Effect Of Activated Protein C Research Articles

Activated protein C resistance in the copresence of emicizumab and activated prothrombin complex concentrates

BackgroundVenous thromboembolic events have been reported in persons with hemophilia A who received emicizumab and activated prothrombin complex concentrate (APCC) concomitantly, but the relevant mechanism(s) remains unclear. We speculated that activated protein C (APC) and antithrombin (AT) resistance might be associated with these adverse events. ObjectivesTo investigate APC and AT resistance in factor (F)VIII–deficient (FVIIIdef) plasma in the presence of emicizumab and APCC. MethodsIn pooled normal plasma or FVIIIdef plasma samples mixed with emicizumab (50 μg/mL) and FVIII-bypassing agents, including recombinant FVIIa (2.2 μg/mL), APCC (1.3 IU/mL), or plasma-derived FVIIa/FX (1.5 μg/mL), the suppression effect of AT (0-2.4 μM) and APC (0-16 nM) was assessed by tissue factor–triggered thrombin generation assay. The APC effects in FVIIIdef plasma with the copresence of emicizumab, FII (1.3 μM), and/or FIXa (280 pM) were also examined. ResultsThe AT resistance in emicizumab and each bypassing agent was not observed. Moreover, APC dose-dependent suppression effect was observed in pooled normal plasma or FVIIIdef plasma mixed with emicizumab and recombinant FVIIa or plasma-derived FVIIa/FX. However, APC-catalyzed inactivation had little effect on thrombin generation assay potential in FVIIIdef plasma spiked with emicizumab and APCC. The addition of FIXa to emicizumab in FVIIIdef plasma could lead to partial APC resistance. Furthermore, FVIIIdef plasma spiked with emicizumab, FIXa, and FII was markedly resistant to APC-mediated inactivation. ConclusionFII and FIXa in APCCs were key clotting factors for APC resistance in FVIIIdef plasma supplemented with emicizumab and APCCs. The APC resistance in persons with hemophilia A receiving emicizumab and APCC may contribute to venous thromboembolic events.

ACTIVATED PROTEIN C (APC) PROMOTES A HEALING PHENOTYPE IN CULTURED MURINE TENOCYTES VIA PROTEASE-ACTIVATED RECEPTOR (PAR)-2, BUT NOT PAR-1

Tendinopathy is a tendon pathology often resulting from a failed healing response to tendon injury. Activated protein C (APC) is a natural anti-coagulant with anti-inflammatory and wound healing promoting functions, which are mainly mediated by its receptors, endothelial protein C receptor (EPCR) and protease activated receptors (PARs). This study aimed to determine whether APC stimulates tenocyte healing and if so, to assess the involvement of the receptors.Mouse-tail tenocytes were isolated from 3-week-old wild type (WT), PAR- 1 knockout (KO) and PAR-2 KO mice. The expression of EPCR, PAR-1 and −2 and the effect of APC on tenocytes tendon healing and the underlying mechanisms were investigated by Reverse transcription real time PCR, western blot, 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, zymography, and scratch wound healing/ migration assay.When compared to WT cells, PAR-1 KO tenocytes showed increased cell proliferation (3.3-fold, p<0.0001), migration (2.7-fold, p<0.0001) and wound healing (3-fold, p<0.0001), whereas PAR-2 KO cells displayed decreased cell proliferation (0.6-fold, p<0.05) and no change in cell migration or wound healing. APC at 1 μg/ml stimulated WT and PAR-1 KO tenocyte proliferation (~1.3, respectively, p<0.05) and wound healing (~1.3-fold, respectively, p<0.05), and additionally promoted PAR1-KO cell migration (1.4-fold, p<0.0001). APC only increased the migration (2-fold, p<0.05) of PAR-2 KO tenocytes. The activation of AKT, extracellular signal-regulated kinase (ERK)-2, and glycogen synthase kinase (GSK)-β3, the intracellular molecules that are associated with cell survival/growth, and matrix metalloproteinase (MMP)-2 that is related to cell migration and wound healing, were increased in all three cell lines in response to APC treatment.These findings show that PAR-1 and PAR-2 act differentially in tenocyte proliferation/migration/wound healing. APC likely promotes tenocyte proliferation/ wound healing via PAR-2, not PAR-1.

Activated protein C inhibits mesothelial-to-mesenchymal transition in experimental peritoneal fibrosis

BackgroundIn addition to its anticoagulant function in downregulating thrombin generation, activated protein C (APC) evokes pleiotropic cytoprotective signaling activities when it binds to endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) in endothelial cells. ObjectivesTo investigate the protective effect of APC in a chlorhexidine gluconate (CG)–induced peritoneal fibrosis model. MethodsPeritoneal fibrosis was induced in wild-type as well as EPCR- and PAR1-deficient mice via daily injection of CG (0.2 mL of 0.1% CG in 15% ethanol and 85% saline) for 21 days with or without concomitant injection of recombinant human APC derivatives (50 μg/kg of bodyweight). The expression of proinflammatory cytokines and profibrotic markers as well as collagen deposition were analyzed using established methods. ResultsCG significantly upregulated the expression of transforming growth factor-β1 in peritoneal tissues, which culminated in the deposition of excessive extracellular matrix proteins, thickening of the peritoneal membrane, and mesothelial-to-mesenchymal transition in damaged tissues. APC potently inhibited CG-induced peritoneal fibrosis and downregulated the expression of proinflammatory cytokines, collagen deposition, Smad3 phosphorylation, and markers of mesothelial-to-mesenchymal transition (α-smooth muscle actin, vimentin, and N-cadherin). APC also inhibited transforming growth factor-β1–mediated upregulation of α-smooth muscle actin, Smad3, and fibronectin in human primary mesothelial cells. Employing signaling-selective and anticoagulant-selective variants of APC and mutant mice deficient for either EPCR or PAR1, we demonstrated that the EPCR-dependent signaling function of APC through PAR1 activation was primarily responsible for its antifibrotic activity in the CG-induced peritoneal fibrosis model. ConclusionAPC and signaling-selective variants of APC may have therapeutic potential for preventing or treating pathologies associated with peritoneal fibrosis.

Activated Protein C-Catalyzed Inactivation Pathway Down-Regulates NXT007-Mediated Hemostatic Function

Activated protein C (APC) together with protein S (PS) inactivates factor (F)Va and FVIIIa and efficient FVIIIa inactivation requires not only PS but also cofactor function of FV. Any impairment in these inactivation pathways is one of the most common risks of venous thrombosis. NXT007, emicizumab (Emi)-based engineered therapeutic bispecific antibody (Ab), is currently undergoing the phase 1 clinical trial. NXT007 increased coagulation potential of FVIII-deficient plasma up to the similar level to healthy controls in tissue factor (TF)-triggered thrombin generation (TG; Yamaguchi, ASH 2020). Additionally, concomitant use of NXT007 and bypassing agents could be useful for improving coagulation potentials in FVIII-deficient samples (Ogiwara, ASH 2021). However, the role of APC-dependent inactivation in NXT007-mediated hemostasis reactions remains to be investigated. Here, we examined the regulatory effects by APC in NXT007-driven hemostasis using a purified FXa generation assay and TF/ellagic acid-triggered thrombin generation assay (TGA). We firstly investigated coagulation potentials in FVIII-deficient plasma (ΔFVIII) spiked with NXT007 (3-100 μg/mL) or Emi (50 μg/mL). Peak thrombin (PeakTh) in ΔFVIII spiked with NXT007 was increased in a NXT007 dose-dependent manner. ΔFVIII spiked with NXT007 (3 μg/mL) showed higher PeakTh (355±74 nM) than emicizumab (50 μg/mL; 274±48 nM). PeakTh in ΔFVIII after the addition of NXT007 (10, 30, and 100 μg/mL) were 419±12, 454±13, and 484±10 nM, respectively. The coagulant effects of NXT007 (30 and 100 μg/mL) were comparable to pooled normal plasma (PNP). To evaluate the impact of APC-mediated down regulation on the hemostatic enhancement by NXT007 in ΔFVIII, TGA in ΔFVIII reacted with NXT007 (10 μg/mL) in the presence of APC (0-16 nM) was examined. PeakTh was decreased in an APC-dose dependent manner. Comparing with PeakTh without and with APC (16 nM), PeakTh was reduced from 457±11 nM to 331±1.4 nM and the %inhibition was 28%. We next evaluated the effects of APC on TG in PNP with or without an anti-FVIII polyclonal Ab (FVIIIAb) (10 BU/mL). The %inhibition of PeakTh in PNP with APC (16 nM) was 30%, indicating that the effect of APC-mediated inactivation was not disturbed by the presence of NXT007. To clarify the association of NXT007 alone with APC-catalyzed inactivation, we examined a purified FXa generation with NXT007 (10 μg/mL) or with FVIIIa (2 nM) after the addition of APC (0-2 nM) and PS (5 nM). FXa generation with FVIIIa was reduced by ~60% at maximum level in an APC dose-dependent manner, whereas that with NXT007 was not impaired, indicating that NXT007 itself was not affected by APC-catalyzed inactivation. To elucidate the contribution of APC-catalyzed inactivation of FV(a) in NXT007-catalyzed hemostatic regulation, we evaluated the effect of APC on the TG with NXT007 (10 μg/mL) and FV (7.5-30 nM; corresponded to 25-100% of physiological concentration) in the FV-deficient plasma with FVIIIAb (10 BU/mL). The PeakTh in the presence of NXT007 and FV (7.5-30 nM) without APC was 310-340 nM and the %inhibition of PeakTh by the addition of APC (16 nM) was ~35%, showing that NXT007 showed sufficient coagulation potential even at low concentration of FV and that the inhibition of TG by APC-catalyzed FV(a) inactivation was not disturbed by the presence of NXT007 even at FV (7.5 nM). In addition, the influence on TG with APC in the FV-Leiden (FV-L) plasma without or with FVIIIAb and NXT007 (10 μg/mL) were investigated. The %inhibition of PeakTh by APC (16 nM) in FV-L plasma was ~20%. While, the %inhibition of PeakTh by APC (16 nM) in FV-L plasma with FVIIIAb and NXT007 was similar to FV-L plasma alone (~20%), indicating that APC could regulate NXT007-catalyzed hemostatic reaction by inactivation of FV(a) under the FV-L condition. We conclude that APC-catalyzed inactivation mechanisms could play a significant role on the down-regulation of coagulation in NXT007-mediated hemostasis.

An Antibody Targeting Human FV Promotes Thrombin Generation and Reduces Bleeding in a Hemophilic Mouse Model

Hemophilia is a bleeding disorder that results from a deficiency of factor VIII (Hemophilia A; HA) or factor IX (Hemophilia B; HB). Protein replacement therapy ameliorates bleeding but can result in the development of neutralizing antibodies. Innovative nonfactor therapies are in development for hemophilia which target anticoagulant proteins such as tissue factor pathway inhibitor (TFPI), antithrombin, and activated protein C (APC). While these approaches show promise, they influence multiple coagulation factors and pathways which could raise safety concerns. Considering its essential role in coagulation in enhancing thrombin generation (TG), we hypothesized that altering the functional properties of FV/FVa could be an attractive approach for HA and HB treatment. Activated FV (FVa) assembles with FXa on activated cellular surfaces and enhances the kinetics of TG by several orders of magnitude. Here we assess whether monoclonal antibodies targeting all forms of FV are effective in promoting TG in hemophilia using both in vitro and in vivo models in hemophilic mice. Using an initial TG assay screen, we identified a procoagulant antibody (GB5) that binds with high affinity (Kd <0.5 nM) to human (h)FV, FVa, and FV-short and investigated its mechanism of action. Using proteolytic fragments of FV and western blotting experiments, we found that GB5 likely binds in the middle of the light chain region. To evaluate the functional effects of GB5 in vitro, we first employed a plasma-based TG assay using HA or HB plasma triggered with low (0.2 pM) tissue factor. The presence of saturating GB5 increased TG about 3-fold in HA plasma. Similar results were observed with HB plasma. Interestingly, when TG was essentially eliminated in HA and HB plasma with addition of exogenous APC, GB5 restored TG. These results suggest that the procoagulant effect of GB5 neutralizes the anticoagulant effect of APC and enhanced TG in hemophilic plasma To begin to assess the mechanism, the effect of GB5 on FV activation in the TG assay was evaluated. TG assay samples were removed over time, quenched, and analyzed by immunoblotting using labeled FV specific antibodies. In the presence of GB5, ~95% of the starting full-length FV was converted to FVa within 3 min compared to 5 min in the absence of GB5. These results show that FV is activated faster in the presence of GB5 in the TG assay. To extend these data, we next assessed the influence of GB5 in the TG assays using FV-deficient plasma supplemented with either FV or FVa and TFPI⍺. The data show that GB5 had essentially no effect on the TG assay when FV deficient plasma was reconstituted with FVa, but enhanced TG at least 3-fold when supplemented with FV. Consistent with this, the kinetics of prothrombin activation with FXa-FVa-membranes in the presence or absence of GB5 were the same. These data suggest that GB5 impacts reactions that influence FV, with no direct effect on FVa procoagulant function. To evaluate whether GB5 has a procoagulant effect in vivo, a tail vein transection (TVT) model was employed in HA mice. In this model, one lateral tail vein is transected, and blood is collected over 40 min with clots at the vessel wall disrupted every 10 min as needed. Since GB5 interacts with human and not mouse FV, plasma derived human FV was infused into HA mice. We found that blood loss in the TVT model using HA mice infused with human FV alone (5 μg/mouse) or GB5 alone (20 μg/mouse) was no different than mice infused with PBS (n = 3-5 mice/group except GB5 alone where n = 1: ~600 μl blood loss). In contrast to these data, when human FV and GB5 were co-administered, blood loss was reduced ~3-fold (~200 μl) which was statistically significant relative to the controls but did not reach the level of wild-type mice (blood loss ~50 μl). Overall, the results show that GB5 is a procoagulant monoclonal antibody that binds with high affinity to human FV and influences reactions that lead to its activation and inactivation. We hypothesize that the net effect of GB5 is the enhanced production of FVa which has a major impact on TG. In plasma-based systems with and without the protein C system and an in vivo hemophilic model, GB5 is procoagulant and can reduce bleeding. Our work shows that targeting the common coagulation pathway at the level of FV has benefit in the context of hemophilia and may be a viable and safe therapeutic approach.

Abstract 10402: Activated Protein C Rescues Sepsis-Associated Cardiac Dysfunction and Renal Injury in Aging

Introduction: Sepsis is a progressive life-threatening condition in which mortality has been consistently linked to increasing organ dysfunction. We have found that activated protein C (APC) has cytoprotective and anti-inflammatory properties in cardiovascular disease. Whether APC is critical for maintaining systemic homeostasis under sepsis in aging remains unknown. Hypothesis: Administration of recombinant APC ameliorates sepsis-associated renal injury and cardiac dysfunction in aging via EPCR (endothelial cell protein C receptor). Methods: Young (3-6 months) and aged (18-21 months) C57BL/6J wild type (WT), and EPCR point mutation (EPCR R84A/R84A ) knock in C57BL/6J mice were subjected to cecal ligation and puncture (CLP) surgery. APC (0.2 μg/g) was injected (i.p.) 1 hour after CLP surgery and every 24 hours to determine the therapeutic effects of APC on CLP-induced sepsis. Cardiac function was assessed daily via echocardiogram. Results: The Kaplan-Meier survival curve showed that the survival rate of young mice is 90% and of aged mice is 0% on day 7 of post-CLP. Moreover, the spleens were significantly enlarged in both young and aged CLP WT mice. Intriguingly, administration of recombinant APC can keep all young animals' survival and increased survival rate of aged animals to 65% on day 7 of post-CLP. The spleen size of young/aged CLP groups were significantly reduced by APC treatment. However, the beneficial effects of APC on the survival rate and spleen enlargement caused by CLP were abolished in EPCR R84A/R84A mice versus EPCR WT/WT littermates. The renal tubules necrosis score and cardiac dysfunctions caused by CLP were worse in aged versus young mice, but APC treatment significantly rescued CLP-caused renal pathology and cardiac dysfunctions of young/aged mice and the rescuing capacity of APC for both renal pathology and cardiac dysfunctions in aged versus young mice was stronger. In addition, APC treatment reduced circulating inflammatory monocytes and neutrophils during CLP-induced sepsis in young/aged WT but not EPCR R84A/R84A mice Conclusions: Sepsis associated mortality is increased in aging. APC treatment can ameliorate age-related sepsis renal injury and cardiac dysfunctions through receptor EPCR to reduce sepsis related mortality.

N-Glycosylation Deficiency Reduces the Activation of Protein C and Disrupts Endothelial Barrier Integrity.

Phosphomannomutase 2 (PMM2) deficiency is the most prevalent congenital disorder of glycosylation. It is associated with coagulopathy, including protein C deficiency. Since all components of the anticoagulant and cytoprotective protein C system are glycosylated, we sought to investigate the impact of an N-glycosylation deficiency on this system as a whole. To this end, we developed a PMM2 knockdown model in the brain endothelial cell line hCMEC/D3. The resulting PMM2low cells were less able to generate activated protein C (APC), due to lower surface expression of thrombomodulin and endothelial protein C receptor. The low protein levels were due to downregulated transcription of the corresponding genes (THBD and PROCR, respectively), which itself was related to downregulation of transcription regulators Krüppel-like factors 2 and 4 and forkhead box C2. PMM2 knockdown was also associated with impaired integrity of the endothelial cell monolayer-partly due to an alteration in the structure of VE-cadherin in adherens junctions. The expression of protease-activated receptor 1 (involved in the cytoprotective effects of APC on the endothelium) was not affected by PMM2 knockdown. Thrombin stimulation induced hyperpermeability in PMM2low cells. However, pretreatment of cells with APC before thrombin simulation was still associated with a barrier-protecting effect. Taken as a whole, our results show that the partial loss of PMM2 in hCMEC/D3 cells is associated with impaired activation of protein C and a relative increase in barrier permeability.

Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging.

APC (activated protein C) is a plasma serine protease with anticoagulant and anti-inflammatory activities. EPCR (Endothelial protein C receptor) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. Young (3-4 months) and aged (24-26 months) wild-type C57BL/6J mice, as well as EPCR point mutation (EPCRR84A/R84A) knockin C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild-type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCRR84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMPK (AMP-activated protein kinase) mediates acute adaptive response while AKT (protein kinase B) is involved in chronic metabolic programming in the hearts with APC treatment. I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Differential Modulation of Plasminogen Activator Mediated Thrombolysis By Recombinant Thrombomodulin and Activated Protein C

Introduction:Urokinase (UK) and tissue plasminogen activator (tPA) mediate thrombolytic actions by activating endogenous plasminogen. Thrombomodulin (TM) complexes with thrombin to activate Protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Activated Protein C (APC) modulates coagulation by digesting factors V and VIII and activates fibrinolysis by decreasing PAI-1 functionality. Since the thrombin/thrombomodulin complex regulates both TAFI functionality and protein C activation, rTM modulates the fibrinolytic process by multiple mechanisms.Aim:To compare the effects of rTM and APC on urokinase and tPA mediated thrombolysis utilizing thromboelastography.Materials and methods:Native whole blood was activated using a diluted intrinsic activator (APTT reagent, Triniclot). The modulation of thrombolysis by tPA and UK (Abbott, Chicago, USA) was studied by supplementing these agents to whole blood and monitoring TEG profiles for 3 hours. To investigate effect of APC (Haematologic Technologies, VT, USA) and rTM (Asahi Kasai Pharma, Tokyo, Japan) these agents were also supplemented to the activated blood at a concentration range of 0.02 - 3.0 ug/ml. The modulation of tPA and UK induced thrombolysis by APC and rTM was studied in terms of thromboelastograph patterns. In addition, the effect of both APC and rTM on plasma based systems supplemented with tPA was also investigated.Results:In comparison to rTM, APC produced a stronger anticoagulant effect in terms of r time, k time, angle and MA. Fibrinolysis was assessed in terms of LY30 (%) and LY60 (%). At concentrations of up to 3.0ug/ml rTM and APC did not produce any direct fibrinolytic effects. APC also produced strong augmentation of the lytic of effects of tPA and urokinase in a concentration dependent fashion. rTM at lower concentrations produced stabilization of clot resisting fibrinolysis. In the plasma based system, APC exhibited a stronger interaction with tPA in comparison to rTM.Conclusion:These studies demonstrate the differential anticoagulant and procoagulant effects of APC and rTM. APC is a stronger anticoagulant than rTM and facilitates thrombolysis. rTM is a weaker anticoagulant and modulates clot stability branding it resistant to fibrinolysis. These observations suggest that while APC may impair hemostasis, rTM restores hemostasis via TAFI activation. The dual mode of action of the rTM may contribute to the regulation of fibrinolysis in facilitating hemostatic balance in both physiologic and pathologic states. [Display omitted] DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

Inhibition of Thrombin and Its Generation By Recombinant Thrombomodulin and Activated Protein C in Whole Blood and Plasma Based Systems

Introduction:Recombinant thrombomodulin (rTM) and activated protein C (APC) are important modulators of coagulation and fibrinolysis. Both agents have been developed for various clinical indications. Beside their direct effect on coagulation, these agents also modulate other components of hemostatic processes to mediate their pharmacologic actions such as the direct inhibition of proteases and their generation.Aim:The aim of this study is to compare the differential inhibition of thrombin and its generation by rTM (ART-123, Asahi Kasei, Tokyo, Japan) and a plasma derived APC (Hematologic Technologies, Burlington, VT, USA).Methods:The inhibitory effects of APC and rTM on thrombin were measured in a plasma based and fluorogenic substrate assays. In the whole blood studies, native blood from healthy volunteers (n=10) was supplemented with rTM and APC at concentrations levels of 0-1.0 ug/ml. Thrombin generation was triggered by adding diluted APTT activator (Triniclot) and the reaction was stopped after 3 minutes. Thrombin generation markers such as F1.2 and TAT were measured using commercially available ELISA methods. In the citrated plasma studies both agents were supplemented at 0-1.0 ug/ml. Thrombin generation studies were carried out using a flouremtric assay (TGA, Techniclone, Vienna, Austria). Lag time peak thrombin generation and area under the curve (AUC) were measured. The IC50 values for each of the agents was calculated.Results:APC did not have any direct inhibitory effects on thrombin in both the clotting and amidolytic assays. However, rTM produced a concentration dependent inhibition of thrombin in both the clot based and amidolytic assays. In the whole blood systems both the APC and rTM produced a concentration dependent inhibition of thrombin generation markers such as F1.2 and TAT. APC produced a slightly stronger inhibition of stronger inhibition of TG as measured by F1.2, IC50 = 0.55 ug/ml compared with thrombomodulin, IC50 = 0.70 ug/ml. In the plasma based system the IC50 for peak thrombin inhibition was 0.46 ug/ml for APC and 0.97 ug/ml for rTM. Additional differences in the lag phase and AUC profiles were also noted. Conclusions: Beside the direct anticoagulant effects both the APC and rTM are capable of inhibiting the TF mediated thrombin generated in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in TG assays in both plasma and whole blood. .Conclusions:In contrast to APC, rTM produces direct inhibitory effects on thrombin in both the amidolytic and clot based assays. Both APC and rTM are capable of inhibiting the TF mediated thrombin generation in whole blood and plasma based systems. APC exhibits slightly stronger inhibitory effects in thrombin generation assays in both plasma and whole blood. These studies suggest that modulation of thrombin and other proteases contribute to the clinical effects of both rTM and APC. DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

Anticoagulant Actions of Recombinant Thrombomodulin and Activated Protein C and Their Neutralization By Factor VIII Inhibitor Bypass Activity (FEIBA)

Introduction:Thromboelastographic (TEG) analysis represents a global approach to monitor the clotability of the native whole blood. rTM (recombinant Thrombomodulin) is a mild anticoagulant with pleiotropic actions which are modulated through its complexation with thrombin. This agent has been developed for various clinical indications including DIC and related disorders. Activated Protein C (APC) represents an anticoagulant which produces its action by regulating activated factors V and VII and modulates fibrinolysis. A recombinant version of this drug was previously approved for the management of Sepsis and this agent is currently withdrawn from the market. FEIBA (Factor VII Inhibitor Bypass Activity) contains non activated factors II, IX and X and activated factor VII and is currently approved for the management of bleeding in hemophilia patients.Aim:The purpose of this study is to compare the relative anticoagulant effects of rTM and APC, employing the thromboelastographic analysis and their neutralization by graded amounts of FEIBA.Methods:Citrated Whole Blood samples were supplemented with rTM and APC at a concentration of 3 ug/mL (n=20). TEG analysis was performed on a TEG 5000 system in which clotting was initiated by recalcification of the whole blood and set parameters as R time, K time, MA and Angle were measured. The relative neutralization profiles of the APC and rTM by FEIBA at 1.0, 0.1, and 0.01 U/ml were investigated. All results were analyzed in terms of Means Values and Standard Deviations.Results:At 3ug/ml rTM produced a mild anticoagulant effect as evident by its TEG profile in terms of prolongation of R and K times and marked decrease in angle and maximum amplitude. APC at 3 ug/mL produced a relatively stronger anticoagulant effects on all of the parameters in the TEG profile. The supplementation of FEIBA at 0.1 U/mL to the whole blood mixtures containing 3 ug/mL completely neutralized rTM and resulted in the partial neutralization of the anticoagulant effects of APC as shown in Table 1. FEIBA at different dosages of 1.0 U/mL and 0.01 U/mL proportionately neutralized the anticoagulant effects of both of these agents.Discussion:These results indicate that on a weight basis, APC is a stronger anticoagulant in comparison to rTM as measured by the thromboelastographic analysis. FEIBA is very effective in the neutralization of the anticoagulant effects of rTM and results in a weaker neutralization of APC in a concentration dependent fashion. FEIBA at a much lower level of less than 0.1 U/mL is effective in neutralizing the effects of rTM. Therefore, this agent can be considered for the neutralization of rTM.Conclusions:These studies clearly indicate that on a comparative basis, rTM is a weaker anticoagulant in comparison to APC. Furthermore, FEIBA at a very low level of less than 0.1 U/mL dosage, projected clinical dosage of 5-10 U/kg, may be effective in the neutralization of the anticoagulant effects rTM. These results suggest that FEIBA can be used for the neutralization of such mild anticoagulants as rTM at much lower levels than the proposed dosing for the bleeding control of hemophiliac patients. [Display omitted] DisclosuresTsuruta:Asahi Kasei Pharma America Corporation: Employment.

Activated Protein C (APC) and 3K3A-APC-Induced Regression of Choroidal Neovascularization (CNV) Is Accompanied by Vascular Endothelial Growth Factor (VEGF) Reduction.

The activated protein C (APC) ability to inhibit choroidal neovascularization (CNV) growth and leakage was recently shown in a murine model. A modified APC, 3K3A-APC, was designed to reduce anticoagulant activity while maintaining full cytoprotective properties, thus diminishing bleeding risk. We aimed to study the ability of 3K3A-APC to induce regression of CNV and evaluate vascular endothelial growth factor (VEGF) role in APC’s activities in the retina. CNV was induced by laser photocoagulation on C57BL/6J mice. APC and 3K3A-APC were injected intravitreally after verification of CNV presence. CNV volume and vascular penetration were evaluated on retinal pigmented epithelium (RPE)-choroid flatmount by fluorescein isothiocyanate (FITC)-dextran imaging. VEGF levels were measured using immunofluorescence anti-VEGF staining. We found that 3K3A-APC induced regression of pre-existing CNV. VEGF levels, measured in the CNV lesion sites, significantly decreased upon APC and 3K3A-APC treatment. Reduction in VEGF was sustained 14 days post a single APC injection. As 3K3A-APC retained APCs’ activities, we conclude that the anticoagulant properties of APC are not mandatory for APC activities in the retina and that VEGF reduction may contribute to the protective effects of APC and 3K3A-APC. Our results highlight the potential use of 3K3A-APC as a novel treatment for CNV and other ocular pathologies.

Abstract 12715: Administration of Activated Protein C Stabilizes Membrane EPCR and Increases Resistance of Heart to Ischemic Insult in Aging

Introduction: Activated protein C (APC) is a circulating protease with anti-coagulant property. Endothelial protein C receptor (EPCR) mediates APC's downstream events. We have revealed that APC exerts cardioprotective effects during ischemia and reperfusion (I/R). Hypothesis: There is an impaired APC signaling in aging and administration of APC restores APC signaling of aged heart to improve cardiac tolerance to ischemic insult caused by I/R stress. Methods: Young C57BL/6J (3-4 months), aged C57BL/6J (24-26 months), and EPCR R84A/R84A (3-4 months, C57BL6/J background) transgenic mice without APC binding affinity were subjected to ischemia (45 min) and reperfusion (24 hrs) by ligation/release of left anterior descending coronary artery. APC, signaling selective APC-2Cys, or anticoagulant selective APC-E170A was injected through jugular vein five-minutes before reperfusion. Results: I/R stress triggers APC signaling in both young and aged hearts, but APC activity was significantly impaired in the aged versus young hearts. Serum EPCR measurement demonstrated that membrane EPCR's shedding into circulation was dramatically promoted during I/R stress. Intriguingly, administration of APC significantly reduced EPCR shedding caused by I/R stress in both young and aged hearts but not EPCR R84A/R84A heart. The echocardiography data showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction by I/R insult in both young and aged mice. Interestingly, APC and APC-2Cys restored aged heart's resistance to a comparable level as young heart to ischemic damage. The cardioprotective effects of APC and ACP-2Cys are diminished in EPCR R84A/R84A mice. Furthermore, metabolomics analysis and working perfusion heart results demonstrated that AMP-activated protein kinase (AMPK) signaling mediates acute adaptive metabolic response while protein kinase B (Akt) signaling pathway is involved in chronic metabolic programming in the heart with APCs treatments. Conclusions: I/R stress causes membrane EPCR shedding in the heart and APC's cardioprotection against I/R injury is not related with its anti-coagulant activity. Administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Neuroprotective Effect of Activated Protein C on Blood-Brain Barrier Injury During Focal Cerebral Ischemia/Reperfusion.

Although the effect of activated protein C (APC) on neuronal injury and neuroinflammatory responses has been extensively studied, the detailed mechanism underlying APC-protective effect in the blood–brain barrier (BBB) injury during ischemia is still not clear. In this study, the APC effect against neuroinflammatory responses was evaluated in the model of right middle cerebral artery occlusion in male Sprague-Dawley rats with 2 hours of ischemia and 22 hours of reperfusion. The results showed that APC can significantly improve the neurological function scoring and reduce the infarct volume and BBB permeability. Moreover, the expression of protein nuclear factor-kappa B (NF-κB), both in cytoplasm and nuclei, was reduced. The downstream of NF-κB activation, including tumor necrosis factor-α and interleukin-1β secretion, was inhibited. In all, APC exerts a neuroprotective effect in focal cerebral ischemia–reperfusion in rats by inhibiting the activation and nuclear translocation of NF-κB. It may indicate a therapeutic approach for ischemic brain injury.

In Vivo hemostatic Significance of Activated Protein C in Factor VIIIa Regulation

Activated factor VIII (FVIIIa) is an essential cofactor in the intrinsic tenase (Xase) enzyme complex that generates factor Xa and propagates clot formation. The FVIIIa heterotrimer is comprised of a metal ion linked dimer (A1/A3-C1-C2 domains) that is associated with the A2 domain by weak non-covalent interactions. Regulation of FXa formation by the intrinsic Xase enzyme complex occurs by FIXa inhibition and mechanisms contributing to FVIIIa inactivation, including: 1) rapid A2 domain dissociation and 2) activated protein C (APC) cleavage of FVIIIa. While FVIIIa inactivation by APC is considered important, there are surprisingly no in vivo studies documenting the hemostatic role of APC in FVIIIa regulation. Further, published data demonstrate APC cleavage of FVIIIa at physiologic protein concentrations occurs over hours while A2 dissociation occurs rapidly over minutes. Thus, it is thought that the predominant mechanism of FVIIIa inactivation is A2 dissociation and APC likely plays a marginal role in FVIIIa regulation. Additionally, unlike described A2 mutations that enhance dissociation and cause hemophilia A (HA), there is no known disease state attributed to altered FVIIIa cleavage by APC. This is in contrast to FVIII's homologous protein, FVa, whereby resistance to APC cleavage is the most common inherited thrombophilia (FV-Leiden [FVL]). Understanding the physiologically relevant mechanisms of FVIIIa inactivation has immediate clinical applicability for understanding safety considerations in HA therapeutics that bypass FVIIIa regulation (FVIII mimetic antibodies, e.g. emicizumab). Further, as evidenced by successful hemophilia B gene therapy trials using a gain of function FIX variant (FIX-Padua), altering FVIIIa inactivation could be exploited for therapeutic benefit in the setting of gene transfer. We aimed to determine the in vivo hemostatic role of APC in FVIIIa regulation and pair these studies with purified system analysis. We introduced Arg to Gln mutations at FVIII APC cleavage sites (R336Q and R562Q, herein called FVIII-QQ) on a B-domain deleted FVIII (FVIII-WT) backbone and produced recombinant FVIII-QQ and FVIII-WT. Unlike FVIII-WT, western blot analysis of FVIII-QQ incubated with APC and phospholipids had no evidence of cleavage. Enzyme kinetic studies using purified components demonstrated no appreciable difference in the Km or Vmax for FX within the intrinsic Xase enzyme complex or A2 dissociation of FVIII-QQ relative to FVIII-WT. These data confirmed no unexpected differences in FVIII-QQ relative to FVIII-WT. To begin to evaluate the role of APC in FVIIIa regulation, we measured thrombin generation in murine and human HA plasma reconstituted with FVIII-QQ or FVIII-WT in the presence of increasing APC concentrations. The IC50 of APC was 2-3-fold higher for FVIII-QQ than FVIII-WT. To evaluate the in vivo hemostatic effect of APC in FVIIIa regulation, HA mice were infused with FVIII-QQ or FVIII-WT and evaluated by tail clip injury and 7.5% FeCl3 carotid artery occlusion models. Required doses of FVIII-QQ to normalize blood loss from a tail clip assay and time to vessel occlusion in a FeCl3 assay were 4-5 fold lower than necessary FVIII-WT doses; the superior hemostatic effect of FVIII-QQ supported the physiologic significance of APC in FVIIIa inactivation. To isolate the role of APC in FVIIIa regulation from APC inactivation of FVa, we backcrossed HA mice with FVL mice to create homozygous HA/FVL mice. HA/FVL mice were infused with FVIII-QQ or FVIII-WT and underwent tail clip assay analysis. Doses of FVIII-QQ required to normalize blood loss were again less than FVIII-WT. To further isolate the enhanced hemostatic effect of FVIII-QQ to APC resistance, we performed the tail clip assay in HA/FVL mice infused with FVIII-QQ or FVIII-WT in the presence or absence of MPC1609, an antibody that blocks murine APC function (Xu et al. J Thromb Haemost 2008). In the presence of MPC1609, the same dose of FVIII-WT and FVIII-QQ was required to normalize blood loss (Figure 1). Collectively, our in vitro and in vivo data support the physiologic significance of APC in FVIIIa regulation. To our knowledge these data are the first to demonstrate the in vivo hemostatic effect of APC in FVIIIa inactivation. Our data may be translated to rationally exploit APC regulation of FVIIIa to develop novel HA therapeutics or further delineate safety considerations in therapies that bypass FVIIIa regulation. Figure 1 Disclosures Camire: Pfizer: Research Funding. George:University of Pennyslvania: Employment; Pfizer: Consultancy; Avrobio: Membership on an entity's Board of Directors or advisory committees.

The Cardioprotective Signaling Activity of Activated Protein C in Heart Failure and Ischemic Heart Diseases.

Activated protein C (APC) is a vitamin-K dependent plasma serine protease, which functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. APC also modulates cellular homeostasis by exhibiting potent cytoprotective and anti-inflammatory signaling activities. The beneficial cytoprotective effects of APC have been extensively studied and confirmed in a number of preclinical disease and injury models including sepsis, type-1 diabetes and various ischemia/reperfusion diseases. It is now well-known that APC modulates downstream cell signaling networks and transcriptome profiles when it binds to the endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) on various cell types. However, despite much progress, details of the downstream signaling mechanism of APC and its crosstalk with other signaling networks are far from being fully understood. In this review, we focus on the cardioprotective properties of APC in ischemic heart disease and heart failure with a special emphasis on recent discoveries related to the modulatory effect of APC on AMP-activated protein kinase (AMPK), PI3K/AKT, and mTORC1 signaling pathways. The cytoprotective properties of APC might provide a novel strategy for future therapies in cardiac diseases.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice.

To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Activated Protein C Induces Stabilization of the Outer Retinal Barrier and Inhibits Laser Induced Choroidal Neovascularization in a Murine Model

Introduction: Age-related macular degeneration (AMD) is considered one of the main causes of severe vision loss in older adults. Wet AMD is characterized by choroidal neovascular membrane developed under or into the sensory retina, breaking the outer blood-retina barrier (oBRB) and causing leakage and bleeding. Protein C (PC) is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts diverse homeostatic cell signaling pathways apart from its anticoagulant activity. APC activities are mediated mainly through endothelial PC receptor (EPCR) and protease activated receptor (PAR)-1. APC was recently shown to act as a novel ligand for Tie2, a tyrosine kinase receptor playing an important role in angiogenesis. Thus, APC may modulate pathological angiogenesis in relevant ocular conditions, such as choroidal neovascularization (CNV), via its interaction with Tie2. To the best of our knowledge, there are no studies addressing the potential use of APC as oBRB stabilizer and CNV inhibitor.Aim: To assess the protective effect of APC as a retinal barrier stabilizer and its ability to reduce CNV growth, leakage and penetration from the choroid into the sensory retina. Moreover, to study the role of Tie2 receptor upon APC mode of action in the retina.Methods: CNV development was induced in C57BL/6J mice by laser applications. Immediately following CNV induction APC (0.1-10 µg/µl) was injected intravitrealy. Seven to 14 days later, the retina was separated from the underlying tissues and the 2 segments were flattened: sclera-choroid-retinal pigment epithelium (RPE) segment and the photoreceptors-sensory retina segment. Blood vessels were stained using CD31 (an endothelial marker) or FITC-dextran and analyzed by using a confocal microscopy. Fluorescein angiography (FA) was performed to detect leakage from CNV. The ability of APC to stabilize the oBRB and prevent leakage was studied using ARPE-19 tissue culture (simulating the oBRB). FITC-Dextran leakage through the cultured cell monolayers was measured and the tight junction protein, Zonula occludens-1 (ZO1) cellular distribution was followed using immunostaining. Tie2 Kinase inhibitor and anti-Tie2 antibodies were used for Tie2 inhibition in the murine and cell culture models, respectively.Results: Quantitative analysis of the CNV area and volume revealed that 7-14 days after laser applications, APC treatment resulted in a statistically significant reduction in CNV growth, area and volume as compared to saline. Moreover, APC inhibited the penetration of CNV from the choroid throughout the RPE into the sensory retina in a dose-dependent manner. Figure 1 summarize the quantitative assessment of APC suppression of blood vessel penetration from the choroid (Fig.1A) to the sensory retina segment (Fig.1B). FA analysis showed that APC significantly suppressed the leakage of fluorescein from the laser lesion and preserved the retinal architecture. Incubation of RPE cells with APC resulted in dose dependent translocation of ZO1 to the cell membrane region, accompanied by reduction in permeability throughout the RPE monolayer. ZO1 translocation formed a typical ZO1 honeycomb pattern that is characteristic for the stabilization of tight junctions (Fig.1C). Inhibition of Tie2, in the laser induced CNV mice model and in RPE cell culture, significantly decreased APC protective activities in both models.Conclusions: Several Chorio-retinal pathologies are accompanied by damage to the oBRB and pathological growth of CNV. Our present data provide evidence that APC can protect the choroid and the retina from CNV growth, can suppress leakage from the newly formed choroidal blood vessels and may act as oBRB stabilizer. The protective function of APC in the eye provides direction for new strategies to treat chorio-retinal diseases affected by abnormal oBRB function and CNV growth. Our preliminary results warrant further investigation to extend the data regarding the potential use of APC as a novel anti-CNV treatment and oBRB protector. DisclosuresNo relevant conflicts of interest to declare.

The effects of oral contraceptive usage on thrombin generation and activated protein C resistance in Saudi women, with a possible impact of the body mass index.

Background and objectivesThe effect of oral contraceptive (OC) usage on coagulation has been studied worldwide. However, no such studies have been conducted in Saudi Arabia on Saudi women using OCs. The aim of this study was to investigate the effects of OC-induced changes of thrombin generation (TG) in the absence and presence of activated protein C (APC) or thrombomodulin (TM) in Saudi women.MethodsA total of 115 adult women, 47 on oral contraception (OC users) and 68 controls (not using OCs) were recruited from the obstetrics-gynecology outpatient clinic in Saudi Arabia. OCs that were used in this study include the following: Marvelon, Gynera, Cerrazetem, Yasmine, Microlut, Gracial and Diane. The plasma calibrated automated thrombinography (CAT) was used to determine TG which was expressed as endogenous thrombin potential (ETP; nM/min), lag time (min), peak (nM) and time-to-peak (ttpeak; min). In the presence of TM or APC, TG parameters were expressed relative to the parameters in the absence of TM or APC.Results and conclusionAs in other populations, our study demonstrated that OC usage induced prothrombotic changes in plasma of Saudi women, including resistance to the inhibitory actions of TM and APC. More specifically, OC usage in our population predominantly influenced TG and APC/TM sensitivity in overweight and obese individuals, a finding that needs confirmation in large cohort studies. The effects of APC and TM on TG parameters showed a positive association, and the correlation coefficients were higher in OC users for both ETP and peak values.