Inhibition Of Activated Protein C Research Articles

Biased agonism of protease-activated receptor-1 regulates thromboinflammation in murine sickle cell disease

AbstractSickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOEs). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOEs, and end-organ damage. Coagulation proteases such as thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein–coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either proinflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation, and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and tumor necrosis factor α challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we used transgenic mice with point mutations at 2 PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared with wild type PAR1 or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.

Carbon Nanofiber-Ionic Liquid Nanocomposite Modified Aptasensors Developed for Electrochemical Investigation of Interaction of Aptamer/Aptamer-Antisense Pair with Activated Protein C.

Selective and sensitive detection of human activated protein C (APC) was performed herein by using carbon nanofiber (CNF) and ionic liquid (IL) composite modified pencil graphite electrode (PGE) and electrochemical impedance spectroscopy (EIS) technique. A carbon nanomaterial-based electrochemical aptasensor was designed and implemented for the first time in this study for the solution-phase interaction of DNA-Apt with its cognate protein APC as well as APC inhibitor aptamer-antidote pair. The applicability of this assay developed for the determination of APC in fetal bovine serum (FBS) and its selectivity against different proteins (protein C, thrombin, bovine serum albumin) was also examined. CNF-IL modified aptasensor specific to APC provided the detection limit as 0.23 μg/mL (equal to 3.83 nM) in buffer medium and 0.11 μg/mL (equal to 1.83 nM) in FBS. The duration of the proposed assay from the point of electrode modification to the detection of APC was completed within only 55 min.

Thrombin spatial distribution determines protein C activation during hemostasis and thrombosis

Rebalancing the hemostatic system by targeting endogenous anticoagulant pathways, like the protein C (PC) system, is being tested as a means of improving hemostasis in patients with hemophilia. Recent intravital studies of hemostasis demonstrated that, in some vascular contexts, thrombin activity is sequestered in the extravascular compartment. These findings raise important questions about the context-dependent contribution of activated PC (APC) to the hemostatic response, because PC activation occurs on the surface of endothelial cells. We used a combination of pharmacologic, genetic, imaging, and computational approaches to examine the relationships among thrombin spatial distribution, PC activation, and APC anticoagulant function. We found that inhibition of APC activity, in mice either harboring the factor V Leiden mutation or infused with an APC-blocking antibody, significantly enhanced fibrin formation and platelet activation in a microvascular injury model, consistent with the role of APC as an anticoagulant. In contrast, inhibition of APC activity had no effect on hemostasis after penetrating injury of the mouse jugular vein. Computational studies showed that differences in blood velocity, injury size, and vessel geometry determine the localization of thrombin generation and, consequently, the extent of PC activation. Computational predictions were tested in vivo and showed that when thrombin generation occurred intravascularly, without penetration of the vessel wall, inhibition of APC significantly increased fibrin formation in the jugular vein. Together, these studies show the importance of thrombin spatial distribution in determining PC activation during hemostasis and thrombosis.

Regulation of Factor VIII By Activated Protein C: Assessment In Vitro and In Vivo

Factor VIII (FVIII) plays an essential role in the activation of factor X (FX), acting as a factor IXa (FIXa) cofactor in the intrinsic Xase complex. Activated protein C (APC) is a serine protease that inactivates FV/Va. Resistance of FV/FVa to APC due to the Leiden mutation is the most common inherited thrombophilia. By cleaving FVIII/FVIIIa at Arg336 and Arg 562, APC can also inactivate FVIII; however, the role of this reaction, especially in vivo, is not well defined. Further, thrombophilic mutations at either of these sites have yet to be identified, calling into question the importance of this reaction. Understanding the role of APC in FVIII regulation is important as there are several novel therapeutics in development for hemophilia A (HA) that either bypass FVIII regulatory pathways or target inhibition of APC. Additionally, while data using a high-specific activity factor IX variant has been successful in a hemophilia B gene transfer trial, there is no such described hyperactive FVIII protein. In principle, manipulation of APC cleavage sites may result in a more procoagulant FVIII protein with enhanced hemostastic function that may serve as a novel therapeutic for HA in the setting of a recombinant protein treatment or gene therapy.We sought to further understand the role of APC regulation of FVIII and to evaluate the in vivo hemostatic effect of an APC-resistant FVIII as a potential treatment strategy for HA. We conducted site-directed mutagenesis to introduce missense arginine to glutamine mutations at FVIII APC cleavage sites (R336Q and R562Q) on a B-domain deleted FVIII (FVIII-WT) backbone. The resulting recombinant, purified FVIII variant protein (FVIII-QQ) was then compared to FVIII-WT. Following incubation with APC, SDS-PAGE analysis of FVIII-QQ demonstrated no apparent cleavage by APC. In contrast, FVIII-WT demonstrated expected cleavage at R336 and R562, thereby confirming FVIII-QQ is resistant to APC cleavage. Surprisingly, one-stage clotting assay determination of FVIII-QQ specific activity was approximately 30% higher than FVIII-WT; this was unexpected given the absence of thrombomodulin or APC. We speculate this may be related to the ability of FXa to cleave FVIIIa at Arg336 and Arg562 inactivating FVIIIa that is likely disrupted in the FVIII-QQ variant (Eaton D et al. Biochemistry 1986 and Plantier JL et al. J Thromb Haemost 2010). To test the role of APC regulation of FVIII, we performed plasma-based in vitro thrombin generation assays in the presence of APC. Human FVIII deficient plasma was reconstituted to 1 nM (normal plasma FVIII concentration) with FVIII-QQ or FVIII-WT wherein increasing concentrations of APC were then added and thrombin generation was measured. Consistent with the known role of APC being an anticoagulant, the amount of thrombin generation decreased with increasing APC concentrations. However, the amount of APC necessary to inhibit 50% peak thrombin generation with FVIII-WT was 5.2 nM ± 0.3 nM versus FVIII-QQ was 11.3 nM ± 0.8 nM, which again demonstrated FVIII-QQ is resistant to APC inhibition. We suspect the observed decrease in thrombin generation with increasing APC concentrations is due to the role of APC in FV/FVa inhibition. Overall, our data support a role for APC inactivation of FVIIIa in this in vitro system. Interestingly, FVIII-QQ reliably demonstrated an approximate 2-fold increase in peak thrombin in the thrombin generation assay. This is being explored, but we again speculate this may be related to the effects of FXa on FVIII-WT at R336 and R562.We next evaluated the in vivo hemostatic efficacy of FVIII-WT vs. FVIII-QQ in HA mice. HA mice were infused with FVIII-WT or FVIII-QQ protein prior to tail clip assay. Comparison of both proteins at a dose of 3 µg/kg demonstrated greater blood loss in HA mice treated with FVIII-WT vs. FVIII-QQ (p=0.006). Further, blood loss in HA mice treated with FVIII-WT was greater than wild type mice (p=0.007); in contrast, there was no difference between wild type and HA mice treated with 3 µg/kg of FVIII-QQ (p=0.26).Our data demonstrate FVIII-QQ has a higher specific activity, requires 2-fold greater APC concentrations to inhibit thrombin generation in vitro and an approximate 2-fold greater in vivo hemostastic efficacy. Our results support that APC has physiologic significance in FVIII/FVIIIa inactivation and provide rationale for the study of APC in FVIII/FVIIIa regulation to develop potential novel HA therapeutics. DisclosuresDavidson:Spark Therapeutics: Consultancy. Camire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Bayer: Consultancy, Research Funding. George:Pfizer: Consultancy; Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC’s anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC’s cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC’s anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

A Refined Plasma Thrombin Generation Assay Identifies Functional Importance of the First Two EGF‐like Domains of the Anticoagulant Protein S

Protein S (PS) is an anticoagulant cofactor for the serine protease Activated Protein C (APC) and the serine protease inhibitor tissue factor pathway inhibitor (TFPI), which inhibit coagulation factors Va (FVa) and Xa (FXa), respectively. FVa and FXa function together to activate thrombin, the enzyme that produces a clot. In addition to these roles, PS also controls the complement system, with ~60% of plasma PS circulating bound to the complement inhibitor C4‐binding protein (C4BP). The remaining ~40% of PS is the anticoagulant pool. Despite its many functions, the physiologic role of PS in coagulation is unclear, because PS concentration does not correlate with plasma thrombin production ex vivo. We sought to develop a thrombin generation assay that is sensitive to PS concentration and to use it to assess the importance of the PS/APC interaction in human plasma.We hypothesized that standard thrombin generation assays are insensitive to PS because APC activation requires the endothelial cell protein thrombomodulin (TM). Thus, we supplemented human plasma with TM. This resulted in a dose‐dependent decrease in thrombin generation when PS was added to PS‐depleted plasma, with a physiologic concentration of 150nM PS causing a 75% reduction. In the absence of TM, only a 12% decrease was observed.We next validated the assay by measuring PS concentration and thrombin generation in plasma samples from 10 healthy controls and 27 patients with Human Immunodeficiency Virus, a condition known to cause PS deficiency. 63% of the patient samples were deficient in PS compared to the control samples (p=0.039). In the absence of TM, plasma PS did not correlate with thrombin generation, consistent with previous reports. In the presence of TM, there was a negative correlation, with decreased PS being associated with increased thrombin peak (p=0.024) and total thrombin produced (p=0.011).Finally, we produced a recombinant protein consisting of the first two epidermal growth factor‐like domains of PS (EGF1‐2), which contain the putative APC binding sites. In a purified protein assay, APC inhibition of thrombin activation was greater in the presence of PS. EGF1‐2 dose‐dependently reversed the effect of PS, with 75% reversal achieved with 200nM EGF1‐2. Unexpectedly, EGF1‐2 had the opposite effect in plasma thrombin generation assays and displayed potent anticoagulant activity. To explain these results, we hypothesize that the EGF1‐2 domains also form part of the C4BP binding site and its addition resulted in release of PS from C4BP, increasing the anticoagulant PS pool. Consistent with this, EGF1‐2 had no effect on thrombin generation in PS‐depleted plasma, which is also depleted of C4BP, but upon PS supplementation, had the expected procoagulant activity.We have developed a PS‐sensitive plasma thrombin generation assay, which we validated using a cohort of PS‐deficient HIV patients. We have also produced a recombinant EGF1‐2 protein which competed with PS for APC binding and inhibited APC function, but had anticoagulant effect in plasma. These data suggest that attempts to pharmacologically inhibit PS must adjust for the C4BP‐bound pool.Support or Funding InformationSupported by NHLBI grants HL056652, HL138179, HL129193.

Design and characterization of α1-antitrypsin variants for treatment of contact system–driven thromboinflammation

AbstractThe contact system produces the inflammatory peptide bradykinin and contributes to experimental thrombosis. C1 esterase-inhibitor (C1INH) deficiency or gain-of-function mutations in factor XII (FXII) cause hereditary angioedema, a life-threatening tissue swelling disease. C1INH is a relatively weak contact system enzyme inhibitor. Although α1-antitrypsin (α1AT) does not naturally inhibit contact system enzymes, a human mutation (M358R; α1AT-Pittsburgh) changes it into a powerful broad-spectrum enzyme inhibitor. It blocks the contact system, but also thrombin and activated protein C (APC), making it an unattractive candidate for therapeutic contact system blockade. We adapted the reactive center loop of α1AT-Pittsburgh (AIPR/S) to overcome these obstacles. Two α1AT variants (SMTR/S and SLLR/S) strongly inhibit plasma kallikrein, activated FXII, and plasmin. α1AT-SMTR/S no longer inhibits thrombin, but residually inhibits APC. In contrast, α1AT-SLLR/S residually inhibits thrombin, but no longer APC. Additional modification at the P1′ position (S→V) eliminates residual inhibition of thrombin and APC for both variants, while retaining their properties as contact system inhibitors. Both α1AT-SMTR/V and -SLLR/V are superior to C1INH in reducing bradykinin production in plasma. Owing to their capacity to selectively block contact system-driven coagulation, both variants block vascular occlusion in an in vivo model for arterial thrombosis. Furthermore, both variants block acute carrageenan-induced tissue edema in mice. Finally, α1AT-SLLR/V, our most powerful candidate, suppresses epithelial leakage of the gut in a mouse model of colitis. Our findings confirm that redesign of α1AT strongly alters its inhibitory behavior and can be used for the treatment of contact system-mediated thrombosis and inflammation.

Identification of serpins specific for activated protein C using a lysate-based screening assay

Activated protein C (APC) is a powerful anticoagulant enzyme that proteolytically inactivates the cofactors of the Xase and prothrombinase complexes, factors VIIIa and Va. A common mutation in factor V, fVLeiden, confers resistance to APC leading to an increased risk of thrombosis in the normal population. However, when coinherited with haemophilia, fVLeiden reduces bleeding severity, suggesting that inhibition of APC may be a useful strategy for treatment of haemophilia. We previously reported on serpins that were rationally designed for improved specificity for APC over other coagulation serine proteases. Based on structural differences in the substrate binding pockets to either side of the P1 Arg, we mutated the P2 and P1′ residues to Lys. Although this approach achieved APC specificity, it resulted in a reduction in the rate of APC inhibition relative to the parent containing only the P1 Arg. Here we conduct site-specific random mutagenesis at the P2 and P1′ positions to determine if improvements could be made in the rate of APC inhibition. In addition to our original Lys mutations, we found that Arg and Gln also confer specificity for APC. However, in all cases specificity for APC resulted in a reduction in inhibition rate.

α2-Macroglobulin Is a Significant In Vivo Inhibitor of Activated Protein C and Low APC:α2M Levels Are Associated with Venous Thromboembolism.

Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α2-macroglobulin (α2M) have been observed by immunoblotting. Here, we report an ELISA for APC:α2M complexes in plasma. Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α2M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α2M, PCI and α1AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls. In all the in vivo experiments, α2M was a significant APC inhibitor. The VTE case-control study showed that VTE patients had significantly lower APC:α2M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α2M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α2M or of APC. The risk increased for individuals with low levels of both parameters. The APC:α2M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α2M level is associated with increased VTE risk.

Modulation of the activated protein C pathway in severe haemophilia A patients: The effects of thrombomodulin and afactor V-stabilizing fab.

The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APC's inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next-generation therapeutic targets for haemophilia treatment. To determine ex vivo the respective concentration-dependent effects of TM and a FV-stabilizing Fab on the APC regulatory pathway in severe FVIII-deficient blood and plasma. Ten severe haemophilia A subjects and one healthy control were enrolled. Blood was spiked with TM (0, 1, 2.5, 5, 10, 20.0nmol/L) and FV-stabilizing Fab (0, 3, 15, 65, 300nmol/L). The respective effects were compared to FVIII concentrations of 3- and 10% using rotational thromboelastometry clotting time (CT) and thrombin generation analysis (TGA). With 1 and 2.5nmol/L TM, 5% FVIII resulted in CT similar to the absence of TM, suggesting it completely reversed the effect of APC. Increasing TM concentrations also reduced peak thrombin generation and ETP. The addition of 300nmol/L FV-stabilizing Fab returned CT to nearly baseline, but for most subjects was less than the effects of 3- or 10% FVIII. The FV-stabilizing Fab produced similar or greater thrombin generation compared to samples with 3- or 10% FVIII. The FV-stabilizing Fab resulted in enhanced CT and TGA parameters consistent with FVIII levels of 3- and 10%. Additional studies need to further characterize how modulating the APC pathway may prove beneficial in developing new haemophilia drug targets.

Targeting activated protein C to treat hemophilia.

Purpose of reviewHemophilia is a debilitating disease, marked by frequent, painful bleeding events, joint deterioration and early death. All current treatments consist of i.v. infusions of replacement factor or other procoagulant factors, and are incompletely effective, due in part to the short half-lives of the proteins. An alternative approach is to rebalance hemostasis by inhibiting natural anticoagulant mechanisms. In this article, we explain why activated protein C (APC) is an appropriate and safe target for the treatment of hemophilia.Recent findingsA serpin (serine protease inhibitor) was engineered to specifically inhibit APC and was found to rescue hemostasis in a hemophilia mouse model, even after a severe tail clip injury. However, APC is also anti-inflammatory and has cytoprotective activities, raising safety concerns over the use of an APC inhibitor to treat hemophilia. We summarize the molecular basis of the anticoagulant and signaling activities of APC to assess the potential impact of targeting APC.SummaryWe conclude that the signaling and anticoagulant functions of APC are in spatially and kinetically distinct compartments, and that it is possible to specifically inhibit the anticoagulant activity of APC. Targeting APC with a serpin is remarkably effective and may be safe for long-term prophylactic use in the treatment of hemophilia.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

Design and characterization of an APC-specific serpin for the treatment of hemophilia

AbstractHemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1′ bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.

Targeted Inhibition of Activated Protein C Anticoagulant Activity By Monoclonal Antibody HAPC1573 for Treatment of Hemophilia

In patients with hemophilia, hereditary defects in coagulation factors result in unstable clots and recurrent bleeding. Although the current standard of care focuses on replacement factor therapy, we propose that inhibition of the anticoagulant pathways may offer novel therapeutic opportunities. Activated protein C (APC) is one of the major anticoagulants, which works by degrading factors Va and VIIIa to maintain hemostatic balance. APC also exhibits cytoprotective effects, which include antiapoptotic effects, endothelial barrier protection, and anti-inflammatory effects. Antibodies are ideally suited to inhibit the anticoagulant activity of APC while preserving its cytoprotective activity.We have developed an anti-APC monoclonal antibody (mAb), HAPC1573 (murine immunoglubulin G1/kappa), using hybridoma technology, that specifically binds to human APC (hAPC) at 3 to 7 nM binding affinity (Kd) but not to its zymogen, protein C (PC) as determined by surface plasmon resonance (SPR) using a Biacore T200 instrument (GE Healthcare, Pittsburgh, PA). To investigate the binding epitope of mAb HAPC1573, APC was inhibited with Phe-Pro-Arg-chloromethylketone (PPACK). PPACK is an irreversible inhibitor of APC and forms a covalent bond with the catalytic triad Ser195 (chymotrypsin numbering).HAPC1573 bound comparably to PPACK-hAPC and untreated hAPC coated on an enzyme-linked immunosorbent assay (ELISA) plate, suggesting that the binding epitope of HAPC1573 is located outside the active site of APC. These ELISA results were also confirmed by SPR analyses. HAPC1573 inhibited the cleavage of a small peptide substrate Spectrozyme PCa (Sekisui Diagnostics, Lexington, MA) by hAPC up to 40%. The antibody protected factors Va and VIIIa from APC-mediated inactivation in a dose-dependent manner. HAPC1573 significantly reduced activated partial thromboplastin time of hemophilic plasma and enhanced thrombin generation (assessed by thrombin generation assay) in the presence of thrombomodulin. HAPC1573 inhibited the anticoagulant activity of APC without affecting its cytoprotective functions, as measured by histone-mediated cytotoxicity assays on human umbilical vein endothelial cells (up to 300 nM HAPC1573). Given its cross-reactivity with monkey APC, the antibody was evaluated in Cynomolgus monkeys for therapeutic efficacy and safety. Intravenous administration of the antibody at 3 and 10 mg/kg significantly shortened bleeding time after injury and restored hemostasis in a dose-dependent manner in an anti-FVIII antibody-induced hemophilia monkey model (Figure). Administration of a sheep-anti-FVIII antibody (Haematologic Technologies, Inc., Essex Junction, VT) reduced plasma FVIIIa activity to below the lower limit of quantification (LLOQ; Figure [left panel]) and led to a significantly longer bleeding time in normal monkeys (Figure; right panel), recapitulating the hemophilia A phenotype. This prolonged bleeding time was partially reduced by 270 µg/kg of recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Plainsboro, NJ) used as a positive control for these studies. There was a statistically significant dose-dependent reduction in bleeding time by HAPC1573 with the 10-mg/kg dose restoring the bleeding time back to normal.This study provides in vivo proof of concept of using anti-APC antibody for hemophilia. HAPC1573 represents an anti-APC antibody with therapeutic utility for patients with hemophilia with inhibitors. [Display omitted] DisclosuresZhao:Bayer Pharmaceuticals: Employment. Yegneswaran:Bayer Healthcare Pharmaceuticals: Employment. Sim:Bayer: Employment. Patel:Bayer Pharmaceuticals: Employment. Schneider:Bayer HealthCare LLC: Employment, Patents & Royalties. McLean:Bayer: Employment, Equity Ownership. Zhu:Bayer Healthcare: Employment. Jiang:Bayer Pharmaceuticals: Employment. Gu:Bayer Pharmaceuticals: Employment. Ivens:Bayer Pharmaceuticals: Employment. Xu:Shanghai RAAS Blood Products Co.Lt: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: Patent. Bringmann:Bayer Corporation, Parmaceuticals Division: Employment. Kauser:Bayer: Employment.

In Vitro Evaluation of Aptamer-Based Reversible Inhibition of Anticoagulant Activated Protein C as a Novel Supportive Hemostatic Approach.

Activated protein C (APC) is a critical regulator of thrombin formation and thereby protects against thrombosis. On the other hand, overwhelming formation of APC increases the risk of bleeding such as in trauma-induced coagulopathy. Thus, pharmacological inhibition of APC activity may improve blood clottability in certain clinical situations. In this study, we demonstrate that the DNA aptamer HS02-52G binds with fast onset (1.118 ± 0.013 × 105 M-1 s-1) to APC and possesses a long residence time of 13.5 min within the aptamer-APC complex. Functional analysis revealed HS02-52G as a highly potent and specific inhibitor of APC in plasma and whole blood with IC50 values ≤30 nM, whose activity can be readily neutralized by the short complementary DNA molecule AD22. These features qualify the novel aptamer-antidote pair as a candidate treatment option for acute APC-related bleedings.

Effects of glycosaminoglycan from Mactra veneriformis on the protein C system and expression of relevant factors in human umbilical vein endothelial cells.

The aim of this study was to examine whether glycosaminoglycan (GAG) from Mactra veneriformis had an impact on the protein C (PC) system in rats and expression of relevant factors in human umbilical vein endothelial cells. Isotonic saline, heparin sodium (4 mg/kg), and GAG (1, 4, and 16 mg/kg) were administered to Wistar rats through caudal vein at the total volume of 0.5 ml for each injection, and the anticoagulant blood was prepared. The activities of PC and protein S, and the concentrations of activated protein C inhibitor (APCI) and thrombomodulin were detected according to the kit instructions. The expression levels of tissue factor, tissue factor pathway inhibitor and thrombomodulin genes in human umbilical vein endothelial cells were detected by the reverse transcription polymerase chain reaction method. GAG could significantly reduce the activity of PC (P < 0.05, P < 0.01) and slightly increase the concentration of APCI, and high concentration of GAG could significantly decrease the activity of protein S (P < 0.05) and significantly increase the concentration of thrombomodulin (P < 0.05). GAG could significantly downregulate the expression level of tissue factor gene (P < 0.05, P < 0.01) and significantly upregulate the expression levels of tissue factor pathway inhibitor and thrombomodulin genes (P < 0.05, P < 0.01). GAG showed the inhibitory effect on PC system in vivo. GAG might play an important role in the resistance to extrinsic coagulation pathway.

Disruption of EPCR-Dependent Functions By Plasmodium Falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Can be Rescued By a Soluble EPCR Variant

Introduction: The endothelial protein C receptor (EPCR) is essential for the functions of the protein C system. EPCR enhances the activation of protein C and facilitates the activation of protease activated receptors 1 and 3 by activated protein C (APC) that are required for its cytoprotective activities. Recently EPCR was implicated in the pathogenesis of cerebral malaria due to Plasmodium falciparum infection. Cerebral malaria results >500,000 deaths annually, and survivors often suffer neurological impairments. Expression of P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) on the surface of infected erythrocytes (IE) allows for IE sequestration on microvascular endothelial cells in the brain, which causes blood-brain barrier dysfunction, vascular leakage, edema, local thrombosis, and inflammation that ultimately results in coma and death. A subset of PfEMP1 variants, associated with cerebral malaria, was found to contain a Cysteine-rich Inter Domain Region (CIDR) subtype alpha 1 (CIDRα1) that binds with high affinity to EPCR. Here we determined the effects of CIDRα1 on EPCR-dependent functions of the protein C system. Moreover, we demonstrate that disruption of EPCR’s cellular functions by CIDRα1.1 can be effectively rescued by soluble E86A-EPCR that is devoid of (A)PC binding.Materials and Methods: EPCR binding FCR3 IT4VAR20 CIDRα1.1 and non-EPCR binding controls FCR3 IT4VAR15 CIDRα3.5, and Dd2 Dd2VAR01 CIDRa3.1 were produced in insect cells. Protein C and APC were plasma-derived and E86A- and wt-soluble EPCR (sEPCR) were produced in HEK293 cells. Cellular EPCR functions were determined on the EA.hy926 endothelial cells and endothelial permeability was determined by TER using the iCelligence (ACEA). IE adhesion assays were done on HBMECs using the P. falciparum FCR3 strain expressing IT4VAR20 PfEMP1.Results: CIDRα1.1 bound to sEPCR with an apparent Kd of 4.3 nM and was a competitive inhibitor of APC binding to sEPCR. Dose-dependent binding of CIDRα1.1 to EA.hy926 cells but not to EPCR knockdown EA.hy926 cells indicated that EPCR was the main receptor for CIDRα1.1 binding on these cells. CIDRα1.1 inhibited APC binding to EA.hy926 cells with an IC50~35 nM. CIDRα1.1 inhibited protein C activation on EA.hy926 by >75% similar to the anti-EPCR blocking antibody rcr252, whereas control CIDRα3.5 did not affect protein C activation. Furthermore, CIDRα1.1 reduced APC-mediated PAR1 cleavage on EA.hy926 cells >3-fold and accordingly abrogated APC-mediated protection against thrombin-induced barrier disruption. Control CIDRα3.1 did not affect APC-mediated barrier protection.To determine whether CIDRα1.1 inhibited APC binding to EPCR by direct competition or by steric hindrance, binding of CIDRα1.1 to E86A-sEPCR that does not bind APC was determined. Remarkably, CIDRα1.1 bound E86A-sEPCR with similar affinity compared to wt-sEPCR. Therefore, the ability of E86A-sEPCR to compete for CIDRα1.1 binding to cellular EPCR was determined. E86A-sEPCR dose-dependently restored APC binding to wt-sEPCR in the presence of CIDRα1.1. Protein C activation on EA.hy926 cells in the presence of CIDRα1.1 was partially restored at 5 nM and completely restored at 50 nM E86A-sEPCR. Likewise, E86A-sEPCR enhanced PAR1 cleavage by APC in the presence of CIDRα1.1 >4-fold and abrogated inhibition of APC’s barrier protective effects by CIDRα1.1. Finally, cytoadhesion of IE expressing full-length CIDRα1.1-containing PfEMP1 to HBMECs was inhibited ~70% by both E86A- and wt-sEPCR (IC50~30 nM).Conclusions: The binding of CIDRα1.1 to EPCR greatly compromised the ability of EPCR to enhance protein C activation and to facilitate APC-mediated PAR1 cleavage and induction of endothelial barrier protective effects. Based on the well-documented neuroprotective effects of the protein C system in the brain it is likely that the PfEMP1-induced loss of EPCR-dependent functions provide a seminal contribution to the mortality and neurological damages associated with cerebral malaria. Although both E86A- and wt-sEPCR can compete for CIDRα1.1 binding to cellular EPCR, E86A-sEPCR does not bind (A)PC and therefore does not interfere with (A)PC binding to cellular EPCR. In summary, these results provide novel insights into the possible pathogenesis of cerebral malaria and present a proof-of-concept strategy for the development of novel adjunct therapies for cerebral malaria. DisclosuresNo relevant conflicts of interest to declare.

Design, synthesis, and SAR of a series of activated protein C (APC) inhibitors with selectivity against thrombin for the treatment of haemophilia

A design strategy was used to identify inhibitors of activated protein C with selectivity over thrombin featured by a basic and/or aromatic functionality for binding to the S2 pocket. Our strongest inhibitor showed an IC50-material value and selectivity for APC vs thrombin similar to a compound previously reported in the literature. However, in contrast to the reference compound, our compound showed a retained coagulant effect of thrombin with increasing substrate concentration in a modified Calibrated Automated Thrombogram (CAT) method. This was likely related to our compound being inactive against FVIIa, while the reference compound showed an IC50 of 8.9μM. Thus, the higher selectivity of our compound against all relevant coagulation factors likely explained its higher therapeutic potential in comparison to the reference compound. The data indicate that at least a 100-fold selectivity over other serine proteases in the coagulation cascade will be required for an effective APC inhibitor.

Endogenous protein C has a protective role during Gram‐negative pneumosepsis (melioidosis)

Activated protein C (APC) exerts anticoagulant effects via inactivation of factors Va and VIIIa and cytoprotective effects via protease activated receptor (PAR)1. Inhibition of endogenous APC in endotoxemia and sepsis results in exacerbation of coagulation and inflammation, with consequent enhanced lethality. We here sought to dissect the distinct roles of the anticoagulant and cytoprotective functions of endogenous APC in severe Gram-negative pneumonia-derived sepsis (melioidosis). We infected wild-type (WT) mice with Burkholderia pseudomallei, a common sepsis pathogen in southeast Asia, and treated them with antibodies inhibiting both the anticoagulant and cytoprotective functions of APC (MPC1609) or the anticoagulant functions of APC (MAPC1591) only. Additionally, we administered SEW2871 (stimulating the S1P1-pathway downstream from PAR1) to control and MPC1609-treated mice. MPC1609, but not MAPC1591, significantly worsened survival, increased coagulation activation, facilitated bacterial growth and dissemination and enhanced the inflammatory response. The effects of MPC1609 could not be reversed by SEW2871, suggesting that S1P1 does not play a major role in this model. These results suggest that the mere inhibition of the anticoagulant function of APC does not interfere with its protective role during Gram-negative pneumosepsis, suggesting a more prominent role for cytoprotective effects of APC .

C0034 A simplified assay for quantification of circulating activated protein C levels

Background: Activated protein C (APC) is an enzyme with anticoagulant and cytoprotective functions. Accumulative data suggest that the number of situations in which in vivo circulating APC will need to be measured will increase over the next few years. Therefore, the availability of a rapid, sensitive and simple assay for quantifying circulating APC is crucial. Available assays are difficult to apply in routine laboratory. Our previously reported assay required an excessive pre-analytical handling: drawing of blood into two citrate tubes; immediate addition of a fresh APC inhibitor to one tube in order to block complexation of circulating APC to protein C inhibitor (PCI), and of heparin to the other tube to force all circulating APC to bind to PCI, and measurement of APC:PCI complexes in both tubes by ELISA (method A). Here we describe a simplifiedmethod based on the measurement of APC:PCI in a unique blood tube anticoagulated with heparin (method B). Methods: We measured APC levels, with both methods, in 125 plasma samples, 69 from patients with venous thrombosis (VT) and 56 from healthy controls. Results: ThemeanAPC level in the 125 sampleswas 1.06±0.46 ng/ml (method A) and 2.21±0.85 ng/ml (method B). The coefficient of correlation between both methods was r=0.849 (Pb0.001). The mean APC level in the 69 VT patients wase 0.84±0.38 (method A) and 1.80± 0.67 (method B), significantly lower (as previously reported) than those in the 56 controls, 1.33±0.39 and 2.72±0.79, respectively, (Pb0.001). In both groups there was a significant correlation between the levels obtained by the two methods (VT, r=0.772; controls, r=0.777 (Pb0.001). Comment: These results show that both assays are equivalent, and confirm that the APC level is lower in VT patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories. (FIS PS09/00610, RECAVA RD06/0014/0004, Prometeo/ 2011/027, y Fundacion para la Investigacion Hospital La Fe; Pilar Medina is a Miguel Servet Researcher, ISCIII CP09/00065).