tested (3.1 - 25 nM). Therefore, mFVIIa-L4F exhibited similar coagulant activity to mFVIIa but gained mEPCR binding capacity, a feature shared with mFVIIa-FMR. Further experiments are underway to determine whether the single substitution in mFVIIa-L4F is sufficient to recapitulate the enhanced hemostatic properties observed in vivo with mFVIIa-FMR. In conclusion, our findings identify a single amino acid residue (Phe4) in the Gla domain of mouse PC that plays a critical role in the binding to its natural receptor. This property can also be transplanted into mFVIIa, without affecting its coagulant activity. These observations reveal another difference between human and mouse systems and may have implications for EPCR-dependent functions or properties of other vitamin K-dependent proteins. Disclosures No relevant conflicts of interest to declare.

Properties of a Recombinant Chimeric Protein in which the gamma-Carboxyglutamic Acid and Helical Stack Domains of Human Anticoagulant Protein C Are Replaced by those of Human Coagulation Factor VII

1997 ◽  
Vol 77 (05) ◽  
pp. 0926-0933 ◽  
Author(s):  
Jie-Ping Geng ◽  
Francis J Castellino
Keyword(s):  
Metal Ion ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Chimeric Protein ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Wild Type ◽  
Coagulation Factor Vii ◽  
Carboxyglutamic Acid ◽  
Wild Type Counterpart

SummaryA chimeric cDNA, encoding residues 1-46 (the γ-carboxyglutamic acid module and its trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419 of human anticoagulant protein C (PC), was constructed and expressed. The resulting protein, r-[∆GD-HSPC/∇GD-HSfVII]PC, was properly processed with regard to signal/ propeptide release, cleavage of the K156R dipeptide, Gla and Hya contents, and the presence of glycosylation.The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to acidic phospholipid vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for the Ca2+-induced conformation of the Gla domain (GD) of PC, but did react with another MAb directed in part to the Ca2+-dependent conformation of the GD of fVII. Further, this chimeric protein possessed similar steady state constants as wild-type r-PC toward activation by thrombin and thrombin/thrombomodulin. The activated form of the chimera was very similar to that of its wild- type counterpart in its whole plasma anticoagulant activity, as well as its activity toward inactivation of coagulation factor VIII. The chimeric protein did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region of fVII is insufficient for that particular interaction.The results demonstrate that the GD/HS of fVII, when present in the PC and APC background, serves to maintain the Ca2+/PL-related functions of these latter proteins, and suggest that the Ca2+ and PL- dependent interactions of the GD-HS of PC are sufficiently general in nature such that the GD-HS regions of other proteins of this type can satisfy most of the requirements of PC and APC. The data presented also offer support for the independent nature of the domain unit consisting of the GD/HS module.

Novel Solution and Membrane-Based Assays Reveal Lack of Interaction Between Murine FVIIa and Murine EPCR

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 531-531
Author(s):  
Giulia Pavani ◽  
Paris Margaritis
Keyword(s):  
Research Funding ◽  
Protein C ◽  
Coagulation Factor ◽  
Healthcare Research ◽  
Published Data ◽  
High Dose ◽  
Endothelial Protein C Receptor ◽  
Single Experiment

Abstract Abstract 531 Generation of anticoagulant activated protein C (aPC) is enhanced by the presentation of protein C (PC) on the endothelial protein C receptor (EPCR) to the thrombin/thrombomodulin complex. Apart from its role in anticoagulation, EPCR also binds human coagulation Factor VIIa (FVIIa). However, the physiological consequences of this interaction either in vitro or in vivo are still unclear. In lieu of the widespread use of high-dose human FVIIa in hemophilic patients with anti Factor VIII or FIX antibodies as well as off-label applications, in vivo experimentation in appropriate mouse models is necessary to further define of the role of the FVIIa-EPCR interaction. Towards this, the aim of this study was to characterize the interaction of murine FVIIa with murine EPCR (mEPCR) in vitro. We used two novel assays that we have developed to study this interaction either in solution (by isothermal titration calorimetry [ITC]) or on the cell surface of mEPCR-expressing cells. The choice of ITC was based on its ability to provide a complete thermodynamic profile of protein-protein interaction in a single experiment: binding constant (K), stoichiometry (n), enthalpy (δH) and entropy (δS), parameters that cannot be determined with other methodologies in a single experiment. We first generated stable cells lines expressing recombinant murine soluble EPCR (msEPCR) or recombinant mFVIIa. The latter was generated as we have previously shown by insertion of a furin intracellular cleavage site between the light and heavy chains of murine FVII (mFVII). This resulted in a molecule secreted and purified in the activated form (mFVIIa) with ∼100% extrinsic activity vs. human FVIIa. Recombinant purified msEPCR had an apparent molecular weight of ∼46 kDa (vs. a predicted ∼26kDa size) which was consistent with extensive carbohydrate modifications. This was confirmed by expressing msEPCR in CHO cells modified to limit the size of the attached N-linked glycan chains: expressed msEPCR from such cells showed a reduction in its apparent molecular size (∼ 38kDa). Next, to validate ITC for the study the FVIIa-EPCR interaction, we used msEPCR and plasma-derived human PC (pd-hPC), a known msEPCR binder, that interacted with msEPCR with a Kd of ∼200nM, a stoichiometry of ∼1:1 and a ΔH and ΔS of −2733 ± 62 cal/mol and 21.3 cal/mol/deg, respectively. Initial ITC experiments with purified recombinant murine PC (mPC) also confirmed its binding to msEPCR. In contrast, we did not observe an interaction between recombinant mFVIIa and msEPCR. This finding was not due to protein degradation, as confirmed by Coomassie protein staining prior to and after ITC. Subsequently, to investigate the mFVIIa-msEPCR interaction in the context of a membrane-anchored murine EPCR, we generated a CHO-K1 cell line that expressed surface-exposed, full-length mEPCR, as verified by flow cytometry. These cells were incubated with pd-hPC or recombinant mFVIIa (50nM) in the presence of physiological Ca2+ (1.6mM) and Mg2+ (0.6mM) ion concentration. The bound protein was eluted with 10mM EDTA, electrophoresed and detected by western blotting. Despite the low femtomolar sensitivity of this assay (corresponding to 0.4% and 0.16% of total mFVIIa and pd-hPC used, respectively), we did not observe mFVIIa binding on mEPCR expressing CHO-K1 cells. Corroborating the ITC data, pd-hPC exhibited detectable binding to mEPCR expressing CHO-K1 cells. As a control, neither pd-hPC nor mFVIIa bound to naïve CHO-K1 cells (no mEPCR expression). In conclusion, we have established two novel assays to study the putative mFVIIa-mEPCR binding and clearly documented the lack of such interaction either in solution or on the cell-membrane. This is in good agreement with recently-published data obtained with surface plasmon resonance. In contrast to human FVIIa-EPCR, our results suggest that extrapolation of certain EPCR-dependent pathologic/physiologic processes using the mouse may be biased by species-specific effects. However, if the mEPCR binding capacity can be engineered to mFVIIa (as seen for human FVIIa-EPCR binding), such mFVIIa molecule(s) may facilitate the dissection of processes involving FVIIa and EPCR using the mouse as a human surrogate in vivo system. The assays developed here can easily assess such possibilities. Disclosures: Pavani: Bayer Healthcare: Research Funding. Margaritis:Bayer Healthcare: Research Funding.

Characterization Of a New Recombinant Human Factor VIIa (LR769)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3583-3583
Author(s):  
Grandoni Jerry ◽  
Gerald Perret ◽  
Cynthia Forier
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Protein C ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Vii ◽  
Endothelial Protein C Receptor ◽  
Binding Studies ◽  
Activation Assay

Abstract Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in the replacement therapies. In such cases factor replacement becomes ineffective, and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB/rEVO Biologics was tested. The goal is to provide patients with Hemophilia A and B who develop inhibitors a cost-effective alternative treatment option. Recombinant Human Factor VII was activated during the purification process to yield a highly homogenous rhFVIIa product (LR769). Kinetic enzyme assays and binding studies were used to characterize LR769. Active site titration demonstrated approximately 1 mole of active site per mole of protein. The Km and kcat for activation of FX and FIX were determined using an assay containing recombinant human tissue factor and phospholipid. The Kd for binding to soluble tissue factor was 22.3 ± 1.7 nM as measured using a FX activation assay. The apparent second order rate constant for inactivation by human plasma antithrombin was 5.9 ± 0.4 x103 M-1 sec-1. In all kinetic assays, LR769 behaved as expected for rhFVIIa. Binding studies with isolated human platelets were performed by FACS and indicated that binding was dependent on Ca+2. Activation of the platelets with thrombin and convulxin increased the binding of LR769. Binding of LR769 to Endothelial Protein C Receptor (EPCR) on the surface of cultured HEK 293 cells was determined by FACS analysis and confirmed that LR769 binds to EPCR. This was further demonstrated with a surface plasmon resonance assay using purified soluble EPCR. Binding to EPCR was dependent on Ca+2, and could be stimulated by Mg+2. Specificity of the binding was confirmed by the fact that it was inhibited by excess Protein C. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Binding of rhFVIIa (LR769) to the Endothelial Protein C Receptor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3509-3509
Author(s):  
Jerry A Grandoni
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Hemophilia A ◽  
Vascular System ◽  
Factor Viia ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Endothelial Protein C Receptor ◽  
Activated Factor Vii ◽  
Alternative Treatment Option

Abstract Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in replacement therapies. In such cases, factor replacement becomes ineffective and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new second generation recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB USA was studied. The goal is to provide patients with Hemophilia A and B who develop inhibitors an alternative treatment option. In work presented previously, we demonstrated that LR769 was similar to NovoSevenRT with respect to activation of FX and FIX, tissue factor binding, and inactivation by antithrombin. LR769 bound better to platelets than NovoSevenRT at saturation but had a similar binding constant(1). It is known that FVIIa binds to Endothelial Protein C Receptor (EPCR) with the same affinity as Protein C (2-4). Several studies suggest EPCR is involved in therapeutic mechanism rhFVIIa (5,6,7,8). In addition to its effect on thrombin generation, binding to EPCR may protect against permeability induced by VEGF and LPS (6). In the work presented here, the binding of LR769 to EPCR on the surface of cultured HUVEC cells was determined and compared to NovoSevenRT. A dose-dependent binding was observed at rhFVIIa concentrations expected in patients undergoing treatment with this product. The specificity for EPCR binding was confirmed by competition with Protein C, soluble EPCR, or anti-EPCR which reduced binding to these cells. Addition of soluble tissue factor increased the binding by approximately 20-30%. Overall, the results show that LR769 binds to primary human endothelial cells via EPCR and that tissue factor increases the binding affinity. Binding was found to be higher with LR769 than NovoSevenRT. The significance of the tissue factor effect on rhFVIIa binding with respect to Hemophilic joint arthropathy is discussed. The methods used will be used to further characterize the interactions of rhFVIIa with important components of the vascular system. 1) Grandoni, J.A. et al. (2013) ASH 2013 poster 2) Gosh et al. (2007) JBC 282 11849-11857 3) Lopez-Sagaseta et al. (2007) JTH 5 1817-1824 4) Preston et al. (2006) JBC 281 28850-28857 5) Pavani, G et al. (2014) Blood 7 1157-65 6) Sundarham et al. J. Thromb and Hemost (2014)12:690-700 7) Monroe DM, et al. (1997) Br J Haematol;99:542-7 Disclosures Grandoni: LFB USA: Employment.

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