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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 -
International Journal of Molecular... Feb 2019Independent of its well-known anticoagulation effects, activated protein C (APC) exhibits pleiotropic cytoprotective properties. These include anti-inflammatory actions,... (Review)
Review
Independent of its well-known anticoagulation effects, activated protein C (APC) exhibits pleiotropic cytoprotective properties. These include anti-inflammatory actions, anti-apoptosis, and endothelial and epithelial barrier stabilisation. Such beneficial effects have made APC an attractive target of research in a plethora of physiological and pathophysiological processes. Of note, the past decade or so has seen the emergence of its roles in cutaneous wound healing-a complex process involving inflammation, proliferation and remodelling. This review will highlight APC's functions and mechanisms, and detail its pre-clinical and clinical studies on cutaneous wound healing.
Topics: Animals; Biomarkers; Clinical Trials as Topic; Disease Models, Animal; Genetic Engineering; Humans; Protein C; Recombinant Proteins; Signal Transduction; Skin; Skin Ulcer; Translational Research, Biomedical; Wound Healing
PubMed: 30791425
DOI: 10.3390/ijms20040903 -
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 -
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 -
American Journal of Veterinary Research Feb 2017OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on...
OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis. SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram. PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti-human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed. RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C. CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.
Topics: Animals; Blood Coagulation Factors; Blood Coagulation Tests; Chemotaxis, Leukocyte; Dog Diseases; Dogs; Flow Cytometry; Neutrophils; Polymerase Chain Reaction; Protein C; Receptors, Cell Surface
PubMed: 28140640
DOI: 10.2460/ajvr.78.2.186 -
Thrombosis and Haemostasis Nov 2021Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective...
Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.
Topics: Animals; Anti-Inflammatory Agents; Cell Line; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Extracellular Traps; Histones; Humans; Immunity, Innate; Inflammation; Macrophages; Male; Mice; Mice, Inbred C57BL; Mutation; Neutrophils; Poly I-C; Protein C; Protein-Arginine Deiminase Type 4; Receptor, PAR-1; Signal Transduction
PubMed: 33706396
DOI: 10.1055/s-0041-1726093 -
Thrombosis Research Mar 2023COVID-19 is associated with an increased thromboembolic risk. However, the mechanisms triggering clot formation in those patients remain unknown.
INTRODUCTION
COVID-19 is associated with an increased thromboembolic risk. However, the mechanisms triggering clot formation in those patients remain unknown.
PATIENTS AND METHODS
In 118 adult Caucasian severe but non-critically ill COVID-19 patients (median age 58 years; 73 % men) and 46 controls, we analyzed in vitro plasma thrombin generation profile (calibrated automated thrombogram [CAT assay]) and investigated thrombophilia-related factors, such as protein C and antithrombin activity, free protein S level, presence of antiphospholipid antibodies and factor V Leiden R506Q and prothrombin G20210A mutations. We also measured circulating von Willebrand factor (vWF) antigen and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) antigen and activity. In patients, blood samples were collected on admission to the hospital before starting any therapy, including heparin. Finally, we examined the relationship between observed alterations and disease follow-up, such as thromboembolic complications.
RESULTS
COVID-19 patients showed 17 % lower protein C activity, 22 % decreased free protein S levels, and a higher prevalence of positive results for IgM anticardiolipin antibodies. They also had 151 % increased vWF, and 27 % decreased ADAMTS13 antigens compared with controls (p < 0.001, all). On the contrary, thrombin generation potential was similar to controls. In the follow-up, pulmonary embolism (PE) occurred in thirteen (11 %) patients. They were characterized by a 55 % elevated D-dimer (p = 0.04) and 2.7-fold higher troponin I (p = 0.002) during hospitalization and 29 % shorter time to thrombin peak in CAT assay (p = 0.009) compared to patients without PE.
CONCLUSIONS
In COVID-19, we documented prothrombotic abnormalities of peripheral blood. PE was characterized by more dynamic thrombin generation growth in CAT assay performed on admittance to the hospital.
Topics: Humans; ADAMTS13 Protein; COVID-19; Protein C; Thrombin; von Willebrand Factor; Protein S
PubMed: 36709678
DOI: 10.1016/j.thromres.2023.01.016 -
Journal of Thrombosis and Haemostasis :... Dec 2022Prothrombin, protein C, and factors VII, IX, and X are vitamin K (VK)-dependent coagulation proteins that play an important role in the initiation, amplification, and...
BACKGROUND
Prothrombin, protein C, and factors VII, IX, and X are vitamin K (VK)-dependent coagulation proteins that play an important role in the initiation, amplification, and subsequent attenuation of the coagulation response. Blood coagulation evolved in the common vertebrate ancestor as a specialization of the complement system and immune response, which in turn bear close evolutionary ties with developmental enzyme cascades. There is currently no comprehensive analysis of the evolutionary changes experienced by these coagulation proteins during the radiation of vertebrates and little is known about conservation of residues that are important for zymogen activation and catalysis.
OBJECTIVES
To characterize the conservation level of functionally important residues among VK-dependent coagulation proteins from different vertebrate lineages.
METHODS
The conservation level of residues important for zymogen activation and catalysis was analyzed in >1600 primary sequences of VK-dependent proteins.
RESULTS
Functionally important residues are most conserved in prothrombin and least conserved in protein C. Some of the most profound functional modifications in protein C occurred in the ancestor of bony fish when the basic residue in the activation site was replaced by an aromatic residue. Furthermore, during the radiation of placental mammals from marsupials, protein C acquired a cysteine-rich insert that introduced an additional disulfide in the EGF1 domain and evolved a proprotein convertase cleavage site in the activation peptide linker that also became significantly elongated.
CONCLUSIONS
Sequence variabilities at functionally important residues may lead to interspecies differences in the zymogen activation and catalytic properties of orthologous VK-dependent proteins.
Topics: Pregnancy; Animals; Female; Vitamin K; Prothrombin; Protein C; Placenta; Blood Coagulation Factors; Sequence Analysis; Mammals
PubMed: 36156849
DOI: 10.1111/jth.15897 -
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 -
Journal of Thrombosis and Haemostasis :... Sep 2014An enhanced risk of cardiovascular mortality has been observed after pneumonia. Epidemiological studies have shown that respiratory tract infections are associated with... (Review)
Review
An enhanced risk of cardiovascular mortality has been observed after pneumonia. Epidemiological studies have shown that respiratory tract infections are associated with an increased risk of thrombotic-related vascular disease such as myocardial infarction, ischemic stroke and venous thrombosis. Myocardial infarction and stroke have been detected essentially in the early phase of the disease (i.e. within 48 h from hospital admission), with an incidence ranging from as low as 1% to as high as 11%. Age, previous cardiovascular events and high pneumonia severity index were independent predictors of myocardial infarction; clinical predictors of stroke were not identified. Deep venous thrombosis and pulmonary embolism may also occur after pneumonia but incidence and clinical predictors must be defined. The biological plausibility of such an association may be deduced by experimental and clinical studies, showing that lung infection is complicated by platelet aggregation and clotting system activation, as documented by up-regulation of tissue factor and down-regulation of activated protein C. The effect of antithrombotic drugs has been examined in experimental and clinical studies but results are still inconclusive.
Topics: Age Factors; Anticoagulants; Blood Coagulation; Clinical Trials as Topic; Fibrinolytic Agents; Humans; Myocardial Infarction; Patient Admission; Platelet Aggregation; Pneumonia; Protein C; Research Design; Stroke; Thromboplastin; Thrombosis; Treatment Outcome; Vascular Diseases
PubMed: 24954194
DOI: 10.1111/jth.12646