Activity Of Activated Protein C Research Articles

Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

Ser252Asn Mutation Introduces a New N-Linked Glycosylation Site and Causes Type IIb Protein C Deficiency.

Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin-thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC. This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the "Paris" mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC). We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation. We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site (252NTT254) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC. The new N-glycosylation site (252NTT254) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient.

Selective modulation of activated protein C activities by a nonactive site-targeting nanobody library.

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the nonactive site of APC was generated using llama antibodies (nanobodies). Twenty-one nanobodies were identified that selectively recognize APC compared with the protein C zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in biolayer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and protease-activated receptor 1 (PAR1) cleavage were used to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential to regulate specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, and LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, and LP20) inhibited only 2 of the 3 functions. Monofunction inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9 and LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41, which results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile.

Safety and efficacy of an anti–human APC antibody for prophylaxis of congenital factor deficiencies in preclinical models

AbstractRebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.

Mechanisms Contributing to Acquired Activated Protein C Resistance in Patients Treated with Thalidomide: A Molecular Dynamics Study.

There is a high incidence of venous thromboembolism (VTE) in patients with Multiple Myeloma (MM), however; until now, the exact mechanisms behind VTE in MM are unknown, and some of the elements that may play a significant role are the treatment with an immunomodulator (IMiD) and acquired resistance to activated protein C (APC). The study aims to reveal the possible mechanisms linked to the reduced antithrombotic activity of APC associated with thalidomide. The molecular docking approach was used to ascertain the in silico inhibitory potential of thalidomide on the APC protease domain in the architecture of the catalytic triad and its interaction with major substrate binding sites. The coupling showed that the inhibitory activity of thalidomide depends on the induction of structural changes in the protease domain of APC, at the level of the Ser/His/Asp catalytic triad, as a result of a significant increase between the distances of CαAsp102 and Cα Ser195 (11.175 angstroms, increase 14.83%) and between CαSer195 and CαHis57 (9.478 angstroms, increase 13.78 %). This can result in an inefficient transfer of the proton between these residues, the other possible mechanism of inhibition, is a potential reduced binding of the substrate as a result of a direct interaction through a carbon-hydrogen bond on His57, an H-bond on Arg306, and a carbon hydrogen bond on Arg506. We demonstrate the in silico inhibitory potential of thalidomide on APC, through two possible inhibition mechanisms, a pathophysiologically relevant finding to understand the factors that can affect the stability and functions of APC in vivo.

Selective Modulation of Activated Protein C Activities By an Exosite-Specific Nanobody Library

Background Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Antibodies that can selectively modulate these APC activities could contribute to the understanding of the regulation of these physiological mechanisms and the development of therapeutics for the pathologies associated with these pathways Aim We sought to construct an antibody library consisting of small-sized nanobodies that could target neo-epitopes on the exosites of the activated form of protein C that are potentially inaccessible or whose three-dimensional structure is not recognized by conventional antibody formats. These APC-specific nanobodies will be used to determine the effect of the blockade of different exosites on APC pleiotropic activities. Methods A nanobody phage library specific for APC exosites was derived from peripheral blood mononuclear cells of llamas immunized with active site-blocked APC (APC-PPACK). The nanobody library was panned for APC-specificity. Biolayer interferometry (BLI) was used to determine the KDs and to epitope bin nanobodies into different clusters. APC functional assays, including Protac-APTT clotting assays, histone H3 cleavage assay, and SEAP-PAR1 cleavage assay with HEK293 cells, were employed to characterize the effects of the nanobodies on APC's pleiotropic activities. Results Of the total 1128 monoclonal phage clones screened in the library, 725 clones were identified as APC-PPACK binders. These clones were sequenced and 158 were found to be unique clones. Among them, 21 nanobodies belonging to 18 families were further characterized by their binding diversity. Overall, 3 clusters of nanobodies were identified based on their competition for APC exosites in BLI studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and SEAP-PAR1 cleavage, were employed to understand the diversity of the nanobodies. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential of regulating specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, LP17) inhibited all 3 APC functions. Five nanobodies (LP1, LP5, LP10, LP16, LP20) inhibited only 2 of the 3 functions. Mono-function inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP 9, LP11). LP 9 and LP11 are 2 of the nanobodies within the library that inhibited APC by >90% in a clotting assay (Table 1). The monospecific inhibition of APC anticoagulant activity could induce a pro-hemostatic effect in hemophilia and traumatic coagulopathy conditions. The molecular basis of APC's cytoprotective functions is based on its biased agonism of PAR1 by cleaving at R46. Interestingly, in R46/R41 cleavage ratio studies on SEAP-PAR1 cell lines, LP11 increased the APC pro-cytoprotective cleavage of R46 by up to 87%. The novel property of LP11 in enhancing R46 cleavage may potentially provide additional cytoprotection signaling and may potentially further benefit the outcome of these bleeding conditions. Conclusions A library of APC exosite-specific llama nanobodies yielded 21 nanobodies that can selectively modulate APC anticoagulation and cytoprotection pathways. The non-competing nature of nanobodies from each cluster allows the possibility of combining nanobodies from different clusters to better understand and study the biology of APC. These nanobodies represent a tool kit of 13 categories of antibodies based on their cluster and activity profile and offer the opportunity to develop novel therapeutics for the treatment of an array of APC-associated indications including acute bleeding, hemophilia, ischemia, and sepsis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blocking human protein C anticoagulant activity improves clotting defects of hemophilia mice expressing human protein C

AbstractHemophilia A and B are hereditary coagulation defects resulting in unstable blood clotting and recurrent bleeding. Current factor replacement therapies have major limitations such as the short half-life of the factors and development of inhibitors. Alternative approaches to rebalance the hemostasis by inhibiting the anticoagulant pathways have recently gained considerable interest. In this study, we tested the therapeutic potential of a monoclonal antibody, HAPC1573, that selectively blocks the anticoagulant activity of human activated protein C (APC). We generated F8−/− or F9−/− hemophilia mice expressing human protein C by genetically replacing the murine Proc gene with the human PROC. The resulting PROC+/+;F8−/− or PROC+/+;F9−/− mice had bleeding characteristics similar to their corresponding F8−/− or F9−/− mice. Pretreating the PROC+/+;F8−/− mice with HAPC1573 shortened the tail bleeding time. HAPC1573 pretreatment significantly reduced mortality and alleviated joint swelling, similar to those treated with either FVIII or FIX, of either PROC+/+;F8−/− or PROC+/+;F9−/− mice in a needle puncture–induced knee-joint bleeding model. Additionally, we found that HAPC1573 significantly improved the thrombin generation of PROC+/+;F8−/− mice but not F8−/− mice, indicating that HAPC1573 enhanced the coagulant activity of hemophilia mice by modulating human APC in vivo. We further documented that HAPC1573 inhibited the APC anticoagulant activity to improve the clotting time of human plasma deficient of FVIII, FIX, FXI, FVII, VWF, FV, or FX. These results demonstrate that selectively blocking the anticoagulant activity of human APC may be an effective therapeutic and/or prophylactic approach for bleeding disorders lacking FVIII, FIX, or other clotting factors.

Selective inhibition of activated protein C anticoagulant activity protects against hemophilic arthropathy in mice

AbstractRecurrent spontaneous or trauma-related bleeding into joints in hemophilia leads to hemophilic arthropathy (HA), a debilitating joint disease. Treatment of HA consists of preventing joint bleeding by clotting factor replacement, and in extreme cases, orthopedic surgery. We recently showed that administration of endothelial cell protein C receptor (EPCR) blocking monoclonal antibodies (mAb) markedly reduced the severity of HA in factor VIII (FVIII)−/− mice. EPCR blocking inhibits activated protein C (APC) generation and EPCR-dependent APC signaling. The present study was aimed to define the role of inhibition of APC anticoagulant activity, APC signaling, or both in suppressing HA. FVIII−/− mice were treated with a single dose of isotype control mAb, MPC1609 mAb, that inhibits anticoagulant, and signaling properties of APC, or MAPC1591 mAb that only blocks the anticoagulant activity of APC. Joint bleeding was induced by needle puncture injury. HA was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections for synovial hypertrophy, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. No significant differences were observed between MPC1609 and MAPC1591 in inhibiting APC anticoagulant activity in vitro and equally effective in correcting acute bleeding induced by the saphenous vein incision in FVIII−/− mice. Administration of MAPC1591, and not MPC1609, markedly reduced the severity of HA. MAPC1591 inhibited joint bleed–induced inflammatory cytokine interleukin-6 expression and vascular leakage in joints, whereas MPC1609 had no significant effect. Our data show that an mAb that selectively inhibits APC's anticoagulant activity without compromising its cytoprotective signaling offers a therapeutic potential alternative to treat HA.

Effects of direct oral anticoagulants dabigatran and rivaroxaban on the blood coagulation function in rabbits.

The aim of this study was to explore the effects of dabigatran and rivaroxaban on the activities of various coagulation factors. To achieve that, 60 rabbits were randomly divided into experimental groups that received different doses of dabigatran or rivaroxaban. The effects of dabigatran and rivaroxaban on the activities of FII, FV, FVIII, FX, and activated protein C (APC) were analyzed. In the dabigatran groups, activated partial thromboplastin time and thromboplastin time (TT) were prolonged after drug administration, and the activities of FII, FV, FVIII, and FX were inhibited as the drug concentration increased. Low doses of dabigatran inhibited APC activity. In the rivaroxaban groups, APTT and TT were not significantly prolonged after drug administration. In contrast, the high-dose rivaroxaban group exhibited prolonged PT, and the degree of inhibition of the activities of FII, FV, FVIII, and FX increased as the drug concentration increased. Rivaroxaban had no significant effect on APC activity regardless of dosage. As the drug concentration increased, both NOACs had more significant inhibitory effects on the activities of FII, FV, FVIII, and FX. Low concentrations of dabigatran generated an inhibitory effect on APC activity, while high concentrations of dabigatran had no significant effect. Rivaroxaban had no significant effect on APC activity.

Protein S: a Central Regulator of Blood Coagulation.

Protein S is a central regulator of coagulation as it critically participates in down-regulation of both extrinsic and intrinsic pathways of the coagulation cascade. In this review, we aim to provide an update on protein S and its anticoagulant functions as a central hemostatic regulator. Electronic databases including, Google, Google Scholar, PMC, PubMed, Science Direct, and Scopus were rigorously searched using the terms protein S, hemostasis, natural anticoagulants, regulators of coagulation, and coagulation inhibitors for the completion of this descriptive review. Literature review shows that protein S is a potent cofactor for activated protein C (APC) in the regulation of the intrinsic pathway and a cofactor for tissue factor pathway inhibitor (TFPI) in the regulation of the extrinsic pathway. The strong association between protein S deficiency either hereditary or acquired and increased risk for venous thrombosis indicates the important and central role of protein S in controlling the initiation and propagation phase of coagulation cascade and that protein S is an important determinant for optimal activity of both APC and TFPI in coagulation regulation. Available evidence suggests that the role of protein S in the down-regulation of blood coagulation is mainly mediated through its high affinity binding to negatively charged phospholipid surfaces. This high affinity binding to negatively charged phospholipids helps bring the anticoagulant proteins to the membranes, resulting in efficient and targeted regulation of coagulation. In the shade of current COVID-19 pandemic, protein S deficiency has been found to be a leading cause of thrombotic complications associated with COVID-19.

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.

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.

Abstract 10237: Activated Protein C Protects Neurons from Alzheimer’s Disease-Related Changes Through Reduction of Tau Hyperphosphorylation

Introduction: Activated protein C (APC) is a blood serine protease that has been shown to have neuroprotective function via its downstream signaling cascade. However, there is no study has investigated how the lack of APC signaling is associated with Alzheimer’s disease (AD)-related neuronal changes. Hypothesis: Diminished APC signaling is related to AD-associated biomarkers and behavioral symptoms. APC ameliorates these symptoms via the activation of the protein kinase B (Akt) signaling pathway which inhibits glycogen synthase kinase 3β (GSK3β) and tau hyperphosphorylation. Methods: Immunofluorescence imaging and proteomic analysis were performed on EPCR R84A/R84A mice, which had blunted APC activity due to mutated endothelial protein C receptor (EPCR), for examining amyloid β (Aβ) deposition. A rodent model of AD, 5xFAD mice, were subjected to either one-time or chronic daily APC injection (100 μg/kg/d i.p.). Morris's water maze test was performed to assess spatial memory and learning. The tissue of the hippocampus and cortex were collected for transcriptomic and proteomic analysis. Results: EPCR R84A/R84A mice showed greater hippocampal and cortical deposition of Aβ comparing to EPCR WT/WT counterparts. This result indicates defective APC signaling might contribute to the development of Aβ accumulation and insoluble plaque formation. A one-time injection of APC significantly activated AKT through phosphorylating the Ser 473 site, as well as inhibited AKT’s downstream target, GSK3β, via phosphorylation at Ser 9 in both wild-type and 5 x FAD mice. Downregulation of GSK3β activity is associated with decreased tau phosphorylation at Ser 202 /Thr 205 but not Ser 396 , indicating a reduction in the hyperphosphorylated form of tau that is prone to neurofibrillary tangle formation. Additionally, chronic APC administration for 3 months was observed to attenuate the cognitive deficits seen in 5 x FAD mice. Conclusions: APC has a crucial neuroprotective role because dampened APC signaling is related to AD-associated neuronal changes. APC has the downstream effect of Akt activation, which in turn inhibits GSK3β and the hyperphosphorylation of tau, ameliorating both the histological and behavioral markers seen in AD.

Could Soluble Endothelial Protein C Receptor Levels Recognize SARS-CoV2-Positive Patients Requiring Hospitalization?

ABSTRACTIntroduction:The endothelial protein C receptor (EPCR) is a protein that regulates the protein C anticoagulant and anti-inflammatory pathways. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits activated protein C (APC) activities. The clinical impact of sEPCR and its involvement in COVID-19 has not been explored. In this study, we investigated whether sEPCR levels were related to COVID-19 patients’ requirement for hospitalization.Methods:Plasma sEPCR levels were measured on hospital admission in 84 COVID-19 patients, and in 11 non-hospitalized SARS-CoV2-positive patients approximately 6 days after reported manifestation of their symptoms. Multiple logistic regression analysis was performed to identify potential risk factors for hospitalization and receiver operating characteristic (ROC) curves were generated to assess their value.Results:In our cohort, hospitalized patients had considerably higher sEPCR levels upon admission compared with outpatients [107.5 (76.7–156.3) vs. 44.6 (12.1–84.4) ng/mL; P < 0.0001)]. The ROC curve using hospitalization as the classification variable and sEPCR levels as the prognostic variable generated an area under the curve at 0.845 (95% CI = 0.710–0.981, P < 0.001). Additionally, we investigated the predictive value of sEPCR combined with BMI, age, or D-dimers.Conclusions:In our cohort, sEPCR levels in COVID-19 patients upon hospital admission appear considerably elevated compared with outpatients; this could lead to impaired APC activities and might contribute to the pro-coagulant phenotype reported in such patients. sEPCR measurement might be useful as a point-of-care test in SARS-CoV2-positive patients.

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.

Molecular Insights and Functional Consequences of the Interaction of Heme with Activated Protein C.

Aims: In hemolysis, which is accompanied by increased levels of labile redox-active heme and is often associated with hemostatic abnormalities, a decreased activity of activated protein C (APC) is routinely detected. APC is a versatile enzyme that exerts its anticoagulant function through inactivation of clotting factors Va and VIIIa. APC has not been demonstrated to be affected by heme as described for other clotting factors and, thus, is a subject of investigation. Results: We report the interaction of heme with APC and its impact on the protein function by employing spectroscopic and physiologically relevant methods. Binding of heme to APC results in inhibition of its amidolytic and anticoagulant activity, increase of the peroxidase-like activity of heme, and protection of human umbilical vein endothelial cells from heme-induced hyperpermeability. To define the sites that are responsible for heme binding, we mapped the surface of APC for potential heme-binding motifs. T285GWGYHSSR293 and W387IHGHIRDK395, both located on the basic exosite, turned out as potential heme-binding sites. Molecular docking employing a homology model of full-length APC indicated Tyr289 and His391 as the Fe(III)-coordinating amino acids. Innovation: The results strongly suggest that hemolysis-derived heme may directly influence the protein C pathway through binding to APC, conceivably explaining the decreased activity of APC under hemolytic conditions. Further, these results extend our understanding of heme as a multifaceted effector molecule within coagulation and may allow for an improved understanding of disease development in hemostasis under hemolytic conditions. Conclusion: Our study identifies APC as a heme-binding protein and provides insights into the functional consequences.

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.

Role of Protease‐activated receptors in Activated protein C‐mediated anti‐inflammatory responses in endothelial cells

Endothelial dysfunction is a hallmark of inflammation is and associated with the pathogenesis of vascular diseases induced by infections, oxidative stress or mechanical stress. This results in endothelial cell activation, disruption of endothelial barrier function, sensitivity to apoptosis and organ failure. While there are very limited options for treating diseases associated with endothelial dysfunction (e.g. sepsis, cardiovascular diseases), Activated protein C (APC) has shown pharmacological benefits in various preclinical settings. APC mediates multiple cytoprotective effects including 1) anti‐inflammatory responses, 2) stabilization of the endothelial barrier, and 3) anti‐apoptotic responses. Protease‐activated receptor‐1 (PAR1), a G protein‐coupled receptor (GPCR), is the central mediator of APC cellular signaling. However, there is limited understanding of the signaling pathways by which APC/PAR1 and APC/PAR1/PAR3 promote endothelial anti‐inflammatory responses. The long‐term goal of these studies is to understand the pathways by which the endothelium can resist inflammation and disruption to facilitate the advancement of new targets for therapeutic development. The subsequent studies aim to provide key mechanistic insight into how PAR1 and PAR3 drive endothelial anti‐inflammatory responses. Endothelial cytoprotection is driven by APC activation of PAR1 through cleavage at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R41. This results in the generation of a tethered ligand that promotes PAR1 biased signaling. We hypothesize that different co‐receptors including PAR3 and S1PR1 distinctly regulate specific APC/PAR1 anti‐inflammatory responses by influencing distinct beta‐arrestin signaling pathways. Here, we will examine the requirement of PAR1, co‐receptors PAR3 and S1PR1, and beta‐arrestin adaptor proteins to modulate APC‐mediated inhibition of TNF‐alpha pro‐inflammatory signaling in endothelial cells. Other studies aimed at elucidating additional mechanisms of APC‐mediated anti‐inflammatory responses, including post‐transcriptional control of pro‐inflammatory genes.Support or Funding InformationBurroughs Welcome Fund: Post Doctoral Enrichment Program awardNIH/NHLB T32 Cardiovascular Physiology and Pharmacology Training Grant ‐NIGMSR01GM116597 (Trejo)