The Endothelial Protein C Receptor (EPCR) Regulates Endogenous Factor VII Levels in Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3629-3629
Author(s):  
Juliana Small ◽  
Shannon Zintner ◽  
Lynn E Dankner ◽  
Paris Margaritis
Keyword(s):  
Mouse Model ◽  
Research Funding ◽  
Protein C ◽  
Binding Capacity ◽  
Factor Vii ◽  
Endothelial Protein C Receptor ◽  
Gain Of Function ◽  
Vessel Occlusion ◽  
Gla Domain

The endothelial protein C receptor (EPCR) has been demonstrated to bind activated FVII (FVIIa) through the Gla domain with equal affinity to Protein C (PC). Mouse studies suggest that EPCR is involved in the extravasation of infused human FVIIa, leading to an extended extravascular tissue persistence, longer than expected based on its circulating half-life. This provides a plausible explanation for the long-term benefits of hemophilic patients on human FVIIa prophylaxis. Collectively, these data suggest that EPCR sequesters administered FVIIa in tissues where it may have a hemostatic effect. However, the role of the endogenous FVII-EPCR interaction in normal conditions is largely unknown. For this, we have developed a mouse model to better understand this interaction in vivo. Endogenous mouse FVII and FVIIa (mFVII/FVIIa) do not bind mouse EPCR. However, our laboratory has demonstrated that L4F, L8M, and T9R substitutions in the Gla domain of mFVIIa enable its interaction with mouse EPCR while retaining full enzymatic activity in vitro. Based on that data, we utilized CRISPR/Cas9 technology to knock-in L4F, L8M, and T9R into the mFVII Gla domain in the mouse F7 locus (F7FMR), thereby developing mice with a chimeric endogenous FVII capable of binding EPCR. Founder animals were generated and capable of producing offspring, indicating that the gain-of-function in mFVII was compatible with life. Animals were subsequently backcrossed to wildtype C57BL/6 mice in order to remove potential off-target effects of the CRISPR/Cas9. Resultant heterozygous animals (F7FMR/WT) from the final cross were bred to generate F7FMR/FMR, F7FMR/WT, and F7WT/WTlittermates. We generated 59 male and 52 female animals and a binomial distribution test demonstrated that sex is equally distributed in the population. Moreover, the genotypes expected from the heterozygous crosses were inherited in a 1:2:1 ratio, further indicating that the gain-of-function in FVII is not lethal during development. As additional metrics of health, we measured weight longitudinally during weeks 1-10 of life and found no differences between the three genotypes for either gender. Complete blood counts (CBCs) revealed no differences between the F7FMR/FMR, F7FMR/WT, and F7WT/WTgenotypes, with the exception of a mild elevation in F7FMR/WTanimals compared to animals with wildtype FVII. Collectively, we found that the gain-of-function in EPCR binding by endogenous FVII is not detrimental to the overall health of the mice. Subsequently, we determined the mFVII levels in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals using an in-house ELISA. We observed that plasmatic mFVII levels were dependent on the EPCR-binding capacity of the endogenous mFVII. Specifically, F7WT/WTmice, whose mFVII does not bind EPCR, had a plasmatic mFVII concentration of ~690 ng/ml. In contrast, F7FMR/FMRhomozygote mice had ~350 ng/ml of mouse FVII, approximately half the plasma levels of the F7WT/WT. Heterozygote animals F7FMR/WThad an intermediate plasmatic mFVII level (~550 ng/ml), suggesting that EPCR may regulate plasmatic FVII levels in vivo. Lastly, we determined the hemostatic response to injury in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals. We did this in two ways, by measuring blood loss following tail clip assay and by determining time to vessel occlusion following ferric chloride injury of the carotid artery. We observed no differences between the three genotypes in response to either injury model. In conclusion, we have generated and characterized a novel mouse model in which endogenous FVII is capable of binding EPCR. Using this model, we demonstrated that EPCR can modulate plasmatic FVII levels in vivo but does not appear to affect hemostasis. Since this model mimics the FVII-EPCR interaction in humans, it can now be used to further investigate how this interaction participates in other normal or pathologic states that depend on FVII and/or EPCR. Disclosures Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol-Myers Squibb: Other: Salary (spouse); CSL Behring: Other: Salary (spouse); NovoNordisk A/S: Research Funding.

scholarly journals A Single Amino Acid in the Gla Domain Mouse FVIIa Allows Its Binding to the Endothelial Protein C Receptor and Enhances Its Coagulant Activity In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3489-3489
Author(s):  
Giulia Pavani ◽  
Lacramioara Ivanciu ◽  
Katherine A. Stafford ◽  
Paris Margaritis
Keyword(s):  
Amino Acids ◽  
Carotid Artery ◽  
Research Funding ◽  
Protein C ◽  
Binding Capacity ◽  
Hemophilia B ◽  
Vessel Occlusion ◽  
Gla Domain ◽  
Program Research

Abstract The human protein C (PC) interaction with the endothelial PC receptor (EPCR) is mediated through the PC Gla domain, via key amino acids Phe4 and Leu8. Specifically, substitution of Leu8 with Val from human prothrombin abolishes the PC-EPCR interaction. The Gla domain of human Factor VII (FVII) shares these positions with PC and, consequently, the EPCR binding capacity. In the mouse, a commonly used in vivo model, the sequence determinants of the Gla domain of mouse PC (mPC) interaction with mouse EPCR (mEPCR) are not known. Remarkably, mouse FVII (mFVII) and its activated form (mFVIIa) have poor affinity for mouse EPCR. We previously described a variant of mFVIIa (mFVIIa-FMR) that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from the mPC Gla domain. We found that this molecule was functionally similar to mFVIIa and could bind mEPCR. Using mFVIIa-FMR as surrogate to study the mPC-mEPCR interaction, we highlighted the importance of the Phe4/Met8/Arg9 in the mPC-mEPCR interaction. We also found that mFVIIa-FMR had enhanced hemostatic properties when infused at 3 mg/kg after FeCl3 carotid artery injury in hemophilic mice (vessel occlusion was 2.5 times faster than mFVIIa). In order to further refine whether the mEPCR binding capacity of mPC is coordinated by any/all of Phe4/Met8/Arg9 positions, we previously generated single variants of mPC at these positions using the corresponding amino acids of mFVIIa (that has poor interaction with mEPCR). We found that Phe4 is the sole determinant of specificity of the mPC-mEPCR interaction. Moreover, when Phe4 was placed in mFVIIa, we found that mFVIIa-Phe4 had activity similar to mFVIIa and bound mEPCR on cells (or in solution to soluble mEPCR) with a Kd of ~350nM. This was of similar magnitude to the mEPCR affinity of a mFVIIa variant with the entire mPC Gla domain (~200 nM), indirectly suggesting that Phe4 determines both the specificity and affinity of mPC to mEPCR. Since mFVIIa-FMR showed improved hemostatic properties in vivo as a result of mEPCR binding, enhancing the EPCR-FVIIa binding may generate improved human FVIIa molecules for the treatment of bleeding. Here we wanted to provide proof-of-concept using limited Gla domain modifications. For this, we utilized mFVIIa-Phe4, a minimally modified mFVIIa molecule, described above. Specifically, hemophilia B animals were subjected to a 7.5% FeCl3 injury of their carotid artery for 2 minutes; after 10 minutes mice were infused with 3 mg/kg of mFVIIa or mFVIIa-Phe4. Time to vessel occlusion was determined by monitoring blood flow. Hemostatically normal mice occluded in 13.3 ± 3.0 min. We found that infusion of mFVIIa resulted in vessel occlusion at 8.9 ± 1.7 min. However, mice that received mFVIIa-Phe4 reached vessel occlusion within 4.5 ± 2.4 min, ~2.5 times faster than mFVIIa-infused mice (P<0.01). This was similar to that we previously observed with mFVIIa-FMR infusion after injury in hemophilia B mice. Our results suggest the following: (1) Phe4 in the mPC Gla domain confers the specificity and affinity to mEPCR; (2) a single Phe4 substitution in mFVIIa is the only requirement for enhancing its clotting function in vivo. These data reveal another difference between human and mouse systems that may affect EPCR-dependent functions of other vitamin K-dependent proteins. Moreover, our results suggest the possibility that minimally modified variants of FVIIa with respect to EPCR binding may have more favorable hemostatic properties for clinical use. Disclosures Ivanciu: Bayer Hemophilia Awards Program: Research Funding. Margaritis:Novo Nordisk A/S: Research Funding; Bayer Hemophilia Awards Program: Research Funding.

FVIIa Binding to the Endothelial Protein C Receptor Reduces Protein Recovery in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 18-18
Author(s):  
Giulia Pavani ◽  
Lacramioara Ivanciu ◽  
Paris Margaritis
Keyword(s):  
Research Funding ◽  
Protein C ◽  
Binding Capacity ◽  
High Dose ◽  
Bolus Administration ◽  
Endothelial Protein C Receptor ◽  
Gla Domain ◽  
Plasmatic Concentration

Abstract Abstract 18 High-dose human activated Factor VII (FVIIa) use is widespread in hemophilic patients with anti Factor VIII or Factor IX antibodies as well as in off-label applications. In addition to tissue factor, the endothelial protein C receptor (EPCR) also binds human FVIIa. However, the physiological consequences of this interaction on the potential hemostatic effects following bolus administration of FVIIa are still unclear. To investigate this in a mouse model, we decided to study the interaction of murine FVIIa (mFVIIa) with murine EPCR (mEPCR) in vitro and, subsequently, in vivo. We have previously shown, either in solution (with murine soluble EPCR) or on cells expressing full-length EPCR, that mFVIIa has a very low affinity for murine EPCR (Kd > 8μM), in contrast to human FVIIa binding to human EPCR. Therefore, to use the mouse as a model to study the FVIIa-EPCR interaction, we engineered mEPCR binding capacity into mFVIIa by partial substitution of its Gla domain, using the murine Protein C Gla domain as a donor. Combined modifications of 3 residues in mFVIIa (L4F, L8M and W9R; FMR-mFVIIa) were sufficient to confer mEPCR binding (Kd ≈ 200 nM, in presence of 1.6 mM Ca2+ and 0.6 mM Mg2+), without impairing the activity of the molecule as measured by a clotting assay. Here, we extend the characterization of FMR-mFVIIa in vitro and in vivo. First, we monitored the affinity of FMR-mFVIIa or wildtype mFVIIa (WT-mFVIIa) for its natural cofactor, murine tissue factor in the context of a cell membrane. For this, we generated CHO-K1 cells stably expressing full-length mTF (CHO-K1-mTF). FMR- or WT-mFVIIa was incubated at 4 degrees C (in presence of 1.6 mM Ca2+ and 0.6 mM Mg2+) on such cells at increasing concentration and, following quantification of the bound fraction, we observed no difference between FMR- and WT-mFVIIa in affinity for mTF (18 ± 13 nM and 17 ± 2 nM, respectively). To begin defining the role of EPCR-FVIIa interaction in vivo, we injected WT-mFVIIa or FMR-mFVIIa (500μg/kg) into C57BL6 wildtype mice (n=5 per protein per timepoint) via tail vein and monitored plasmatic concentration at different timepoints. The decrease in plasmatic levels over time followed a biphasic pattern. At 5 and 15 minutes post injection, plasmatic concentration of FMR-mFVIIa was significantly lower than WT-mFVIIa (41 ± 9% [FMR-mFVIIa] vs. 66 ± 7% [WT-mFVIIa] of the initial dose at 5 min, p=0.001; 18.5 ± 2.8% [FMR-mFVIIa] vs. 40.5 ± 9.0% [WT-mFVIIa] of the initial dose at 15 min, p=003). No differences were observed at later timepoints (up to 2 hours post protein infusion). Moreover, there were no changes in either platelet counts or hematocrit over the period of observation. Next, we wanted to confirm that the differences in recovery between the infused proteins were the result of mEPCR binding. For this, we infused an EPCR-blocking (RCR-252) or isotype control antibody (50μg/mouse) prior to administration of FMR- or WT-mFVIIa. We assessed plasmatic concentration at 5 min post protein infusion. In accordance with our previous data, mice that received isotype control IgG showed reduced recovery for the FMR-mFVIIa chimera vs. WT-mFVIIa (p=0.001). In contrast, antibody blocking of mEPCR prior to protein infusion increased the recovery of FMR-mFVIIa to that observed for WT-mFVIIa. These data suggest that the reduced recovery observed by bolus administration of FMR-mFVIIa vs. WT-mFVIIa was attributable to the mEPCR binding capacity of FMR-mFVIIa. In conclusion, we have now characterized a mFVIIa chimeric molecule indistinguishable from WT-mFVIIa in terms of mTF binding and clotting activity, but bearing the capacity to interact with mEPCR in vitro and, more importantly, in vivo. These features mimic those found in human FVIIa, thereby allowing the study of EPCR-dependent mechanisms in the clearance and/or biodistribution of FVII/FVIIa in vivo. Our observations suggest, for the first time in a homologous system, that EPCR-binding capacity has a specific negative effect on the recovery of the mFVIIa chimera. This molecule can now be utilized in the context of bolus protease administration in hemophilic mice following injury, to test any potential hemostatic effects from a FVIIa-EPCR interaction in vivo. This may provide additional insight into the mechanism of action of high-dose FVIIa administration in hemophilia. Disclosures: Pavani: Bayer: Research Funding. Margaritis:Novo Nordisk A/S: Research Funding; Bayer: Research Funding.

scholarly journals Characterization of the Interaction of Activated FVII with the Endothelial Protein C Receptor (EPCR) in the Rat

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2455-2455
Author(s):  
Juliana Small ◽  
Shannon Zintner ◽  
Lynn E Dankner ◽  
Paris Margaritis
Keyword(s):  
Rat Model ◽  
Research Funding ◽  
Protein C ◽  
Hemophilia A ◽  
Binding Capacity ◽  
Stable Cell Line ◽  
The Novel ◽  
Endothelial Protein C Receptor ◽  
Gla Domain

Abstract Activated FVII (FVIIa) has been shown to interact with EPCR resulting in long-term extravascular tissue presence in mice, well beyond its short circulating half-life. In addition, data in hemophilia patients on short term FVIIa prophylaxis indicate that clinical improvements can persist even in the post-prophylaxis period. Taken together, these data suggest that EPCR may sequester administered FVIIa in the extravascular space that retains activity, potentially explaining the hemostatic improvements that persist long after its infusion has ceased. As such, the EPCR-FVIIa interaction may have mechanistic and translational ramifications in the treatment of bleeds in hemophilia which needs to be further investigated. Unfortunately, hemophilic mice do not model the human disorder in terms of bleeding diathesis. In contrast, the novel rat model of hemophilia A (Nielsen LN et al, 2014) exhibits spontaneous bleeds that, if untreated, can be fatal. Therefore, this model can be utilized to better understand the FVIIa-EPCR interaction in the on-demand and prophylactic action of FVIIa administration in hemophilia. As an initial step towards that goal, here we characterize for the first time the rat FVIIa-EPCR interaction in vitro. We generated rat FVIIa by introduction of a PACE/Furin intracellular cleavage site (RKRRKR) between the light and heavy chains of rat FVII, an approach we have previously shown to result in secretion of two-chain activated FVII protease of various species (human, mouse and canine) in vitro. Recombinant rat FVIIa was purified in a two-chain form using conditioned medium from a HEK-293 stable cell line. The affinity of rat FVIIa to rat EPCR was assessed by incubation of the protease on engineered CHO-K1 cells that stably expressed full length rat EPCR. We found that rat FVIIa exhibited minimal binding to rat EPCR; in contrast human FVIIa bound rat EPCR with a Kd of ~400 nM. The lack of EPCR binding of rat FVIIa, similar to what we previously observed with mouse FVIIa (Pavani G et al., 2014 and 2016), complicates studies aimed at deciphering the hemostatic effects of the FVIIa-EPCR interaction in hemophilia using the rat. Therefore, we decided to engineer a rat FVIIa molecule with EPCR binding capacity so that it can be compared to rat FVIIa (non-EPCR binder) in subsequent in vivo experiments. For this, a rat FVIIa chimeric protease was generated using the first 11 amino acids of the Gla domain of rat protein C (rat FVIIa-FVRAG, with substitutions L4F, L8V, W9R, S10A, S11G). Using CHO-K1 expressing rat EPCR, we found that purified recombinant rat FVIIa-FVRAG bound rat EPCR with a Kd of ~500 nM, comparable to human FVIIa. Considering that the Gla domain, where the modifications are located, is essential for protease function, we also determined the procoagulant activity of rat FVIIa-FVRAG vs. FVIIa. We performed a thrombin generation assay in which each protease was added at increasing concentration (0-500 nM) and we measured the lag time, peak thrombin, endogenous thrombin potential. We found that rat FVIIa, rat FVIIa-FVRAG and human FVIIa (control) behaved similarly in all measured parameters for each concentration tested. In conclusion, we have defined the lack of EPCR binding of rat FVIIa and characterized a recombinant rat FVIIa molecule that can bind rat EPCR in vitro as a gain of function, but is otherwise similar to rat FVIIa. This molecule can now be used to probe the FVIIa-EPCR interaction in the novel and relevant hemophilia A rat model. Disclosures Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol Myers Squibb: Other: Salary (spouse); Novo Nordisk A/S: Research Funding.

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.

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.

scholarly journals Prothrombin Membrane Binding and Gla-Dependent Function Are Not Required for Effective Hemostasis In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 124-124
Author(s):  
Lindsey A. Greene ◽  
Nabil K. Thalji ◽  
Harlan Bradford ◽  
Sriram Krishnaswamy ◽  
Rodney M. Camire
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Membrane Binding ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Full Length ◽  
Vessel Occlusion ◽  
Gla Domain

Abstract Prothrombin, like other vitamin K-dependent coagulation factors, undergoes γ-carboxylation of its Gla domain, a posttranslational modification critical for membrane binding. In patients on anticoagulant treatment with warfarin, the INR has historically been correlated with the degree of des-gamma-carboxy-prothrombin (DCP or PIVKA-II). PIVKA-II can be measured readily and used as a marker for vitamin K deficiency or warfarin therapy and is thought to be useful in detecting subclinical disease. Long-standing dogma suggests prothrombin γ-carboxylation is necessary for prothrombin membrane binding facilitating engagement with prothrombinase leading to rapid thrombin generation and effective hemostasis. However, recent studies indicate that despite an inability to bind membranes, uncarboxylated (desGla) full-length prothrombin demonstrated an unexpected modest decrease in the rate of thrombin generation (J Biol Chem 2013, 288:27789-800). Thus, it is possible loss of prothrombin γ-carboxylation, and thereby membrane binding, is far less significant for prothrombin activation than previously appreciated. Instead warfarin's effect on other coagulation factors (FX, FIX, and FVII) may be the primary causative determinant impairing hemostasis in these anticoagulated patients. To test these ideas, we first analyzed thrombin generation using recombinant full-length fully carboxylated and desGla prothrombin in vitro. Human prothrombin deficient plasma (Factor II activity <4%) was reconstituted to normal levels (100 μg/mL) with desGla or carboxylated prothrombin. DesGla prothrombin generated approximately half the amount of thrombin observed in carboxylated prothrombin plasma and normal human plasma controls. We next analyzed full-length desGla prothrombin's in vivo hemostatic function. A prothrombin anti-sense oligonucleotide (ASO) was administered to hemostatically normal mice to knock down endogenous murine prothrombin expression (<0.1-1μg/mL, 0.1-1%) and confirmed by ELISA analysis. Hemostasis was analyzed by the ferric chloride (FeCl3) carotid artery injury model. In mice treated with an ASO control, vessel occlusion occurred at approximately 8 minutes while mice treated with the prothrombin ASO did not clot during the 30-minute post injury observation period. In additional experiments two minutes following injury, prothrombin ASO treated mice were administered either carboxylated or desGla recombinant prothrombin to restore plasma concentrations to the normal range (100 μg/mL). Remarkably, administration of either desGla or carboxylated prothrombin restored vessel occlusion to ASO control findings, with minimal variability observed between desGla and carboxylated prothrombin treated mice (Figure 1). Warfarin treatment results in impaired prothrombin γ-carboxylation. However, if prothrombin γ-carboxylation, is, in fact, not necessary for prothrombin activation, fully carboxylated Factor Xa (FXa) should reverse the effects of warfarin by efficiently activating the un/under-carboxylated prothrombin thereby bypassing the other warfarin-affected factors. To study this, we used a "zymogen-like" factor Xa (FXaI16L) molecule previously developed by our group (Nat. Biotech 2011, 29:1028-33) that has a greater half-life than the wild-type protein. In thrombin generation assays, addition of 1nM FXaI16L to plasma from patients anticoagulated with warfarin, irrespective on INR (2.8, 4,4 7.1), resulted in thrombin generation comparable to that of normal human plasma. Importantly, similar results were obtained in vivo in warfarin-anticoagulated mice (INR 2-3). Administration of 3 mg/kg FXaI16L to 8 out of 8 warfarin mice corrected the time to carotid artery occlusion in the FeCl3 injury model. In two separate in vitro and in vivo model systems, we demonstrated that prothrombin membrane binding is not absolutely required for thrombin generation. Thrombin is unique among the coagulation serine proteases in that it does not have a Gla domain once fully processed by prothrombinase; thus, the absence of a Gla domain in the protease (thrombin) may explain the lack of a requirement for membrane binding by the zymogen (prothrombin) precursor. Our findings may also have clinical relevance, since they suggest that FXa (or a variant) could be used as a novel warfarin bypass strategy to rapidly achieve hemostasis in the setting of warfarin anticoagulation. Figure 1. Figure 1. Disclosures Greene: Baxter: Research Funding. Camire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding.

scholarly journals Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo

2010 ◽  
Vol 103 (06) ◽  
pp. 1239-1244 ◽  
Author(s):  
Miguel Centelles ◽  
Cristina Puy ◽  
Jacinto Lopez-Sagaseta ◽  
Kenji Fukudome ◽  
Ramón Montes ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombus Formation ◽  
Activated Protein C ◽  
Endothelial Protein C Receptor ◽  
Vessel Occlusion ◽  
Total Occlusion ◽  
Thrombosis Risk ◽  
Injury Model ◽  
First Time

SummaryThe endothelial protein C receptor (EPCR) plays an anticoagulant role by improving protein C activation. Although low levels of activated protein C (APC) constitute a thrombosis risk factor, the relationship between modulating EPCR function and thrombosis has not been addressed so far. Monoclonal antibodies (mAb) against murine EPCR were raised, and their ability to block protein C/APC binding was tested. The ferric chloride carotid artery injury model in mice was chosen to test the effect of anti-EPCR mAb on thrombus formation. The time to total occlusion of the vessel was analysed in three groups, given an isotype control mAb (IC), a blocking (RCR-16) or a non-blocking (RCR-20) anti-EPCR mAb. RCR-16 prevented the interaction between protein C/APC and EPCR as demonstrated by surface plasmon resonance and flow cytometry, and inhibited the activation of protein C on the endothelium. IC and RCR-20 were unable to induce such effects. In vivo, RCR-16 shortened the time to total vessel occlusion with respect to IC [13.4 ± 1.0 (mean ± SD) and 17.8 ± 3.2 minutes, respectively, p<0.001]. Occlusive thrombi lasting for more than one hour were observed in all RCR-16-treated animals, but only in 43% of IC-treated ones. Results with RCR-20 were indistinguishable from those observed with IC. For the first time, a direct relationship between blocking EPCR and thrombosis is demonstrated. Blocking anti-EPCR autoantibodies can predispose to thrombosis episodes and may constitute a new therapeutic target.

Sulforaphane inhibits endothelial protein C receptor shedding in vitro and in vivo

2014 ◽  
Vol 63 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Sae-Kwang Ku ◽  
Min-Su Han ◽  
Jong-Sup Bae
Keyword(s):  
Protein C ◽  
Endothelial Protein C Receptor ◽  
Receptor Shedding

Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sanjana Dayal ◽  
Sean X Gu ◽  
Katinan M Wilson ◽  
Ryan Hutchins ◽  
Steven R Lentz
Keyword(s):  
Superoxide Dismutase ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Oxidative Modification ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Experimental Thrombosis

In vitro studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo . We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that Sod1-/- mice formed stable occlusions significantly faster than Sod1+/+ mice (P<0.05). In an inferior vena cava (IVC) stasis model Sod1- /- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs. Sod1+/+ mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from Sod1-/- and Sod1+/+ mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in Sod1-/- and Sod1 +/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in Sod1 -/- mice (P<0.05 vs. Sod1 +/+ mice). Further, in vivo generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in Sod1-/- mice (P<0.05 vs. Sod1+/+ mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1 -/- mice vs. Sod1 +/+ mice, suggesting that altered generation of activated protein C in Sod1-/- mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.

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