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British Journal of Haematology Mar 2010Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In... (Review)
Review
Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti-inflammatory, anti-apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as 'Xigris') was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro- and anti-inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis.
Topics: Blood Coagulation; Cytoprotection; Humans; Inflammation; Mutagenesis; Protein C; Sepsis; Signal Transduction; Structure-Activity Relationship
PubMed: 19995397
DOI: 10.1111/j.1365-2141.2009.08020.x -
American Journal of Veterinary Research Jul 2023To establish normal values for pre- and post-prandial bile acids and protein C in Pacific harbor seal (Phoca vitulina richardsi) pups.
OBJECTIVE
To establish normal values for pre- and post-prandial bile acids and protein C in Pacific harbor seal (Phoca vitulina richardsi) pups.
ANIMALS
45 harbor seals undergoing rehabilitation at the Vancouver Aquarium Marine Mammal Rescue Centre, 0 to 16 weeks, and deemed healthy aside from malnutrition or maternal separation.
PROCEDURES
Venous blood was collected from the intervertebral extradural sinus in fasted seals and again 2 hours after a fish meal.
RESULTS
The reference interval (90% CL, confidence limit) for pre-prandial (fasting) bile acids was 17.2 μmol/L to 25.4 μmol/L, post-prandial bile acids were 36.9 μmol/L to 46.4 μmol/L, and protein C was 72.3% to 85.4%, across ages. For comparison between developmental ages, pups were grouped into 3 age classes: < 14 days, 5 to 8 weeks, and 10 to 16 weeks. Age affected pre- and post-prandial bile acids; pups < 14 days had significantly higher pre-prandial bile acids (36.0 μmol/L ± 16.5 μmol/L; P < .0001) than other age groups and pups 5 to 8 weeks had significantly higher post-prandial bile acids (50.4 μmol/L ± 21.9 μmol/L; P < .001). Protein C was also affected by age, with seals < 14 days having significantly lower values (mean, 51.8% ± 16.7%; P < .0001).
CLINICAL RELEVANCE
This study established normal reference intervals for bile acids in harbor seal pups and offered a preliminary investigation into protein C in pinnipeds. The bile acid values from 0- to 16-week-old seal pups were well above established normal ranges for domestic species, highlighting the utility of age- and species-specific reference ranges. The values presented here and the differences across age classes will aid clinicians in accurately diagnosing hepatobiliary disease in harbor seal pups.
Topics: Animals; Phoca; Protein C; Maternal Deprivation
PubMed: 37187459
DOI: 10.2460/ajvr.23.03.0057 -
Journal of Thrombosis and Haemostasis :... Apr 2023The complex reactions of blood coagulation are balanced by several natural anticoagulants resulting in tuned hemostasis. During several decades, the knowledge base of... (Review)
Review
The complex reactions of blood coagulation are balanced by several natural anticoagulants resulting in tuned hemostasis. During several decades, the knowledge base of the natural anticoagulants has greatly increased and we have also learned about antiinflammatory and cytoprotective activities expressed by antithrombin and activated protein C (APC). Some coagulation proteins have also been found to function as anticoagulants; e.g., thrombin when bound to thrombomodulin activates protein C. Another example is factor V (FV), which in addition to being a procofactor to FVa has emerged as an anticoagulant. The discovery of APC resistance, caused by FVLeiden, as a thrombosis risk factor resulted in the identification of FV as an APC cofactor working in synergy with protein S in the regulation of FVIIIa in the Xase complex. More recently, a natural anticoagulant FV splice isoform (FV-Short) was discovered when investigating the East Texas bleeding disorder. In FV-Short, the truncated B domain exposes a high-affinity binding site for tissue factor pathway inhibitor alpha (TFPIα), and together with protein S a high-affinity trimolecular complex is generated. The FXa-inhibitory activity of TFPIα is synergistically stimulated by FV-Short and protein S. The circulating FV-Short/protein S/TFPIα complex concentration is normally low (≈0.2 nM) but provides an anticoagulant threshold. In the East Texas bleeding, the concentration of the complex, and thus the threshold, is increased 10-fold, which results in bleeding manifestations. The anticoagulant properties of FV were discovered during investigations of individual patients and follow the great tradition of bed-to-bench and bench-to-bed research in the coagulation field.
Topics: Humans; Anticoagulants; Protein C; Factor V; Protein S; Blood Coagulation
PubMed: 36746318
DOI: 10.1016/j.jtha.2023.01.033 -
Scientific Reports Apr 2022Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic...
Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca-dependent binding of APC to HS02-52G, an efficient capturing of APC was applied in the presence of Ca ions, while a gentle release of captured APC was achieved in the elution buffer containing low EDTA concentration (5 mM). The captured and eluted APC showed more than 95% purity according to SDS-PAGE gel analysis and an enzyme-linked fluorescent assay (VIDAS Protein C). The purification yield of 45% was calculated when 4.2 µg APC was used, however this yield reduced to 21% if the starting amount of APC increased to 28.5 µg. Altogether, this method is recommended for rapid and efficient PC activation and APC purification. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.
Topics: Anticoagulants; Blood Coagulation Tests; Magnetic Iron Oxide Nanoparticles; Oligonucleotides; Protein C
PubMed: 35490167
DOI: 10.1038/s41598-022-11198-5 -
Journal of Thrombosis and Haemostasis :... Dec 2023Extracellular histone H3 is implicated in several pathologies including inflammation, cell death, and organ failure. Neutralization of histone H3 is a strategy that was...
BACKGROUND
Extracellular histone H3 is implicated in several pathologies including inflammation, cell death, and organ failure. Neutralization of histone H3 is a strategy that was shown beneficial in various diseases, such as rheumatoid arthritis, myocardial infarction, and sepsis. It was shown that activated protein C (APC) can cleave histone H3, which reduces histone cytotoxicity. However, due to the anticoagulant properties of APC, the use of APC is not optimal for the treatment of histone-mediated cytotoxicity, in view of its associated bleeding side effects.
OBJECTIVES
This study aimed to investigate the detailed molecular interactions between human APC and human histone H3, and subsequently use molecular docking and molecular dynamics simulation methods to identify key interacting residues that mediate the interaction between APC and histone H3 and to generate novel optimized APC variants.
METHODS
After molecular simulations, the designed APC variants 3D2D-APC (Lys37-39Asp and Lys62-63Asp) and 3D2D2A-APC (Lys37-39Asp, Lys62-63Asp, and Arg74-75Ala) were recombinantly expressed and their abilities to function as anticoagulant, to bind histones, and to cleave histones were tested and correlated with their cytoprotective properties.
RESULTS
Compared with wild type-APC, both the 3D2D-APC and 3D2D2A-APC variants showed a significantly decreased anticoagulant activity, increased binding to histone H3, and similar ability to proteolyze histone H3.
CONCLUSIONS
Our data show that it is possible to rationally design APC variants that may be further developed into therapeutic biologicals to treat histone-mediated disease, by proteolytic reduction of histone-associated cytotoxic properties that do not induce an increased bleeding risk.
Topics: Humans; Anticoagulants; Hemorrhage; Histones; Molecular Docking Simulation; Protein C; Proteolysis
PubMed: 37657561
DOI: 10.1016/j.jtha.2023.08.023 -
Journal of Thrombosis and Haemostasis :... Jul 2009Alterations in expression of protein C (PC) pathway components have been identified in patients with active inflammatory disease states. While the PC pathway plays a... (Review)
Review
Alterations in expression of protein C (PC) pathway components have been identified in patients with active inflammatory disease states. While the PC pathway plays a pivotal role in regulating coagulation and fibrinolysis, activated PC (aPC) also exhibits cytoprotective properties. For example, PC-deficient mice challenged in septic/endotoxemic models exhibit phenotypes that include hypotension, disseminated intravascular coagulation, elevated inflammatory mediators, neutrophil adhesion to the microvascular endothelium, and loss of protective endothelial and epithelial cell barriers. Further, inflammatory bowel disease has been correlated with diminished endothelial PC receptor and thrombomodulin levels in the intestinal mucosa. Downregulated expression of the cofactor, protein S, as well as PC, is also associated with ischemic stroke. Studies to elucidate further the structural elements that differentiate the various functions of PC will serve to identify novel therapeutic approaches toward regulating these and other diseases.
Topics: Animals; Cardiovascular Diseases; Humans; Inflammation; Protein C
PubMed: 19630787
DOI: 10.1111/j.1538-7836.2009.03410.x -
British Journal of Pharmacology Oct 2009The protein C system is an important natural anticoagulant mechanism mediated by activated protein C (APC) that regulates the activity of factors VIIIa and Va. Besides... (Review)
Review
The protein C system is an important natural anticoagulant mechanism mediated by activated protein C (APC) that regulates the activity of factors VIIIa and Va. Besides well-defined anticoagulant properties, APC also demonstrates anti-inflammatory, anti-apoptotic and endothelial barrier-stabilizing effects that are collectively referred to as the cytoprotective effects of APC. Many of these beneficial effects are mediated through its co-receptor endothelial protein C receptor, and the protease-activated receptor 1, although exact mechanisms remain unclear and are likely pleiotropic in nature. Increased insight into the structure-function relationships of APC facilitated design of APC variants that conserve cytoprotective effects and reduce anticoagulant features, thereby attenuating the risk of severe bleeding with APC therapy. Impairment of the protein C system plays an important role in acute lung injury/acute respiratory distress syndrome and severe sepsis. The pathophysiology of both diseases states involves uncontrolled inflammation, enhanced coagulation and compromised fibrinolysis. This leads to microvascular thrombosis and organ injury. Administration of recombinant human APC to correct the dysregulated protein C system reduced mortality in severe sepsis patients (PROWESS trial), which stimulated further research into its mechanisms of action. Several other clinical trials evaluating recombinant human APC have been completed, including studies in children and less severely ill adults with sepsis as well as a study in acute lung injury. On the whole, these studies have not supported the use of APC in these populations and challenge the field of APC research to search for additional answers.
Topics: Adult; Child; Clinical Trials as Topic; Cytoprotection; Humans; Inflammation; Protein C; Recombinant Proteins; Sepsis; Severity of Illness Index
PubMed: 19466992
DOI: 10.1111/j.1476-5381.2009.00251.x -
Journal of Neuroinflammation Jun 2022Inflammation and coagulation are linked and pathogenic in neuroinflammatory diseases. Protease-activated receptor 1 (PAR1) can be activated both by thrombin, inducing...
BACKGROUND
Inflammation and coagulation are linked and pathogenic in neuroinflammatory diseases. Protease-activated receptor 1 (PAR1) can be activated both by thrombin, inducing increased inflammation, and activated protein C (aPC), inducing decreased inflammation. Modulation of the aPC-PAR1 pathway may prevent the neuroinflammation associated with PAR1 over-activation.
METHODS
We synthesized a group of novel molecules based on the binding site of FVII/aPC to the endothelial protein C receptor (EPCR). These molecules modulate the FVII/aPC-EPCR pathway and are therefore named FEAMs-Factor VII, EPCR, aPC Modulators. We studied the molecular and behavioral effects of a selected FEAM in neuroinflammation models in-vitro and in-vivo.
RESULTS
In a lipopolysaccharide (LPS) induced in-vitro model, neuroinflammation leads to increased thrombin activity compared to control (2.7 ± 0.11 and 2.23 ± 0.13 mU/ml, respectively, p = 0.01) and decreased aPC activity (0.57 ± 0.01 and 1.00 ± 0.02, respectively, p < 0.0001). In addition, increased phosphorylated extracellular regulated kinase (pERK) (0.99 ± 0.13, 1.39 ± 0.14, control and LPS, p < 0.04) and protein kinase B (pAKT) (1.00 ± 0.09, 2.83 ± 0.81, control and LPS, p < 0.0002) levels indicate PAR1 overactivation, which leads to increased tumor necrosis factor-alpha (TNF-α) level (1.00 ± 0.04, 1.35 ± 0.12, control and LPS, p = 0.02). In a minimal traumatic brain injury (mTBI) induced neuroinflammation in-vivo model in mice, increased thrombin activity, PAR1 activation, and TNF-α levels were measured. Additionally, significant memory impairment, as indicated by a lower recognition index in the Novel Object Recognition (NOR) test and Y-maze test (NOR: 0.19 ± 0.06, -0.07 ± 0.09, p = 0.03. Y-Maze: 0.50 ± 0.03, 0.23 ± 0.09, p = 0.02 control and mTBI, respectively), as well as hypersensitivity by hot-plate latency (16.6 ± 0.89, 12.8 ± 0.56 s, control and mTBI, p = 0.01), were seen. FEAM prevented most of the molecular and behavioral negative effects of neuroinflammation in-vitro and in-vivo, most likely through EPCR-PAR1 interactions.
CONCLUSION
FEAM is a promising tool to study neuroinflammation and a potential treatment for a variety of neuroinflammatory diseases.
Topics: Animals; Endothelial Protein C Receptor; Factor VII; Inflammation; Lipopolysaccharides; Mice; Neuroinflammatory Diseases; Protein C; Receptor, PAR-1; Signal Transduction; Thrombin; Tumor Necrosis Factor-alpha
PubMed: 35690769
DOI: 10.1186/s12974-022-02505-y -
Journal of Thrombosis and Haemostasis :... May 2020Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein...
BACKGROUND
Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein C-deficient patients both of whom carry a heterozygous Gly197 to Arg (G197R) mutation in PROC and experience venous thrombosis.
OBJECTIVE
The objective of this study was to determine the molecular basis of the clotting defect in patients carrying the G197R mutation.
METHODS
We expressed protein C-G197R in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems.
RESULTS
The activation of protein C-G197R by thrombin was improved ~10-fold; however, its activation by thrombin was not promoted by thrombomodulin (TM). In a tissue factor-mediated thrombin generation assay, the addition of soluble TM to protein C-deficient plasma, supplemented with protein C-G197R, did not have a significant inhibitory effect on thrombin generation parameters. APC-G197R did not exhibit a significant anticoagulant activity in either purified or plasma-based assay systems. APC-G197R was essentially inactive because it showed no activity in an aPTT assay. Anti-inflammatory activity of APC-G197R was also dramatically impaired as determined by an endothelial cell permeability assay. Structural modeling predicted that the side-chain of Arg cannot be accommodated at this site of APC without a major distortion of the local structure that appears to propagate and adversely affect the reactivity/folding of the catalytic pocket.
CONCLUSION
The G197R mutation in patients appears to be functionally equivalent to a heterozygous protein C knockout with half of the protein having no significant activity and thus causing thrombosis.
Topics: Animals; Blood Coagulation Tests; Heterozygote; Humans; Mutation; Protein C; Thrombin; Thrombosis
PubMed: 32078247
DOI: 10.1111/jth.14777 -
Blood Coagulation & Fibrinolysis : An... Jun 2024The global incidence of thrombosis is increasing. However, research on thrombosis in the context of Korea is scarce. We aimed to analyze the relationship between factor...
OBJECTIVE
The global incidence of thrombosis is increasing. However, research on thrombosis in the context of Korea is scarce. We aimed to analyze the relationship between factor V and protein C test results and thrombosis in Koreans through a domestic commissioned testing institution conducting mass examinations.
METHODS
Results of factor V and protein C tests of 1386 individuals referred simultaneously to EONE Laboratories (Incheon, Republic of Korea) from January 2017 to July 2023 were analyzed retrospectively to identify the association with thrombotic disease. The tests were performed using a STAR MAX (Diagnostica Stago, Asnieres, France) automatic blood coagulation analyzer. The results were analyzed by age and sex.
RESULTS
The inspection rate increased gradually from 2017 to 2022. Women (70.0%) demonstrated a higher test rate than did men (30.0%). Young women reported high test rates; the test rate and age distribution differed by sex. Women aged between 20 and 49 years reported lower factor V and higher protein C concentrations than did men between 20 and 49 years of age.
CONCLUSIONS
The tests were more commonly performed in women than in men. Women aged between 20 and 49 years reported lower factor V concentrations and higher protein C concentrations than men between 20 and 49 years of age. This study will facilitate recognizing and preventing thrombotic diseases in women.
Topics: Humans; Female; Protein C; Male; Adult; Middle Aged; Republic of Korea; Young Adult; Retrospective Studies; Factor V; Thrombosis; Blood Coagulation Tests; Aged; Sex Factors
PubMed: 38477838
DOI: 10.1097/MBC.0000000000001296