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Frontiers in Cell and Developmental... 2022High-throughput profiling of protein C-termini is still a challenging task. Proteomics provides a powerful technology for systematic and high-throughput study of protein...
High-throughput profiling of protein C-termini is still a challenging task. Proteomics provides a powerful technology for systematic and high-throughput study of protein C-termini. Various C-terminal peptide enrichment strategies based on chemical derivatization and chromatography separation have been reported. However, they are still costly and time-consuming, with low enrichment efficiency for C-terminal peptides. In this study, by taking advantage of the high reaction selectivity of 2-pyridinecarboxaldehyde (2-PCA) with an α-amino group on peptide N-terminus and high affinity between biotin and streptavidin, we developed a 2-PCA- and biotin labeling-based C-terminomic (PBC) strategy for a high-efficiency and high-throughput analysis of protein C-terminome. Triplicates of PBC experiments identified a total of 1,975 C-terminal peptides corresponding to 1,190 proteins from 293 T cell line, which is 180% higher than the highest reported number of C-terminal peptides identified from mammalian cells by chemical derivatization-based C-terminomics study. The enrichment efficiency (68%) is the highest among the C-terminomics methods currently reported. In addition, we not only uncovered 50 proteins with truncated C-termini which were significantly enriched in extracellular exosome, vesicle, and ribosome by a bioinformatic analysis but also systematically characterized the whole PTMs on C-terminal in 293 T cells, suggesting PBC as a powerful tool for protein C-terminal degradomics and PTMs investigation. In conclusion, the PBC strategy would benefit high-efficiency and high-throughput profiling of protein C-terminome.
PubMed: 36120566
DOI: 10.3389/fcell.2022.995590 -
International Journal of Molecular... Jun 20233K3A-Activated Protein C (APC) is a recombinant variant of the physiological anticoagulant APC with cytoprotective properties and reduced bleeding risks. We studied the...
3K3A-Activated Protein C Inhibits Choroidal Neovascularization Growth and Leakage and Reduces NLRP3 Inflammasome, IL-1β, and Inflammatory Cell Accumulation in the Retina.
3K3A-Activated Protein C (APC) is a recombinant variant of the physiological anticoagulant APC with cytoprotective properties and reduced bleeding risks. We studied the potential use of 3K3A-APC as a multi-target therapeutic option for choroidal neovascularization (CNV), a common cause of vision loss in age-related macular degeneration. CNV was induced by laser photocoagulation in a murine model, and 3K3A-APC was intravitreally injected. The impact of 3K3A-APC treatment on myeloid and microglia cell activation and recruitment and on NLRP3 inflammasome, IL-1β, and VEGF levels was assessed using cryosection, retinal flat-mount immunohistochemistry and vascular imaging. Additionally, we evaluated the use of fluorescein angiography as a surrogate marker for in vivo evaluation of the efficacy of 3K3A-APC treatment against leaking CNV lesions. Our results demonstrated that 3K3A-APC treatment significantly reduced the accumulation and activation of myeloid cells and microglia in the CNV area and decreased the NLRP3 and IL-1β levels at the CNV site and the surrounding retina. Furthermore, 3K3A-APC treatment resulted in leakage regression and CNV growth suppression. These findings indicate that the anti-inflammatory activities of 3K3A-APC contribute to CNV inhibition. Our study suggests the potential use of 3K3A-APC as a novel multi-target treatment for CNV.
Topics: Mice; Animals; Protein C; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Vascular Endothelial Growth Factor A; Retina; Choroidal Neovascularization; Disease Models, Animal; Mice, Inbred C57BL
PubMed: 37445820
DOI: 10.3390/ijms241310642 -
Thrombosis Research Oct 2023Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa)....
INTRODUCTION
Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation.
MATERIALS AND METHODS
We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems.
RESULTS
In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS.
CONCLUSIONS
We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.
Topics: Humans; Anticoagulants; Blood Coagulation; Factor V; Factor Va; Factor Xa; Protein C; Protein S; Thrombin; Thromboplastin
PubMed: 37660436
DOI: 10.1016/j.thromres.2023.08.012 -
Autoimmunity Reviews Sep 2019Studies on last genetic and epigenetic predisposition to APS are summarized. It is well known that genetic predisposition is in HLA system (DR4 and DRw53) and that lupus... (Review)
Review
Studies on last genetic and epigenetic predisposition to APS are summarized. It is well known that genetic predisposition is in HLA system (DR4 and DRw53) and that lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are both associated with the same HLA antigens. Other genes, outside the MHC, give their contribution to the development of this autoimmune syndrome, such as IRF5, STAT4 and those related to inherited thrombophilia - factor V Leiden and G20210A prothrombin polymorphisms. Finally, post-transcriptional modifications of anti-beta2GPI antibodies could be implicated too. The most important discovery of last years is that altered microRNAs' expression is linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in APS.
Topics: Activated Protein C Resistance; Antibodies, Anticardiolipin; Antiphospholipid Syndrome; Epigenesis, Genetic; Genetic Predisposition to Disease; Genotype; HLA Antigens; Humans; Lupus Coagulation Inhibitor; Polymorphism, Genetic; Thrombosis; beta 2-Glycoprotein I
PubMed: 31323355
DOI: 10.1016/j.autrev.2019.102352 -
FASEB Journal : Official Publication of... Dec 2022Placental dysfunction is the leading cause of both preeclampsia and fetal growth restriction. This study aimed to characterize endothelial protein C receptor (EPCR) in...
Placental dysfunction is the leading cause of both preeclampsia and fetal growth restriction. This study aimed to characterize endothelial protein C receptor (EPCR) in preterm preeclampsia, term preeclampsia, and fetal growth restriction (defined by delivery of a small for gestational age [SGA] infant [<10% birthweight centile]) and examine its regulation in primary syncytiotrophoblast. Placental EPCR mRNA and protein were significantly increased in patients with preterm preeclampsia (<34 weeks gestation) compared to gestation-matched controls (p < .0001). In the plasma, EPCR was also significantly elevated (p = .01) in established preterm preeclampsia while its substrate, protein C (PC) was significantly reduced (p = .0083). Placentas from preterm small for gestational age (SGA) cases, had elevated EPCR mRNA expression (p < .0001) relative to controls. At 36 weeks, no significant changes in plasma EPCR were detected in samples from patients destined to develop preeclampsia or deliver an SGA infant at term. In terms of syncytiotrophoblast, hypoxia significantly increased EPCR mRNA expression (p = .008), but Tumor Necrosis Factor Alpha (TNF-α) decreased EPCR mRNA. Interleukin-6 (IL-6) had no significant effect on EPCR mRNA expression. When isolated syncytiotrophoblast was treated with metformin under hypoxia (1% O ) or normoxia (8% O ), EPCR mRNA expression was significantly reduced (p = .008) relative to control. In conclusion, EPCR is markedly elevated in the placenta and the circulation of patients with established preterm preeclampsia and placental increases may be associated with hypoxia. Additionally, fetal growth-restricted pregnancies (as defined by the delivery of an SGA infant) also demonstrated elevated placental EPCR.
Topics: Infant, Newborn; Humans; Female; Pregnancy; Pre-Eclampsia; Fetal Growth Retardation; Endothelial Protein C Receptor; Placenta; Hypoxia; RNA, Messenger
PubMed: 36394528
DOI: 10.1096/fj.202201150R -
The Lancet. Respiratory Medicine May 2024Glucocorticoids probably improve outcomes in patients hospitalised for community acquired pneumonia (CAP). In this a priori planned exploratory subgroup analysis of the... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
Glucocorticoids probably improve outcomes in patients hospitalised for community acquired pneumonia (CAP). In this a priori planned exploratory subgroup analysis of the phase 3 randomised controlled Activated Protein C and Corticosteroids for Human Septic Shock (APROCCHSS) trial, we aimed to investigate responses to hydrocortisone plus fludrocortisone between CAP and non-CAP related septic shock.
METHODS
APROCCHSS was a randomised controlled trial that investigated the effects of hydrocortisone plus fludrocortisone, drotrecogin-alfa (activated), or both on mortality in septic shock in a two-by-two factorial design; after drotrecogin-alfa was withdrawn on October 2011, from the market, the trial continued on two parallel groups. It was conducted in 34 centres in France. In this subgroup study, patients with CAP were a preselected subgroup for an exploratory secondary analysis of the APROCCHSS trial of hydrocortisone plus fludrocortisone in septic shock. Adults with septic shock were randomised 1:1 to receive, in a double-blind manner, a 7-day treatment with daily administration of intravenous hydrocortisone 50 mg bolus every 6h and a tablet of 50 μg of fludrocortisone via the nasogastric tube, or their placebos. The primary outcome was 90-day all-cause mortality. Secondary outcomes included all-cause mortality at intensive care unit (ICU) and hospital discharge, 28-day and 180-day mortality, the number of days alive and free of vasopressors, mechanical ventilation, or organ failure, and ICU and hospital free-days to 90-days. Analysis was done in the intention-to-treat population. The trial was registered at ClinicalTrials.gov (NCT00625209).
FINDINGS
Of 1241 patients included in the APROCCHSS trial, CAP could not be ruled in or out in 31 patients, 562 had a diagnosis of CAP (279 in the placebo group and 283 in the corticosteroid group), and 648 patients did not have CAP (329 in the placebo group and 319 in the corticosteroid group). In patients with CAP, there were 109 (39%) deaths of 283 patients at day 90 with hydrocortisone plus fludrocortisone and 143 (51%) of 279 patients receiving placebo (odds ratio [OR] 0·60, 95% CI 0·43-0·83). In patients without CAP, there were 148 (46%) deaths of 319 patients at day 90 in the hydrocortisone and fludrocortisone group and 157 (48%) of 329 patients in the placebo group (OR 0·95, 95% CI 0·70-1·29). There was significant heterogeneity in corticosteroid effects on 90-day mortality across subgroups with CAP and without CAP (p=0·046 for both multiplicative and additive interaction tests; moderate credibility). Of 1241 patients included in the APROCCHSS trial, 648 (52%) had ARDS (328 in the placebo group and 320 in the corticosteroid group). There were 155 (48%) deaths of 320 patients at day 90 in the corticosteroid group and 186 (57%) of 328 patients in the placebo group. The OR for death at day 90 was 0·72 (95% CI 0·53-0·98) in patients with ARDS and 0·85 (0·61-1·20) in patients without ARDS (p=0·45 for multiplicative interaction and p=0·42 for additive interaction). The OR for observing at least one serious adverse event (corticosteroid group vs placebo) within 180 days post randomisation was 0·64 (95% CI 0·46-0·89) in the CAP subgroup and 1·02 (0·75-1·39) in the non-CAP subgroup (p=0·044 for multiplicative interaction and p=0·042 for additive interaction).
INTERPRETATION
In a pre-specified subgroup analysis of the APROCCHSS trial of patients with CAP and septic shock, hydrocortisone plus fludrocortisone reduced mortality as compared with placebo. Although a large proportion of patients with CAP also met criteria for ARDS, the subgroup analysis was underpowered to fully discriminate between ARDS and CAP modifying effects on mortality reduction with corticosteroids. There was no evidence of a significant treatment effect of corticosteroids in the non-CAP subgroup.
FUNDING
Programme Hospitalier de Recherche Clinique of the French Ministry of Health, by Programme d'Investissements d'Avenir, France 2030, and IAHU-ANR-0004.
Topics: Humans; Hydrocortisone; Shock, Septic; Community-Acquired Infections; Male; Female; Fludrocortisone; Aged; Middle Aged; Pneumonia; Double-Blind Method; Drug Therapy, Combination; Anti-Inflammatory Agents; Treatment Outcome; Protein C
PubMed: 38310918
DOI: 10.1016/S2213-2600(23)00430-7 -
The Egyptian Journal of Internal... 2023The COVID-19 pandemic has been associated with millions of deaths around the world. One of the important causes of death associated with COVID-19 was pulmonary...
BACKGROUND
The COVID-19 pandemic has been associated with millions of deaths around the world. One of the important causes of death associated with COVID-19 was pulmonary thromboembolism. The risk for venous thromboembolism was markedly increased in patients with COVID-19 especially those admitted to the intensive care unit. The aims of our study were to measure the protein C and S levels in COVID-19-infected patients in comparison with the normal population and to assess the correlation of protein C and S levels in the plasma to the severity of infection.
METHODS
This was a case-control study measuring the protein C and S levels in patients infected with COVID-19 at the time of diagnosis compared to the normal population. The study included one hundred participants, sixty of them are patients with COVID-19, and forty of them are normal healthy adults. The patient group was subclassified into three subgroups according to disease severity: mild, moderate, and severe COVID-19 infections.
RESULTS
The activity of protein C in the patient group serum was significantly lower than that in the control group serum (79.35 ± 26.017 vs 97.43 ± 15.007; < ). Protein S is also significantly decreased in patients' serum when compared to the control group (70.233 ± 22.476 vs 91 ± 14.498; < ). There was a statistically significant decrease in the levels of protein C and S associated with the increase in disease severity ( < 0.05). However, protein S showed no statistically significant difference between the moderate and severe disease subgroups.
CONCLUSION
The study concluded that the levels of protein C and S activities were both decreased in patients with COVID-19 when compared to the healthy population. It also concluded that the decrease in their levels is statistically significant in relation to the disease severity.
PubMed: 36845330
DOI: 10.1186/s43162-023-00195-3 -
Methods in Molecular Biology (Clifton,... 2023Protein C (PC) is a vitamin K-dependent zymogen synthesized in the liver that plays a major role in regulating the coagulation pathway. Upon interaction with the...
Protein C (PC) is a vitamin K-dependent zymogen synthesized in the liver that plays a major role in regulating the coagulation pathway. Upon interaction with the thrombin-thrombomodulin complex, PC is converted to its active form, activated PC (APC). APC complexes with protein S and regulates thrombin generation by the inactivation of Factors Va and VIIIa. The role of PC as a key regulator of the coagulation process is highlighted in the deficiency state, in which heterozygous deficiency of PC predisposes to an increased risk of venous thromboembolism (VTE), while in the homozygous deficiency state, potentially fatal complications in the fetus including purpura fulminans and disseminated intravascular coagulation (DIC) can occur. Protein C is often measured with other factors such as protein S and antithrombin as a screen in the investigation of VTE. The chromogenic PC assay, the protocol described in this chapter, quantitates the amount of functional PC in the plasma using an activator of PC with the degree of color change proportional to the amount of PC present in the sample. Other methods, including functional clotting-based assays and antigenic assays, are available; however, protocols for these assays will not be provided in this chapter.
Topics: Humans; Protein C; Thrombin; Venous Thromboembolism; Anticoagulants; Blood Coagulation; Protein C Deficiency
PubMed: 37204713
DOI: 10.1007/978-1-0716-3175-1_13 -
Preparative Biochemistry & Biotechnology 2020Protein C (PC) plays an important role in the balance of coagulation and anticoagulation. Thus, the detection of PC activity is diagnostically significant for patients...
Protein C (PC) plays an important role in the balance of coagulation and anticoagulation. Thus, the detection of PC activity is diagnostically significant for patients with cardiovascular diseases. Presently, the key methods to detect PC activity are the chromogenic assay and activated partial thromboplastin time (APTT) test. PROTAC used in the chromogenic assay is isolated from venom as protein C activator (PCA). However, the use of the chromogenic assay is limited because of the high price of PROTAC. In this study, PCA was successfully purified from venom (AAV) by ion-exchange and gel chromatography. PCA from AAV has a relative molecular mass of 24 kD, calculated from the measurement of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The components of PCA were identified by MALDI-TOF/TOF-MS and mascot searches revealed that the coverage rate between PCA and zinc metalloproteinase AaPA from AAV was 21%. The chromogenic assay and APTT test were used to measure the enzymatic activity of PCA, and the results showed that PCA from AAV could specifically activate PC. In summary, the chromogenic assay described herein is highly sensitive and easy to perform.
Topics: Agkistrodon; Animals; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Activators; Protein C; Rabbits; Snake Venoms; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 32478599
DOI: 10.1080/10826068.2020.1768541 -
A&A Practice May 2020Critically ill patients with coronavirus disease 2019 (COVID-19) have been observed to be hypercoagulable, but the mechanisms for this remain poorly described. Factor...
Critically ill patients with coronavirus disease 2019 (COVID-19) have been observed to be hypercoagulable, but the mechanisms for this remain poorly described. Factor VIII is a procoagulant factor that increases during inflammation and is cleaved by activated protein C. To our knowledge, there is only 1 prior study of factor VIII and functional protein C activity in critically ill patients with COVID-19. Here, we present a case series of 10 critically ill patients with COVID-19 who had severe elevations in factor VIII activity and low normal functional protein C activity, which may have contributed to hypercoagulability.
Topics: Acute Kidney Injury; Adult; Aged; Aged, 80 and over; Antithrombins; Betacoronavirus; C-Reactive Protein; COVID-19; Comorbidity; Coronavirus Infections; Critical Illness; Diabetes Mellitus; Dyslipidemias; Extracorporeal Membrane Oxygenation; Factor VIII; Female; Ferritins; Fibrin Fibrinogen Degradation Products; Fibrinogen; Humans; Hypertension; International Normalized Ratio; Male; Middle Aged; Obesity; Pandemics; Partial Thromboplastin Time; Pneumonia, Viral; Protein C; Prothrombin Time; Renal Dialysis; Renal Insufficiency, Chronic; Respiration, Artificial; Respiratory Distress Syndrome; SARS-CoV-2; Thrombophilia
PubMed: 32539272
DOI: 10.1213/XAA.0000000000001236