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

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.

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

Blood ◽

10.1182/blood-2019-130596 ◽

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.

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

Blood ◽

10.1182/blood.v120.21.18.18 ◽

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.

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

Blood ◽

10.1182/blood.v126.23.124.124 ◽

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.

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

Blood ◽

10.1182/blood-2018-99-116892 ◽

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.

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Five novel mutations located in exons III and IX of the protein C gene in patients presenting with defective protein C anticoagulant activity

Blood ◽

10.1182/blood.v82.1.159.bloodjournal821159 ◽

1993 ◽

Vol 82 (1) ◽

pp. 159-168 ◽

Cited By ~ 20

Author(s):

S Gandrille ◽

M Alhenc-Gelas ◽

P Gaussem ◽

MF Aillaud ◽

E Dupuy ◽

...

Keyword(s):

Amino Acids ◽

Protein C ◽

Denaturing Gradient Gel Electrophoresis ◽

Anticoagulant Activity ◽

Direct Sequencing ◽

Catalytic Triad ◽

Activity Levels ◽

Novel Mutations ◽

Protein C Deficiency

We describe five families presenting with type II hereditary protein C deficiency characterized by normal antigen and amidolytic activity levels but low anticoagulant activity. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining amplification by the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis of the amplified fragments, and direct sequencing of fragments displaying altered melting behavior. We detected five novel mutations. Three were located in the C-terminal part of the propeptide encoded by exon III: Arginine (Arg)-5 to tryptophan (Trp), Arg-1 to histidine (His), and Arg-1 to cysteine (Cys) mutations. The two others, located in exon IX, affected Arg 229 and serine (Ser) 252, which were respectively replaced by glutamine (Gln) and asparagine (Asn). DNA studies of the other exons from affected individuals showed no other abnormalities. These novel mutations provide further insight into the importance of the affected amino acids located close to the active site, near Asp 257, one of the three amino acids of the catalytic triad. The low anticoagulant activity of the abnormal protein C indicated that Arg 229 and Ser 252 play a key role during the interaction between protein C and its cofactor protein S, phospholipids, or factors Va and VIIIa. The Arg-1 to Cys mutation led to the dimerization of protein C with another plasmatic component, as evidenced by the presence in the plasma of a high molecular weight form of protein C that disappeared after reduction. No molecular mass abnormalities were observed in heavy and light chains of all other protein C mutants. In the five families explored, 9 (64%) of the 14 subjects bearing the mutations reported thrombotic events. This suggests that the protein C amino acids affected by the mutations are very important for the in vivo expression of the antithrombotic properties of protein C.

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Correlates of Lenalidomide Induced Immune Stimulation and Response in CLL: Analysis in Patients on Treatment

Blood ◽

10.1182/blood.v118.21.979.979 ◽

2011 ◽

Vol 118 (21) ◽

pp. 979-979 ◽

Cited By ~ 1

Author(s):

Georg Aue ◽

Stefania Pittaluga ◽

Delong Liu ◽

Larry Stennett ◽

Susan Soto ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

Expression Profiling ◽

Research Funding ◽

Immune Stimulation ◽

Intramural Research Program ◽

Pre Treatment ◽

Program Research

Abstract Abstract 979 Lenalidomide's mechanism of action in chronic lymphocytic leukemia (CLL) is not well understood. In vitro data suggest that anti-leukemic immune responses are important. Tumor flare reactions during treatment have been associated with response in some but not other studies. In vivo data that mechanistically link immune stimulation to clinical responses are lacking. We designed an independent, single center, phase II trial of lenalidomide in relapsed/refractory CLL (clinicaltrials.gov: NCT00465127). Here we report final clinical data and results of multiple translational analyses that indicate that an IFNy centered immune response is critical for response. A 3 week on, 3 weeks off treatment scheme (42 day cycles) was chosen to pulse immune stimulation while trying to minimize myelosuppression. The starting dose was 20 mg daily for the first 10 patients and 10 mg for the subsequent 23. Response was measured at 24 weeks. 5 patients, 4 with del 17p, achieved a PR by IWCLL criteria (16%) and were eligible to continue drug for 4 more cycles; the PFS in these patients was 16 months compared to 7 months for all other (p<0.001). Myelosupression remained the limiting side effect. A cytokine release syndrome often accompanied by tumor flare reactions was seen in 78% of patients in cycle 1 and often recurred in subsequent cycles. Compared to other studies it appears that the long treatment free period increased the inflammatory reaction upon restarting of L. All correlative analyses reported here were performed on PBMCs, lymph node (LN) core biopsies and serum obtained from patients during cycle 1 and 2 and included flow cytometry, gene expression profiling (Affymetrix arrays), and cytokine measurements. Nine patients with decreased lymphadenopathy ≥10% (10–85%) on CT after 4 cycles were considered responders (R) for correlative studies. There was a significant decrease in CLL count (median 14% on day 8 and 49% on day 22, p<0.01) and in the number of circulating T (CD3, CD4, CD8) and NK-cells (n=22, p<0.05) with no difference between R and non-responders (NR). In contrast, the CD3 count in LN core biopsies increased 1.4 fold in R compared to matched pre-treatment biopsies (p<0.05) with no change in NR (0.95 fold). In the L free interval CLL cells rebounded to pre-treatment levels. A rapid rebound of CLL counts during treatment interruptions has been previously described but its mechanism is not well understood. In migration assays we observed a 3-fold increased migration towards SDF-1 for L compared to control cells (p=0.03), indicating that increased homing of lymphocytes to tissue sites may be responsible for the rapid decrease in peripheral counts. The cell surface molecules CD40, 54, 86, 95, DR5 were upregulated (p<0.05) while CD5 and 20 were downregulated (p<0.001) on circulating CLL cells. Effects on CD54 and CD5 were stronger in R than NR (p<0.05). Next we performed gene expression profiling on purified PB-CLL cells and LN core biopsies obtained on day 8. L induced upregulation of 95 genes, many of which are known to be regulated by interferon gamma (IFNγ). The comparison with a gene expression signature induced by recombinant IFNγ in CLL cells cultured in vitro confirmed the significant induction of a typical IFNγ response by L in vivo (n=24, p<0.0001). The IFNγ response in PB-CLL cells was no different in R vs NR (n=12, p=0.78), but in LN biopsies it was more prominent in R (n=7) than NR (n=5) (p<0.05). Consistently the IFNG gene was upregulated in LN biopsies of R but actually decreased in NR (p=0.001). Serum IFNγ levels were elevated on L (n=14 at all time points, day 4 p=0.03, day 8 p=0.01, day 22 p=0.02, day 49 p<0.01), but off drug returned to pretreatment levels. Next we sought to determine the source of IFNγ. The tumor cells are ruled out as IFNG was not expressed in purified CLL cells. By flow cytometry the number of IFNγ secreting CD4 T-cells increased on day 8 from 0.8% to 1.5%, p=0.006), an effect that was stronger in R had than NR (p<0.05). IFNγ positive NK cells did not increase on L. These data provide a first mechanistic link between the degree of Lenalidomide induced immune activation to clinical response in CLL. Based on our experience we suggest that continued dosing of L may be superior to dose interruptions. Disclosures: Aue: NHLBI, Intramural Research Program: Research Funding. Off Label Use: Lenalidomide is not FDA approved for CLL. Wiestner:NHLBI, Intramural Research Program: Research Funding.

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Effect Of Combined Heparin- and Antithrombin-Binding Exosite Mutations On Human Factor IX(a) Coagulant Activity, Thrombin Generation, and Protease Half-Life In Human Plasma

Blood ◽

10.1182/blood.v122.21.2333.2333 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2333-2333

Author(s):

Pamela R. Westmark ◽

Pansakorn Tanratana ◽

John P. Sheehan

Keyword(s):

Human Plasma ◽

Half Life ◽

Research Funding ◽

Thrombin Generation ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Coagulant Activity ◽

Plasma Half Life

Abstract Introduction Hemophilia B is an X-linked genetic disorder characterized by defective factor IX activity. Recombinant factor IX (rFIX) is employed as protein replacement for the treatment and prophylaxis of bleeding episodes. Antithrombin is the primary plasma inhibitor of activated factor IX (FIXa), and inhibition is enhanced by heparin/heparan sulfate. We hypothesize that selective disruption of protease interactions with heparin and antithrombin via mutations in the respective heparin- and antithrombin-binding exosites may enhance rFIX(a) efficacy by prolonging protease half-life in vivo. Aim To assess the effect of mutations in the FIX(a) heparin- and antithrombin-binding exosites on traditional coagulant activity, thrombin generation, and protease half-life in human plasma. Methods Human FIX cDNA constructs with alanine substitutions (chymotrypsinogen numbering) in the heparin exosite (K126A, K132A, K126A/K132A), antithrombin exosite (R150A), or both (K126A/R150A, K132A/R150A, K126A/K132A/R150A) were expressed in HEK293 cell lines. Recombinant zymogens were purified from conditioned media, and a portion activated to protease with human factor XIa. Zymogen and protease forms were characterized in APTT-based clotting assays, and tissue factor (TF) and FIXa-initiated thrombin generation (TG) assays in pooled human FIX-deficient plasma, respectively. Comparisons were made with human plasma-derived factor IX (pFIX) and recombinant FIX wild type (WT). Protease half-life in pooled, citrated human plasma was determined using a novel assay that detects FIXa activity by TG response. Results Zymogen coagulant activities (% WT ± S.E) were: pFIX 105.2 ± 2.8, WT 100 ± 7.1, K132A/R150A 75.8 ± 3.4, K126A 63.3 ± 2.3, R150A 62.4 ± 4.0, K132A 30.9 ± 1.0, K126A/R150A 27.0 ± 2.1, K126A/K132A 20.6 ± 9.2, and K126A/K132A/R150A 7.3 ± 3.8. Similarly, protease coagulant activities were: WT 100 ± 6.1, pFIXa 98.4 ± 11.4, K132A 91.4 ± 1.6, K132A/R150A 84.9 ± 2.8, R150A 77.1 ± 5.8, K126A 39.5 ± 2.4, K126A/R150A 25.3 ± 2.8, K126A/K132A/R150A 10.9 ± 0.6, and K126A/K132A 9.3 ± 0.6. In contrast to their relative coagulant activities, FIX K126A (1.9-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased peak thrombin concentrations during TF-triggered TG; pFIX, FIX K132A and K126A/R150A were similar to WT; and FIX K126A/K132A/R150A (0.6-fold) and K126A/K132A (0.2-fold) demonstrated marked reductions in peak thrombin relative to WT. In the FIXa-initiated TG assay, FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased peak thrombin concentrations; pFIXa, FIXa K132A, R150A, and K126A (0.8-1.0 fold) were similar to WT; while FIXa K126A/K132A and K126A/K132A/R150A demonstrated markedly reduced (0.2-0.3 fold) and delayed peak thrombin concentrations. In pooled, citrated FIX-deficient plasma, FIXa WT (40.9 ± 1.4 min) and K126A/K132A (37.2 ± 0.7 min) demonstrated similar half-lives, while FIXa R150A, K126A/R150A, and K132A/R150A all had half-lives > 2 hr. Conclusions Single exosite mutations resulted in mild to moderate reductions in coagulant activity, while the double mutation in the heparin exosite (K126A/K132A) markedly reduced activity, likely due to a synergistic effect on cofactor binding. Traditional coagulant activity did not accurately represent the ability of the mutant proteins to support thrombin generation. Despite variable reductions in coagulant activity, FIX K126A, K132A, R150A, K126A/R150A and K132A/R150A supported levels of plasma thrombin generation that were equal to or greater than FIX WT. The plasma half-life of FIXa WT activity was remarkably lengthy, and while mutations in the heparin exosite had negligible effects, R150A in the antithrombin exosite substantially increased protease half-life, consistent with a primary role for antithrombin in the plasma inhibition of FIXa. Thus, single exosite mutations did not significantly disrupt the procoagulant function of human FIX(a), and combined exosite mutations (K126A/R150A and K132A/R150A) maintain or enhance plasma thrombin generation while disrupting exosite-mediated regulatory mechanisms. The combination of intact procoagulant function with disruption of antithrombin- and heparin-mediated regulation of FIX(a) will potentially enhance in vivo recovery, prolong plasma half-life, and enhance the efficacy of hemophilia B replacement therapy. Disclosures: Sheehan: Novo Nordisk Access to Insight Basic Research Grant: Research Funding; Bayer Hemophilia Awards Program: Research Funding; Diagnostica Stago: reagents, reagents Other.

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Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement

Blood ◽

10.1182/blood.v112.11.20.20 ◽

2008 ◽

Vol 112 (11) ◽

pp. 20-20

Author(s):

Roger JS Preston ◽

Jennifer A Johnson ◽

Fionnuala Ni Ainle ◽

Shona Harmon ◽

Owen P. Smith ◽

...

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Anticoagulant Activity ◽

Protein S ◽

Endothelial Barrier ◽

Platelet Factor ◽

Barrier Permeability ◽

Gla Domain ◽

Anti Inflammatory

Abstract Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

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Single-Stranded Oligonucleotide Binding to P-Selectin Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to Endothelial Cells in Sickle Cell Model Mice: Novel Therapeutics for Vaso-Occlusive Episodes.

Blood ◽

10.1182/blood.v114.22.1545.1545 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1545-1545

Author(s):

Diana Gutsaeva ◽

James Parkerson ◽

Robert G Schaub ◽

Jeffrey Kurz ◽

Alvin Head

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Endothelial Cells ◽

Carotid Artery ◽

Blood Cells ◽

Research Funding ◽

Leukocyte Rolling ◽

Adhesive Interactions

Abstract Abstract 1545 Poster Board I-568 To date, a variety of therapeutic approaches to sickle cell disease (SCD) have been explored, however, knowledge about the efficacy of anti-adhesive agents in this disorder is limited. In SCD, P-selectin expressed on endothelial cells plays a key role in leukocyte recruitment as well as in the adhesion of sickle red blood cells (sRBC) to the endothelium. The interaction of P-selectin and its ligands is thus likely to contribute to impairment of the microvascular flow and thereby to the development of painful vaso-occlusive episodes. Studies with anti-P-selectin antibody and P-selectin-deficient mice support the notion that P-selectin-directed interventions may be a potential approach to the treatment of vaso-occlusive episodes in SCD. Aptamers, short single-stranded oligonucleotides, have been developed for a wide range of therapeutic targets (Keefe & Schaub, 2008). Aptamers bind molecular targets with high affinity and specificity, do not elicit immune responses, and can be readily delivered to the vascular compartment intravenously or subcutaneously. Although anti-P-selectin aptamers have been shown to inhibit leukocyte rolling in vitro and to display efficacy in mouse models for inflammation, the anti-adhesion activity of anti-P-selectin aptamers has never been evaluated in SCD. The purpose of this study was to determine in vivo whether the anti-P-selectin aptamer ARC5690 can inhibit adhesion of sickle RBC and leukocytes to vascular endothelial cells in the bone marrow microvasculature of SCD model mice. Knockout-transgenic SCD mice generated by Ryan et al. (Ryan et al., 1997) were used in this study. SCD mice were injected with 20 mg/kg ARC5690, scrambled aptamer ARC5694 (negative control), or vehicle (saline). Anti-P-selectin monoclonal antibody was also used as a positive control. After 2.5 hours, the mice were subjected to 1 hour of hypoxia (12% O2 in air) followed by 1 hour of reoxygenation at room air. Intravital observations of the bone marrow microcirculation were performed to monitor adhesive interactions between blood cells and endothelial cells. Leukocytes were labeled in vivo with rat anti mouse CD45 antibody conjugated with PE which were infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery. Effects of ARC5690 on sRBC and leukocyte adhesive interactions were compared to those of ARC5694 and vehicle. Administration of the anti-P-selectin aptamer ARC5690 significantly reduced sRBC adhesion (p<0.05) in SCD mice compared to those treated with vehicle. The anti-adhesive activity of ARC5690 for sRBC was comparable to anti-P-selectin monoclonal antibody, whereas the scrambled aptamer did not show any anti-adhesive activity. We also studied the effects of the anti-P-selectin-specific ARC5690 on leukocyte flow dynamics. Mice pretreated with ARC5690, but not those treated with scrambled aptamer ARC5694, showed a greater than four-fold decrease in leukocyte rolling (p<0.001) and a six-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Again, the anti-adhesive activity of ARC5690 and its effect on leukocyte rolling and adhesion were similar to those of the anti-P-selectin antibody. Furthermore, ARC5690 improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and d wall shear rates. In conclusion, our study demonstrates significant anti-adhesive activities of ARC5690, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in SCD model mice. ARC5690 may represent a novel therapeutic strategy that can be used to treat vaso-occlusive episodes in SCD Disclosures Gutsaeva: Archemix Corporation: Research Funding. Parkerson:Archemix Corporation: Research Funding. Schaub:Archemix: Research Funding. Kurz:Archemix Corporation: Employment. Head:Archemix Corporation: Research Funding.

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

Blood ◽

10.1182/blood.v118.21.531.531 ◽

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.

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