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Seminars in Thrombosis and Hemostasis Sep 2023Thrombophilia is a complex disease process, clinically manifesting in various forms of venous thromboembolism. Although both genetic and acquired (or environmental)...
Thrombophilia is a complex disease process, clinically manifesting in various forms of venous thromboembolism. Although both genetic and acquired (or environmental) risks factors have been reported, the presence of a genetic defect (antithrombin [AT], protein C [PC], protein S [PS]) is considered three of the major contributing factors of thrombophilia. The presence of each of these risk factors can be established by clinical laboratory analysis; however, the clinical provider and laboratory personnel must understand the testing limitations and shortcomings associated with the assays for these factors to be able to ensure an accurate diagnosis. This article will describe the major pre-analytical, analytical, and post-analytical issues associated with the various types of assays and discuss evidence-based algorithms for analyzing AT, PC, and PS in plasma.
Topics: Humans; Antithrombins; Protein C; Anticoagulants; Thrombophilia; Antithrombin III; Protein S
PubMed: 36940716
DOI: 10.1055/s-0043-1764468 -
Journal of Thrombosis and Haemostasis :... Jan 2023In addition to its anticoagulant function in downregulating thrombin generation, activated protein C (APC) evokes pleiotropic cytoprotective signaling activities when it...
BACKGROUND
In 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.
OBJECTIVES
To investigate the protective effect of APC in a chlorhexidine gluconate (CG)-induced peritoneal fibrosis model.
METHODS
Peritoneal 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.
RESULTS
CG 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.
CONCLUSION
APC and signaling-selective variants of APC may have therapeutic potential for preventing or treating pathologies associated with peritoneal fibrosis.
Topics: Humans; Animals; Mice; Peritoneal Fibrosis; Transforming Growth Factor beta1; Endothelial Protein C Receptor; Endothelial Cells; Protein C; Actins; Receptor, PAR-1; Cytokines; Anticoagulants
PubMed: 36695376
DOI: 10.1016/j.jtha.2022.10.012 -
Methods in Enzymology 2023Protein termini are critical for protein functions. They are often more accessible than internal regions and thus are frequently subjected to various modifications that...
Protein termini are critical for protein functions. They are often more accessible than internal regions and thus are frequently subjected to various modifications that affect protein function. Protein termini also contribute to regulating protein lifespan. Recent studies have revealed a series of degradation signals located at protein C-termini, termed C-degrons or C-end degrons. C-degrons have been implicated as underlying a protein quality surveillance system that eliminates truncated, cleaved and mislocalized proteins. Despite the importance of C-degrons, our knowledge of them remains sparse. Here, we describe an established framework for the characterization of C-degrons by Global Protein Stability (GPS) profiling assay, a fluorescence-based reporter system for measuring protein stability in cellulo. Furthermore, we apply an approach that couples GPS with random peptide libraries for unbiased and context-independent characterization of C-degron motifs. Our methodology provides a robust and efficient platform for analyzing the degron potencies of C-terminal peptides, which can significantly accelerate our understanding of C-degrons.
Topics: Proteolysis; Protein C; Proteins; Peptides
PubMed: 37532407
DOI: 10.1016/bs.mie.2023.02.009 -
Journal of Clinical Medicine Mar 2024Endothelial Protein C Receptor (EPCR) is a key regulator of the activated protein C anti-coagulation pathway due to its role in the binding and activation of this... (Review)
Review
Endothelial Protein C Receptor (EPCR) is a key regulator of the activated protein C anti-coagulation pathway due to its role in the binding and activation of this protein. EPCR also binds to other ligands such as Factor VII and X, γδ T-cells, plasmodium falciparum erythrocyte membrane protein 1, and Secretory group V Phospholipases A2, facilitating ligand-specific functions. The functions of EPCR can also be regulated by soluble (s)EPCR that competes for the binding sites of membrane-bound (m)EPCR. sEPCR is created when mEPCR is shed from the cell surface. The propensity of shedding alters depending on the genetic haplotype of the gene that an individual may possess. EPCR plays an active role in normal homeostasis, anti-coagulation pathways, inflammation, and cell stemness. Due to these properties, EPCR is considered a potential effector/mediator of inflammatory diseases. Rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus are autoimmune/inflammatory conditions that are associated with elevated EPCR levels and disease activity, potentially driven by EPCR. This review highlights the functions of EPCR and its contribution to rheumatic diseases.
PubMed: 38610795
DOI: 10.3390/jcm13072030 -
Clinical Laboratory Oct 2021Myocardial infarction (MI) or acute myocardial infarction (AMI), commonly referred to as a heart attack, happens when the blood flow to part of the heart stops, causing...
BACKGROUND
Myocardial infarction (MI) or acute myocardial infarction (AMI), commonly referred to as a heart attack, happens when the blood flow to part of the heart stops, causing damage to the heart muscle. Chest pain or discomfort that may flow into the shoulder, arm, back, neck, or jaw is the most common symptom. Most MIs occur due to coronary artery disease. High blood pressure, smoking, diabetes, lack of exercise, obesity, high blood pressure, poor diet, excessive alcohol use, etc. are risk factors. Antithrombin III (AT III) is a glycoprotein produced by the liver and consists of 432 amino acids. Protein C, also referred to as autoprothrombin IIA and factor XIV of blood coagulation, is a zymogen. In regulating anticoagulation, inflammation, cell death, and maintaining the permeability of blood vessel walls in humans and other animals, the activated form of protein C plays an important role.
METHODS
A case control study was conducted in Saudi Arabia to determine the levels of AT III and protein C in Saudi MI patients. Samples (n = 150) from MI patients as well as healthy controls (n = 50) were collected (2.5 mL of venous blood for sandwich ELISA).
RESULTS
This study showed that the mean AT III and protein C levels were within normal levels in patients (86 ± 19.63 and 76.20 ± 30.64, respectively). A comparison of mean AT III and protein C levels in patient and control groups showed no significant difference (p-value = 0.26, 0.2, and 0.19, respectively). The results also showed that some of the samples had low levels of AT III (8.7%) and protein C (11.3%).
CONCLUSIONS
A deficiency of AT III and protein C were not strong significant risk factors for myocardial infarction.
Topics: Anticoagulants; Antithrombin III; Case-Control Studies; Humans; Myocardial Infarction; Protein C; Saudi Arabia
PubMed: 34655191
DOI: 10.7754/Clin.Lab.2021.201206 -
Biochimica Et Biophysica Acta. General... Jun 2021We previously demonstrated that heterozygous Gly197 to Arg mutation in PROC is associated with venous thrombosis due to the mutation abrogating both zymogenic and...
We previously demonstrated that heterozygous Gly197 to Arg mutation in PROC is associated with venous thrombosis due to the mutation abrogating both zymogenic and enzymatic activities of protein C and activated protein C (APC). In this study, we investigated the role of Gly197 on the structure and function of protein C by replacing it with Ala, Lys and Glu in separate constructs. Characterization of protein C mutants indicated their activation by thrombin is improved ~5-20-fold with the order of PC-G197K > PC-G197E > PC-G197A > PC-WT. Interestingly, the cofactor function of thrombomodulin (TM) in promoting the activation of zymogens by thrombin followed the reverse order of PC-WT > PC-G197A > PC-G197E > PC-G197K. The thrombin-generation inhibitory profiles of zymogens in a tissue factor-mediated thrombin generation assay using protein C-deficient plasma with or without supplementation with TM followed the same order of zymogen activation in the purified system. Evaluation of anticoagulant activities of APC derivatives by prothrombinase and aPTT assays revealed a normal activity for APC-G197A but dramatically impaired activity for the other two mutants. In the endothelial cell permeability assay, APC-G197A exhibited normal antiinflammatory activity, but the other two mutants were nearly inactive. These results suggest that Gly197 plays a key role in TM cofactor-dependent protein C activation by thrombin. It facilitates the recognition of protein C by thrombin in the presence of TM but impedes it in the absence of the cofactor. In APC, a small residue at this position is required for the proper folding/reactivity of the active-site pocket of the protease, a hypothesis supported by structural modeling.
Topics: Anti-Inflammatory Agents; Anticoagulants; Factor V; Glycine; Humans; Mutagenesis, Site-Directed; Mutation; Protein C; Protein Conformation; Structure-Activity Relationship; Thrombin; Thrombomodulin
PubMed: 33722640
DOI: 10.1016/j.bbagen.2021.129892 -
Journal of Thrombosis and Haemostasis :... Jan 2021Essentials Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective effects. We tested whether APC or non-canonical PAR-derived peptides...
Essentials Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective effects. We tested whether APC or non-canonical PAR-derived peptides suppress inflammasome activity. APC or PAR1- and PAR3-derived peptides restrict inflammasome-dependent caspase-1 activity. Combined PAR1-derived and PAR3-derived peptides synergistically suppress caspase-1 activity. ABSTRACT: Background Activated protein C (APC) has been shown to restrict murine inflammasome activity. However, whether APC can exert anti-inflammatory activity in part through suppression of inflammasome activation in human systems is unknown. Objectives Studies were made to determine whether either APC or protease activated receptor (PAR)-derived peptides can reduce NLRP3 inflammasome activity in differentiated human THP-1 macrophage-like cells or in primary human monocytes stimulated to activate the inflammasome. Methods Human THP-1 cells or primary human monocytes were differentiated, treated with APC or PAR-derived peptides, and then stimulated with lipopolysaccharide and ATP to induce caspase-1 activity, a product of inflammasome activation. Results Activated protein C or noncanonical PAR1-derived or PAR3-derived peptides significantly reduced caspase-1 activity, detection of fluorescent NLRP3, and IL-1β release from THP-1 cells. At low concentrations where no effect was observed for each individual peptide, combinations of the PAR1-derived peptide and the PAR3-derived peptide resulted in a significant synergistic decrease in caspase-1 and IL-1β release. Caspase-1 activity was also reduced in primary human monocytes. Studies using blocking antibodies and small molecule PAR1 inhibitors suggest that EPCR, PAR1, and PAR3 each play roles in the observed anti-inflammatory effects. Several shortened versions of the PAR1- and PAR3-derived peptide reduced caspase-1 activity and exhibited synergistic anti-inflammatory effects. Conclusions The results indicate that both APC and certain PAR1- and PAR3-derived peptides, which are biased agonists for PAR1 or PAR3, can reduce inflammasome activity in stimulated human monocytes as measured by caspase-1 activity and IL-1β release and that PAR-derived biased peptide agonist combinations are synergistically anti-inflammatory.
Topics: Adaptor Proteins, Signal Transducing; Anti-Inflammatory Agents; Caspase 1; Cell Cycle Proteins; Endothelial Protein C Receptor; Humans; Inflammasomes; Interleukin-1beta; Macrophages; NLR Family, Pyrin Domain-Containing 3 Protein; Peptides; Protein C; Receptor, PAR-1; Signal Transduction; THP-1 Cells
PubMed: 33049092
DOI: 10.1111/jth.15133 -
Journal of Thrombosis and Haemostasis :... Feb 2024Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood.
BACKGROUND
Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood.
OBJECTIVES
We aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation.
METHODS
We used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease.
RESULTS
Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice.
CONCLUSION
Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.
Topics: Animals; Mice; Protein C; Endothelial Protein C Receptor; Myeloid Cells; Inflammation; Thrombophilia; Glycolysis; Mice, Inbred C57BL
PubMed: 37865288
DOI: 10.1016/j.jtha.2023.10.006 -
Cellular and Molecular Biology... Jul 2020Oxygen is transported in the blood through red blood cells and a protein called hemoglobin. The protein consists of two alpha and two beta chains. The lack of any of...
Oxygen is transported in the blood through red blood cells and a protein called hemoglobin. The protein consists of two alpha and two beta chains. The lack of any of these chains is caused by the malfunction of the genes that produce them, and can lead to a genetic disease called thalassemia. In β-thalassemia, hemoglobin does not produce enough beta protein. According to mild to severe effects on the body, β-thalassemia is divided into three types minor, interstitial and major thalassemia. There are increasing risks for thrombosis complications in thalassemia major. The purpose of this study was to evaluate protein C and protein S levels in β-thalassemia major and their association to the hypercoagulable state. Seventy patients with β-thalassemia major and 35 apparently healthy subjects as a control group were investigated for protein C and protein S. The mean of protein C (71.31%) and protein S (34.3%) levels were significantly reduced in β- thalassemia major patients in comparison with control subjects (p-value <0.001). Mean of fibrinogen level (2.42) g/l was significantly decreased in β-thalassemia major patients while the mean of D dimer level (0.43) μg/ml was significantly increased in comparison to control subjects (p-value 0.001). This study demonstrates a chronic hypercoagulable state in B- thalassemia major patients.
Topics: Adult; Case-Control Studies; Erythrocytes; Female; Fibrinogen; Hemoglobins; Humans; Male; Protein C; Protein S; Thrombophilia; Thrombosis; Young Adult; beta-Thalassemia
PubMed: 33040808
DOI: No ID Found -
Journal of Thrombosis and Haemostasis :... Sep 2023The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against...
BACKGROUND
The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against β2-glycoprotein I (β2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against β2GPI and prothrombin cause APC resistance is currently unclear.
OBJECTIVES
To investigate how anti-β2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance.
METHODS
The effects of anti-β2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies.
RESULTS
APC resistance was observed in LA-positive patients with anti-β2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-β2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-β2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-β2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306.
CONCLUSION
Anti-β2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.
Topics: Humans; Activated Protein C Resistance; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Factor V; Lupus Coagulation Inhibitor; Phosphatidylserines; Protein C; Prothrombin; Thrombosis
PubMed: 37290588
DOI: 10.1016/j.jtha.2023.05.024