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Nature Communications Nov 2023The precise modification or functionalization of the protein C-terminus is essential but full of challenges. Herein, a chemical approach to modify the C-terminus is...
The precise modification or functionalization of the protein C-terminus is essential but full of challenges. Herein, a chemical approach to modify the C-terminus is developed by fusing a cysteine protease domain on the C-terminus of the protein of interest, which could achieve the non-enzymatic C-terminal functionalization by InsP-triggered cysteine protease domain self-cleavage. This method demonstrates a highly efficient way to achieve protein C-terminal functionalization and is compatible with a wide range of amine-containing molecules and proteins. Additionally, a reversible C-terminal de-functionalization is found by incubating the C-terminal modified proteins with cysteine protease domain and InsP, providing a tool for protein functionalization and de-functionalization. Last, various applications of protein C-terminal functionalization are provided in this work, as demonstrated by the site-specific assembly of nanobody drug conjugates, the construction of a bifunctional antibody, the C-terminal fluorescent labeling, and the C-terminal transpeptidation and glycosylation.
Topics: Protein C; Proteins; Glycosylation; Cysteine Proteases; Cysteine
PubMed: 37935692
DOI: 10.1038/s41467-023-42977-x -
Journal of Medicine and Life Nov 2022This study was conducted to assess the level of proteins C and S in patients with thalassemia intermedia from the Thalassemia Center in Erbil, Iraq. This study aimed to...
This study was conducted to assess the level of proteins C and S in patients with thalassemia intermedia from the Thalassemia Center in Erbil, Iraq. This study aimed to evaluate protein C and S levels in patients with β-thalassemia intermedia and correlate them to different clinical and laboratory parameters. This comprehensive descriptive case-control study was conducted in 2021. Twenty-three thalassemia intermedia patients were recruited. After the participants' demographic data were recorded, plasma levels of both proteins were measured. The acquired files were examined for the 23 patients studied, 48% of whom were female. The mean age of the patients was 16.32 years. The findings show that the proportion of protein C in males was greater than in females, while this percentage contrasts when compared with protein S (ranging between 89-99% and 85-96%, respectively). Concerning age, these two types of protein in children have more value compared to older ages. Only seven people had less than 1,000 ferritins, while the others had higher values. A decrease in proteins C and S was observed in the thalassemia intermediate compared to the control group. There was a significant relationship between the decreased protein C and S levels with splenectomy. Given the significant reduction in protein C and S levels among patients with thalassemia intermediate compared to the control group, there is an increased risk of thromboembolic events in patients with thalassemia intermediate.
Topics: Adolescent; Child; Female; Humans; Male; beta-Thalassemia; Case-Control Studies; Ferritins; Protein C; Protein S; Thromboembolism
PubMed: 36567848
DOI: 10.25122/jml-2021-0316 -
Journal of the American Heart... Feb 2022Background Five classic thrombophilias have been recognized: factor V Leiden (rs6025), the prothrombin G20210A variant (rs1799963), and protein C, protein S, and...
Background Five classic thrombophilias have been recognized: factor V Leiden (rs6025), the prothrombin G20210A variant (rs1799963), and protein C, protein S, and antithrombin deficiencies. This study aimed to determine the thrombotic risk of classic thrombophilias in a cohort of middle-aged and older adults. Methods and Results Factor V Leiden, prothrombin G20210A and protein-coding variants in the (protein C), (protein S), and (antithrombin) anticoagulant genes were determined in 29 387 subjects (born 1923-1950, 60% women) who participated in the Malmö Diet and Cancer study (1991-1996). The Human Gene Mutation Database was used to define 68 disease-causing mutations. Patients were followed up from baseline until the first event of venous thromboembolism (VTE), death, or Dec 31, 2018. Carriership (n=908, 3.1%) for disease-causing mutations in the , , and genes was associated with incident VTE: Hazard ratio (HR) was 1.6 (95% CI, 1.3-1.9). Variants not in Human Gene Mutation Database were not linked to VTE (HR, 1.1; 95% CI, 0.8-1.5). Heterozygosity for rs6025 and rs1799963 was associated with incident VTE: HR, 1.8 (95% CI, 1.6-2.0) and HR, 1.6 (95% CI, 1.3-2.0), respectively. The HR for carrying 1 classical thrombophilia variant was 1.7 (95% CI, 1.6-1.9). HR was 3.9 (95% CI, 3.1-5.0) for carriers of ≥2 thrombophilia variants. Conclusions The 5 classic thrombophilias are associated with a dose-graded risk of VTE in middle-aged and older adults. Disease-causing variants in the , , and genes were more common than the rs1799963 variant but the conferred genetic risk was comparable with the rs6025 and rs1799963 variants.
Topics: Aged; Anticoagulants; Antithrombins; Cohort Studies; Factor V; Female; Humans; Male; Middle Aged; Mutation; Protein C; Protein S; Prothrombin; Risk Factors; Thrombophilia; Thrombosis; Venous Thromboembolism
PubMed: 35112923
DOI: 10.1161/JAHA.121.023018 -
Blood May 2015The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a... (Review)
Review
The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a cytoprotective on the basis of endothelial barrier stabilization and anti-inflammatory and antiapoptotic actions; and (3) a regenerative on the basis of stimulation of neurogenesis, angiogenesis, and wound healing. Pharmacologic therapies using recombinant human and murine APCs indicate that APC provides effective acute or chronic therapies for a strikingly diverse range of preclinical injury models. APC reduces the damage caused by the following: ischemia/reperfusion in brain, heart, and kidney; pulmonary, kidney, and gastrointestinal inflammation; sepsis; Ebola virus; diabetes; and total lethal body radiation. For these beneficial effects, APC alters cell signaling networks and gene expression profiles by activating protease-activated receptors 1 and 3. APC's activation of these G protein-coupled receptors differs completely from thrombin's activation mechanism due to biased signaling via either G proteins or β-arrestin-2. To reduce APC-associated bleeding risk, APC variants were engineered to lack >90% anticoagulant activity but retain normal cell signaling. Such a neuroprotective variant, 3K3A-APC (Lys191-193Ala), has advanced to clinical trials for ischemic stroke. A rich data set of preclinical knowledge provides a solid foundation for potential translation of APC variants to future novel therapies.
Topics: Animals; Anticoagulants; Cytoprotection; Humans; Protein C
PubMed: 25824691
DOI: 10.1182/blood-2015-02-355974 -
Surfactant protein C mutations and familial pulmonary fibrosis: stuck in a loop on the scenic route.The European Respiratory Journal Jan 2022
Topics: AAA Domain; Humans; Idiopathic Pulmonary Fibrosis; Mutation; Protein C; Surface-Active Agents
PubMed: 35086844
DOI: 10.1183/13993003.02147-2021 -
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 -
Blood Aug 2014In this issue of , Omarova et al show that fibrinogen, particularly the γ′ variant, increases the anticoagulant effect of activated protein C in plasma.
In this issue of , Omarova et al show that fibrinogen, particularly the γ′ variant, increases the anticoagulant effect of activated protein C in plasma.
Topics: Factor V; Female; Fibrinogens, Abnormal; Humans; Male; Protein C
PubMed: 25170115
DOI: 10.1182/blood-2014-07-583898 -
Blood Jul 2018Protein C is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts potent anticoagulant activity. In addition to its antithrombotic role... (Review)
Review
Protein C is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts potent anticoagulant activity. In addition to its antithrombotic role as a plasma protease, pharmacologic APC is a pleiotropic protease that activates diverse homeostatic cell signaling pathways via multiple receptors on many cells. Engineering of APC by site-directed mutagenesis provided a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks >90% anticoagulant activity but retains normal cell signaling activities. This 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein-coupled receptor, protease activated receptor 1. Potent neuroprotection in murine ischemic stroke models is linked to 3K3A-APC-induced signaling that arises due to APC's cleavage in protease activated receptor 1 at a noncanonical Arg46 site. This cleavage causes biased signaling that provides a major explanation for APC's in vivo mechanism of action for neuroprotective activities. 3K3A-APC appeared to be safe in ischemic stroke patients and reduced bleeding in the brain after tissue plasminogen activator therapy in a recent phase 2 clinical trial. Hence, it merits further clinical testing for its efficacy in ischemic stroke patients. Recent studies using human fetal neural stem and progenitor cells show that 3K3A-APC promotes neurogenesis in vitro as well as in vivo in the murine middle cerebral artery occlusion stroke model. These recent advances should encourage translational research centered on signaling selective APC's for both single-agent therapies and multiagent combination therapies for ischemic stroke and other neuropathologies.
Topics: Animals; Biomarkers; Blood-Brain Barrier; Combined Modality Therapy; Disease Susceptibility; Enzyme Activation; Gene Expression Regulation; Humans; Neurons; Protein C; Receptor, PAR-1; Regeneration; Signal Transduction; Stem Cells; Stroke
PubMed: 29866816
DOI: 10.1182/blood-2018-02-769026 -
Thrombosis Research May 2016Of the five Plasmodium species that infect humans, infection with P. falciparum is the most lethal, causing severe malaria syndromes, that result in over half a million... (Review)
Review
Of the five Plasmodium species that infect humans, infection with P. falciparum is the most lethal, causing severe malaria syndromes, that result in over half a million annual deaths. With parasites becoming increasingly resistant to artemisinin there is an urgent need for new preventative and therapeutic options, for which understanding of the mechanisms that cause death and disability in malaria is essential. The recent discoveries that certain variants of P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on infected erythrocytes are intimately linked to the precipitation of severe malaria syndromes and that these PfEMP1 variants contain EPCR binding domains provides new opportunities to improve our understanding of the molecular mechanisms responsible for the pathogenesis of severe malaria. EPCR is known for its essential role in the protein C (PC) system and for its ability to support the cytoprotective effects of activated protein C (APC) that result in vascular and tissue protective effects in many organ systems of the body, including the brain, lung, kidney, and liver. Observations that binding of PfEMP1 to EPCR results in an acquired functional PC system deficiency support the new paradigm that EPCR plays a central role in the pathogenesis of severe malaria. Thus, targeting of the PfEMP1-EPCR interaction and restoring the functionality of the PC system may provide new strategies for the development of novel adjuvant therapies for severe malaria.
Topics: Animals; Antigens, CD; Antimalarials; Drug Discovery; Endothelial Protein C Receptor; Host-Parasite Interactions; Humans; Malaria, Falciparum; Molecular Targeted Therapy; Plasmodium falciparum; Protein C; Protozoan Proteins; Receptors, Cell Surface
PubMed: 27207424
DOI: 10.1016/S0049-3848(16)30364-4 -
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