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1.
Rheumatology (Oxford) ; 60(3): 1376-1386, 2021 03 02.
Article in English | MEDLINE | ID: mdl-32964932

ABSTRACT

OBJECTIVES: Risk factors for thromboembolism in SLE are poorly understood. We hypothesized a possible role for protein C, based on its dual activity in inflammation and haemostasis and on the evidence of an association between acquired activated protein C (APC) resistance (APCR) and high-avidity anti-protein C antibodies (anti-PC) with a severe thrombotic phenotype in venous thrombosis APS patients. METHODS: In a cross-sectional study of 156 SLE patients, the presence and avidity of IgG anti-PC was established by in house-ELISA, and APCR to exogenous recombinant human APC (rhAPC) and Protac (which activates endogenous protein C) was assessed by thrombin generation-based assays. Associations with aPL profile, thrombotic history and disease activity (BILAG and SLEDAI-2K) were also established. RESULTS: Anti-PC were detected in 54.5% of patients and APCR in 59%. Anti-PC positivity was associated with APCR to both rhAPC (P <0.0001) and Protac (P =0.0001). High-avidity anti-PC, detected in 26.3% of SLE patients, were associated with APCR in patients with thrombosis only (P <0.05), and with the development of thrombosis over time (range: 0-52 years; P =0.014). High-avidity anti-PC levels correlated with SLEDAI-2K (P =0.033) and total BILAG (P =0.019); SLEDAI-2K correlated inversely with APCR to Protac (P =0.004). CONCLUSION: Anti-PC occur in patients with SLE, independently of aPL profile, and are associated with APCR. High-avidity anti-PC are associated with thrombosis and with active disease and might prove a novel marker to monitor the risk of thrombosis and disease progression in SLE.


Subject(s)
Activated Protein C Resistance/immunology , Autoantibodies/immunology , Lupus Erythematosus, Systemic/immunology , Protein C/immunology , Thromboembolism/immunology , Activated Protein C Resistance/blood , Activated Protein C Resistance/complications , Activated Protein C Resistance/etiology , Biomarkers/blood , Cross-Sectional Studies , Female , Humans , Lupus Erythematosus, Systemic/complications , Male , Middle Aged , Risk Factors , Thrombin/metabolism , Thromboembolism/etiology
2.
Nat Commun ; 11(1): 2992, 2020 06 12.
Article in English | MEDLINE | ID: mdl-32532974

ABSTRACT

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.


Subject(s)
Antibodies, Monoclonal/pharmacology , Hemophilia A/prevention & control , Immunoglobulin Fab Fragments/immunology , Protein C Inhibitor/pharmacology , Protein C/antagonists & inhibitors , Animals , Antibodies, Monoclonal/classification , Antibodies, Monoclonal/immunology , Bleeding Time , Cell Membrane Permeability/drug effects , Cells, Cultured , Crystallography, X-Ray , Hemophilia A/blood , Hemorrhage/prevention & control , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Immunoglobulin Fab Fragments/metabolism , Macaca fascicularis , Male , Protein C/chemistry , Protein C/immunology , Protein C/metabolism , Protein C Inhibitor/blood , Protein C Inhibitor/pharmacokinetics
3.
Blood Coagul Fibrinolysis ; 30(8): 393-400, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31490209

ABSTRACT

: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers.


Subject(s)
Plasma/chemistry , Protein C/genetics , Protein S/genetics , Adult , Antigens/blood , Asian People , Female , Gene Frequency , Heterozygote , Humans , Japan , Mutation , Phenotype , Protein C/immunology , Protein S/immunology , Thrombophilia/genetics , Young Adult
4.
Rinsho Shinkeigaku ; 58(12): 764-766, 2018 Dec 21.
Article in Japanese | MEDLINE | ID: mdl-30487363

ABSTRACT

We reported a 31-year-old man with recurrent cerebral venous thrombosis caused by congenital protein C deficiency. He was diagnosed with cerebral venous thrombosis before 7 months. He was transferred to our hospital with numbness of right hand and right side of face, and dysarthria. The blood examination showed that his protein C antigen level and protein C activity were decreased than the lower limits of normal. Brain magnetic resonance venography showed poor visualization of the superior sagittal sinus and cortical veins. Genetic analysis revealed a single-base substitution (C>T) at the codon 811 (Arg to Trp) in the 9th exon portion of the protein C gene. Taking those results, he was diagnosed with recurrent cerebral venous thrombosis due to congenital protein C deficiency. Cerebral venous sinus thrombosis that occurred in the absence of an incidents of disease or internal history when there is a juvenile onset, a past history, or a family history, is suspected of congenital thrombophilia and needs blood tests and genetic tests.


Subject(s)
Cerebral Veins , Intracranial Thrombosis/etiology , Intracranial Thrombosis/genetics , Mutation , Protein C Deficiency/congenital , Protein C Deficiency/genetics , Protein C/genetics , Adult , Autoantigens/blood , Exons/genetics , Humans , Intracranial Thrombosis/diagnostic imaging , Male , Protein C/immunology , Recurrence
5.
Semin Thromb Hemost ; 44(2): 167-175, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29232721

ABSTRACT

Tissue injury prompts the initiation of host defense responses to limit blood loss, restrict pathogen entry, and promote repair. Biochemical and cellular pathways that lead to blood coagulation serve a fundamental role in generating a physical barrier at the wound site, but have also evolved to promote immune response to injury. Similarly, anticoagulant pathways that attenuate clot formation also regulate innate and adaptive immune responses. Of particular importance is activated protein C (APC) which serves as a principal regulator of thrombin generation, shapes the innate immune response to infection, and has been shown to contribute to the adaptive immune response in several preclinical models of autoimmune disease. APC controls blood coagulation by proteolytic degradation of procoagulant activated cofactors essential for fibrin clot development, but also cleaves multiple additional substrates and interacts with cell surface receptors to exert additional physiologically important roles. In this review, we focus on the molecular mechanisms utilized by APC to limit inflammation and, in particular, current understanding of the basis for APC anticoagulant and signaling activities. In particular, we provide an overview of established and emerging signaling pathways initiated by APC on endothelial cells, monocytes, neutrophils, dendritic cells, and T cells to control and regulate immune cell physiology. Finally, we consider the impact of APC activity in the context of both acute and chronic inflammatory disease, and the continuing efforts to harness the immunoregulatory properties of recombinant APC for therapeutic use.


Subject(s)
Inflammation/blood , Monocytes/metabolism , Protein C/immunology , Humans , Inflammation/immunology , Inflammation/pathology
6.
Microb Pathog ; 112: 30-37, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28942173

ABSTRACT

Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4+ T cells in effective immunity against S. aureus infection was shown; however, CD4+ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cell-proliferation rate as compared with controls. Additionally, CD4+ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-γ and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/isolation & purification , Manganese/metabolism , Protein C/metabolism , Staphylococcus aureus/immunology , Staphylococcus aureus/metabolism , Algorithms , Amino Acid Sequence , Animals , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Cytokines/metabolism , Epitope Mapping , Escherichia coli/genetics , Female , Interferon-gamma/metabolism , Interleukin-17/metabolism , Major Histocompatibility Complex , Mice , Mice, Inbred BALB C , Protein C/genetics , Protein C/immunology , Protein Structure, Secondary , Recombinant Proteins/immunology , Staphylococcal Infections/immunology , Th1 Cells/immunology , Th17 Cells/immunology
7.
Nat Commun ; 8(1): 311, 2017 08 21.
Article in English | MEDLINE | ID: mdl-28827518

ABSTRACT

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4+FOXP3+). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4-CD4+) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD.Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand Treg cells, mitigating the disease in mice.


Subject(s)
Graft vs Host Disease/immunology , Protein C/immunology , Receptor, PAR-2/immunology , Receptors, Proteinase-Activated/immunology , Receptors, Thrombin/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Graft vs Host Disease/etiology , Hematopoietic Stem Cell Transplantation/adverse effects , Hematopoietic Stem Cell Transplantation/methods , Humans , Kaplan-Meier Estimate , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Protein C/metabolism , Protein Multimerization , Receptor, PAR-2/chemistry , Receptor, PAR-2/metabolism , Receptors, Proteinase-Activated/chemistry , Receptors, Proteinase-Activated/metabolism , Receptors, Thrombin/chemistry , Receptors, Thrombin/metabolism , Signal Transduction/immunology , T-Lymphocytes, Regulatory/metabolism , Transplantation, Homologous
8.
Sheng Li Xue Bao ; 69(2): 172-182, 2017 Apr 25.
Article in Chinese | MEDLINE | ID: mdl-28435976

ABSTRACT

The present study is designed to explore the role of plasma cells in the change of protein C system (PCS) in ulcerative colitis (UC). Dextran sulfate sodium (DSS, 4% in concentration) was used to induce mouse UC model. The plasma cells and the type of immune complex in colon were observed by immunofluorescence. The amount and type of plasma cells separated from colonic mucosal lamina propria were detected by flow cytometry using anti-CD54+CD38+ and IgA/M/G antibodies, respectively. After stimulation of macrophages by IgG type immune complex, TNF-α and IL-6 levels were evaluated by ELISA. After co-incubation of microvascular endothelial cells with TNF-α or IL-6, the expressions of endothelial protein C receptor (EPCR) and thrombomodulin (TM), and the activity of activated protein C (APC) were examined. As the results showed, the IgG type plasma cells infiltration and the quantity of IgG type immune complex were increased in DSS group in comparison with control group. After incubation with IgG type immune complex, the levels of TNF-α and IL-6 in the supernatant of macrophages were increased (P < 0.01) in a concentration-dependent manner. Meanwhile, after incubation with TNF-α or IL-6, the expressions of EPCR and TM in the microvascular endothelial cells were decreased (P < 0.05 or P < 0.01), while the activity of APC was reduced (P < 0.05 or P < 0.01). These results suggested that the quantity of IgG type plasma cells increases in UC and forms immune complexes, which affect the secretion of cytokines from macrophage, thereby affecting the function of endothelial cells and finally inhibiting PCS in UC. Therefore, plasma cell may be a novel target for the treatment of UC.


Subject(s)
Antigen-Antibody Complex/immunology , Colitis, Ulcerative/immunology , Immunoglobulin G/immunology , Plasma Cells/immunology , Protein C/immunology , Animals , Colitis, Ulcerative/chemically induced , Colon/cytology , Dextran Sulfate , Disease Models, Animal , Interleukin-6/immunology , Intestinal Mucosa/cytology , Macrophages/immunology , Mice , Receptors, Cell Surface , Recombinant Proteins/immunology , Tumor Necrosis Factor-alpha/immunology
9.
J Biol Chem ; 292(21): 8616-8629, 2017 05 26.
Article in English | MEDLINE | ID: mdl-28408624

ABSTRACT

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.


Subject(s)
Extracellular Traps/immunology , Neutrophil Activation/immunology , Neutrophils/immunology , Protein C/immunology , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , Disease Models, Animal , Endothelial Protein C Receptor , Escherichia coli , Escherichia coli Infections/blood , Escherichia coli Infections/immunology , Extracellular Traps/metabolism , Female , Humans , Macrophage-1 Antigen/immunology , Macrophage-1 Antigen/metabolism , Male , Neutrophil Activation/drug effects , Neutrophils/metabolism , Papio anubis , Protein C/metabolism , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , Sepsis/blood , Sepsis/immunology , Tetradecanoylphorbol Acetate/pharmacology
10.
Menopause ; 24(4): 360-370, 2017 04.
Article in English | MEDLINE | ID: mdl-27922933

ABSTRACT

OBJECTIVE: Vasomotor symptoms (VMS) may be a marker of cardiovascular risk. We aimed to evaluate the cross-sectional association of VMS presence and severity with hemostatic parameter levels measured at baseline among Women's Health Initiative (WHI) Hormone Therapy trial postmenopausal participants. METHODS: This cross-sectional analysis included 2,148 postmenopausal women with measures of VMS presence and severity reported in the 4 weeks before WHI baseline, who were not using warfarin or hormone therapy and for whom the following baseline hemostatic parameters were measured within the WHI Cardiovascular Disease Biomarker Case-Control Study: antithrombin, plasminogen activator inhibitor-1, protein C antigen, total and free protein S antigen, total and free tissue factor pathway inhibitor, D-dimer, normalized activated protein C sensitivity ratio, and thrombin generation. Using multiple linear regression, we estimated the adjusted average difference in each hemostatic parameter associated with VMS presence and severity. A multiple comparisons-corrected P value was computed using the P-min procedure to determine statistical significance of our smallest observed P value. RESULTS: Women were 67 years of age on average and 33% reported VMS presence at baseline. There was some suggestion that VMS presence may be associated with a -0.34 adjusted difference in normalized activated protein C sensitivity ratio compared with no VMS (95% CI, -0.60 to -0.087; P = 0.009), but this association was not significant after correction for multiple comparisons (P = 0.073). VMS presence or severity was not significantly associated with the other hemostatic parameters. CONCLUSIONS: We found no convincing evidence that VMS presence or severity was associated with levels of hemostatic parameters among postmenopausal women.


Subject(s)
Hot Flashes/blood , Postmenopause/blood , Sweating , Aged , Antigens/blood , Antithrombin Proteins/metabolism , Cross-Sectional Studies , Female , Fibrin Fibrinogen Degradation Products/metabolism , Hemostasis , Humans , Lipoproteins/blood , Middle Aged , Plasminogen Activator Inhibitor 1/blood , Protein C/immunology , Protein S/immunology , Severity of Illness Index , Symptom Assessment , Thrombin/biosynthesis
11.
Exp Neurol ; 283(Pt A): 396-403, 2016 09.
Article in English | MEDLINE | ID: mdl-27412766

ABSTRACT

Activated protein C (APC) is a serine protease that promotes favorable changes in vascular barrier integrity and post-ischemic angiogenic remodeling in animal models of ischemic stroke, and its efficacy is currently being investigated in clinical ischemic stroke trials. Interestingly, application of sub-clinical chronic mild hypoxia (CMH) (8% O2) also promotes angiogenic remodeling and increased tight junction protein expression, suggestive of enhanced blood-brain barrier (BBB) integrity, though the role of APC in mediating the influence of CMH has not been investigated. To examine this potential link, we studied CMH-induced cerebrovascular remodeling after treating mice with two different reagents: (i) a function-blocking antibody that neutralizes APC activity, and (ii) exogenous recombinant murine APC. While CMH promoted endothelial proliferation, increased vascular density, and upregulated the angiogenic endothelial integrins α5ß1 and αvß3, these events were almost completely abolished by functional blockade of APC. Consistent with these findings, addition of exogenous recombinant APC enhanced CMH-induced endothelial proliferation, expansion of total vascular area and further enhanced the CMH-induced right-shift in vessel size distribution. Taken together, our findings support a key role for APC in mediating physiological remodeling of cerebral blood vessels in response to CMH.


Subject(s)
Brain Ischemia/pathology , Brain Ischemia/physiopathology , Brain/pathology , Gene Expression Regulation/physiology , Neovascularization, Physiologic/physiology , Protein C/metabolism , Animals , Antibodies/pharmacology , Disease Models, Animal , Endothelium, Vascular/metabolism , Integrin alpha5/metabolism , Ki-67 Antigen/metabolism , Male , Mice , Mice, Inbred C57BL , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Protein C/immunology , Time Factors
12.
Crit Care ; 20(1): 151, 2016 May 23.
Article in English | MEDLINE | ID: mdl-27215212

ABSTRACT

BACKGROUND: Altered plasma levels of protein C, thrombomodulin, and the endothelial protein C receptor are associated with poor clinical outcomes in patients with acute respiratory distress syndrome (ARDS). We hypothesized that common variants in these genes would be associated with mortality as well as ventilator-free and organ failure-free days in patients with ARDS. METHODS: We genotyped linkage disequilibrium-based tag single-nucleotide polymorphisms in the ProteinC, Thrombomodulin and Endothelial Protein C Reptor Genes among 320 self-identified white patients of European ancestry from the ARDS Network Fluid and Catheter Treatment Trial. We then tested their association with mortality as well as ventilator-free and organ-failure free days. RESULTS: The GG genotype of rs1042580 (p = 0.02) and CC genotype of rs3716123 (p = 0.002), both in the thrombomodulin gene, and GC/CC genotypes of rs9574 (p = 0.04) in the endothelial protein C receptor gene were independently associated with increased mortality. An additive effect on mortality (p < 0.001), ventilator-free days (p = 0.01), and organ failure-free days was observed with combinations of these high-risk genotypes. This association was independent of age, severity of illness, presence or absence of sepsis, and treatment allocation. CONCLUSIONS: Genetic variants in thrombomodulin and endothelial protein C receptor genes are additively associated with mortality in ARDS. These findings suggest that genetic differences may be at least partially responsible for the observed associations between dysregulated coagulation and poor outcomes in ARDS.


Subject(s)
Protein C/genetics , Respiratory Distress Syndrome/epidemiology , Adult , Aged , Antigens, CD/metabolism , Blood Coagulation/genetics , Blood Coagulation/immunology , Endothelial Protein C Receptor , Female , Genetic Variation/immunology , Humans , Male , Middle Aged , Protein C/immunology , Receptors, Cell Surface/metabolism , Respiratory Distress Syndrome/drug therapy , Respiratory Distress Syndrome/genetics , Thrombomodulin/metabolism , White People/genetics
13.
Biomaterials ; 80: 96-105, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26708087

ABSTRACT

Many neurological disorders are initiated by blood-brain barrier breakdown, which potentiates spinal neuroinflammation and neurodegeneration. Peripheral neuropathic injuries are known to disrupt the blood-spinal cord barrier (BSCB) and to potentiate inflammation. But, it is not known whether BSCB breakdown facilitates pain development. In this study, a neural compression model in the rat was used to evaluate relationships among BSCB permeability, inflammation and pain-related behaviors. BSCB permeability increases transiently only after injury that induces mechanical hyperalgesia, which correlates with serum concentrations of pro-inflammatory cytokines, IL-7, IL-12, IL-1α and TNF-α. Mammalian thrombin dually regulates vascular permeability through PAR1 and activated protein C (APC). Since thrombin protects vascular integrity through APC, directing its affinity towards protein C, while still promoting coagulation, might be an ideal treatment for BSCB-disrupting disorders. Salmon thrombin, which prevents the development of mechanical allodynia, also prevents BSCB breakdown after neural injury and actively inhibits TNF-α-induced endothelial permeability in vitro, which is not evident the case for human thrombin. Salmon thrombin's production of APC faster than human thrombin is confirmed using a fluorogenic assay and APC is shown to inhibit BSCB breakdown and pain-related behaviors similar to salmon thrombin. Together, these studies highlight the impact of BSCB on pain and establish salmon thrombin as an effective blocker of BSCB, and resulting nociception, through its preferential affinity for protein C.


Subject(s)
Chronic Pain/drug therapy , Endothelium, Vascular/drug effects , Hyperalgesia/drug therapy , Protective Agents/therapeutic use , Protein C/immunology , Spinal Cord/blood supply , Thrombin/therapeutic use , Amino Acid Sequence , Animals , Capillary Permeability/drug effects , Chronic Pain/blood , Chronic Pain/immunology , Chronic Pain/pathology , Cytokines/blood , Cytokines/immunology , Endothelium, Vascular/immunology , Endothelium, Vascular/pathology , Human Umbilical Vein Endothelial Cells , Humans , Hyperalgesia/blood , Hyperalgesia/immunology , Hyperalgesia/pathology , Inflammation/blood , Inflammation/drug therapy , Inflammation/immunology , Inflammation/pathology , Male , Models, Molecular , Molecular Sequence Data , Protective Agents/chemistry , Protective Agents/isolation & purification , Rats , Salmon/metabolism , Spinal Cord/drug effects , Spinal Cord/immunology , Spinal Cord/pathology , Thrombin/chemistry , Thrombin/isolation & purification
14.
Ukr Biochem J ; 87(2): 88-94, 2015.
Article in English | MEDLINE | ID: mdl-26255342

ABSTRACT

The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2∙10(-9) M. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.


Subject(s)
Antibody Specificity , Oligopeptides/chemistry , Protein C/analysis , Single-Chain Antibodies/isolation & purification , Animals , Cattle , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/virology , Female , Hemocyanins/chemistry , Hemocyanins/immunology , Humans , Immunoconjugates/chemistry , Immunoconjugates/immunology , Mice , Mice, Inbred BALB C , Mollusca , Oligopeptides/immunology , Peptide Library , Protein Binding , Protein C/chemistry , Protein C/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/immunology , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/chemistry
15.
Microbiol Immunol ; 59(7): 381-8, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25891444

ABSTRACT

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti-apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF-α and IL-6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL-12 and anti-inflammatory cytokine IL-10. Interestingly, treatment with 5 µg/mL, but not 25 µg/mL, of APC significantly enhanced production of IL-10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co-stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti-inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF-α and/or IL-6 production.


Subject(s)
Dendritic Cells/immunology , Protein C/immunology , Cell Differentiation , Cells, Cultured , Dendritic Cells/cytology , Humans , Interleukin-10/immunology , Interleukin-12/immunology , Interleukin-6/immunology , Monocytes/cytology , Monocytes/immunology
16.
Thromb Res ; 135(5): 963-9, 2015 May.
Article in English | MEDLINE | ID: mdl-25813362

ABSTRACT

INTRODUCTION: The role of leukocyte and its death in the progression in inflammation attracts attention nowadays. The purpose of this study is to examine the effects of activated protein C (APC) on leucocyte cell death and vascular endothelial damage in sepsis. METHODS: Wistar rats were infused with lipopolysaccharide (8.0mg/kg) concomitantly with either a low dose (0.5mg/kg), a high dose (5.0mg/kg) of plasma-derived APC or albumin. One and 3hours after the injections, the mesenteric microcirculation was observed by intravital microscopy. The serum levels of nucleosome and High Mobility Group Box 1 (HMGB1) were measured in each group. In another series, cultured leukocyte cell-death in the medium supplemented with serum obtained from each group was examined in vitro. RESULTS: Microcirculatory disturbance was significantly suppressed in both the high-dose and low-dose groups compared to the control group (P<0.01, 0.05, respectively). The bleeding area was significantly increased in the control and high-dose groups (P<0.05, 0.01, respectively). Serum levels of cell death markers such as nucleosome and HMGB1 were significantly decreased in the treatment groups (P<0.01), and the protective effect was more pronounced in high-dose group. Cell death suppression was most prominent in high-dose group and the formation of neutrophil extracellular traps (NETs) was significantly suppressed in the treatment groups. CONCLUSION: Low-dose plasma-derived APC exerted protective effects on the microcirculation without increasing the risk of bleeding. The protective effect against leukocyte cell death and the suppressive effect on NETs formation of APC might be related to its beneficial effects.


Subject(s)
Endothelium, Vascular/pathology , Leukocytes/pathology , Protein C/immunology , Sepsis/pathology , Animals , Cell Death , Endothelium, Vascular/immunology , Leukocytes/immunology , Lipopolysaccharides/immunology , Microcirculation , Protein C/therapeutic use , Rats, Wistar , Sepsis/blood , Sepsis/immunology , Sepsis/therapy
17.
J Thromb Haemost ; 12(11): 1801-9, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25196808

ABSTRACT

BACKGROUND: Antiphospholipid antibodies may interfere with the anticoagulant activity of activated protein C (APC) to induce acquired APC resistance (APCr). AIMS: To investigate the frequency and characteristics of APCr by using recombinant human APC (rhAPC) and endogenous protein C activation in antiphospholipid syndrome (APS). METHODS: APCr was assessed in APS and non-APS venous thromboembolism (VTE) patients on warfarin and normal controls with rhAPC or Protac by thrombin generation. IgG anti-protein C and anti-protein S antibodies and avidity were assessed by ELISA. RESULTS: APS patients showed greater resistance to both rhAPC and Protac than non-APS patients and normal controls (median normalized endogenous thrombin potential inhibition): APS patients with rhAPC, 81.3% (95% confidence interval [CI] 75.2-88.3%; non-APS patients with rhAPC, 97.7% (95% CI 93.6-101.8%; APS patients with Protac, 66.0% (95% CI 59.5-72.6%); and non-APS patients with Protac, 80.7 (95% CI 74.2-87.2%). APS patients also had a higher frequency and higher levels of anti-protein C antibodies, with 60% (15/25) high-avidity antibodies. High-avidity anti-protein C antibodies were associated with greater APCr and with a severe thrombotic phenotype (defined as the development of recurrent VTE while patients were receiving therapeutic anticoagulation or both venous and arterial thrombosis). Twelve of 15 (80%) patients with high-avidity anti-protein C antibodies were classified as APS category I. CONCLUSION: Thrombotic APS patients showed greater APCr to both rhAPC and activation of endogenous protein C by Protac. High-avidity anti-protein C antibodies, associated with greater APCr, may provide a marker for a severe thrombotic phenotype in APS. However, in patients with category I APS, it remains to be established whether anti-protein C or anti-ß2 -glycoprotein I antibodies are responsible for APCr.


Subject(s)
Activated Protein C Resistance/immunology , Antibodies, Antiphospholipid/blood , Antiphospholipid Syndrome/complications , Protein C/immunology , Venous Thromboembolism/immunology , Activated Protein C Resistance/blood , Activated Protein C Resistance/diagnosis , Activated Protein C Resistance/drug therapy , Adult , Aged , Anticoagulants/therapeutic use , Antiphospholipid Syndrome/blood , Antiphospholipid Syndrome/diagnosis , Antiphospholipid Syndrome/drug therapy , Antiphospholipid Syndrome/immunology , Biomarkers/blood , Blood Coagulation Tests , Case-Control Studies , Cross-Sectional Studies , Enzyme-Linked Immunosorbent Assay , Female , Fibrinolytic Agents/therapeutic use , Humans , Intercellular Signaling Peptides and Proteins , Male , Middle Aged , Peptides/therapeutic use , Phenotype , Protein C/therapeutic use , Recombinant Proteins/therapeutic use , Severity of Illness Index , Venous Thromboembolism/blood , Venous Thromboembolism/diagnosis , Venous Thromboembolism/prevention & control , Warfarin/therapeutic use
18.
Thromb Res ; 133 Suppl 1: S32-4, 2014 May.
Article in English | MEDLINE | ID: mdl-24759138

ABSTRACT

The inflammation-induced activation of the protein C pathway provides negative feedback inhibition of coagulation and exerts coagulation-independent anti-inflammatory and cytoprotective effects. The balance between these activities of aPC modulates the outcome of diverse inflammatory diseases such as encephalitis, diabetes, and sepsis; and is affected by naturally occurring aPC-resistance of coagulation factor V Leiden.


Subject(s)
Blood Coagulation , Inflammation/complications , Protein C/immunology , Activated Protein C Resistance/complications , Activated Protein C Resistance/immunology , Animals , Brain Diseases/immunology , Diabetes Complications/complications , Diabetes Complications/immunology , Encephalitis , Factor V/immunology , Hashimoto Disease/immunology , Humans , Inflammation/immunology , Sepsis/complications , Sepsis/immunology , Signal Transduction
19.
PLoS Negl Trop Dis ; 8(4): e2819, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24762740

ABSTRACT

BACKGROUND: During severe (pneumo)sepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM) is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC) into active protein C (APC). APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia caused by Burkholderia (B.) pseudomallei. METHODOLOGY/PRINCIPAL FINDINGS: (WT) mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice) were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination. CONCLUSION/SIGNIFICANCE: TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumo)sepsis.


Subject(s)
Burkholderia pseudomallei/immunology , Melioidosis/immunology , Mutant Proteins/genetics , Pneumonia, Bacterial/immunology , Protein C/immunology , Sepsis/immunology , Thrombomodulin/immunology , Animals , Male , Melioidosis/pathology , Mice , Mice, Inbred C57BL , Mutant Proteins/immunology , Pneumonia, Bacterial/complications , Pneumonia, Bacterial/pathology , Point Mutation , Protein C/metabolism , Sepsis/pathology , Survival Analysis , Thrombomodulin/genetics
20.
J Rheumatol ; 40(12): 2042-6, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24128780

ABSTRACT

OBJECTIVES: The risk of venous thromboembolism (VTE) is increased in patients with antineutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) as compared to healthy subjects. The mechanisms underlying this increased occurrence of VTE are not completely understood. We hypothesize that AAV patients in remission are more procoagulant than healthy controls. METHODS: Patients with AAV in remission and no VTE for the last 6 months were included. Patients with severe renal impairment (serum creatinine > 250 µmol/l) were excluded. Age and sex matched healthy controls were included. The endogenous thrombin potential (ETP) was determined together with hemostatic variables: fibrinogen, D-dimers, factor VIII (FVIII), tissue factor pathway inhibitor (TFPI), protein C, and free protein S. RESULTS: Thirty-one patients were included. In 27 patients not taking anticoagulants, ETP was measured and found to be elevated: 137.1% as compared to a median of 90.0% for healthy controls (p < 0.01). Fibrinogen and D-dimer levels were not elevated in patients (median 3.5 g/l and 279 µg/l, respectively). FVIII and TFPI levels were also significantly increased in patients as compared to healthy controls (159% vs 137%; 122.5% vs 101%, respectively), whereas protein C and free protein S levels were not elevated (126.5% vs 118.6% and 124.6% vs 118.3%, respectively). CONCLUSION: Patients with AAV in remission are more procoagulant than healthy controls, as indicated by an increased ETP. The increased FVIII level measured in these patients suggests persistence of endothelial activation and/or dysfunction. This endothelial dysfunction may cause a continuous low-grade procoagulant state.


Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Antibodies, Antineutrophil Cytoplasmic/immunology , Thrombophilia/immunology , Venous Thromboembolism/immunology , Adult , Aged , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/blood , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/epidemiology , Antibodies, Antineutrophil Cytoplasmic/blood , Factor VIII/immunology , Factor VIII/metabolism , Female , Fibrin Fibrinogen Degradation Products/immunology , Fibrin Fibrinogen Degradation Products/metabolism , Fibrinogen/immunology , Fibrinogen/metabolism , Follow-Up Studies , Humans , Lipoproteins/immunology , Lipoproteins/metabolism , Male , Middle Aged , Protein C/immunology , Protein C/metabolism , Protein S/immunology , Protein S/metabolism , Remission Induction , Risk Factors , Thrombin/immunology , Thrombin/metabolism , Thrombophilia/blood , Thrombophilia/epidemiology , Venous Thromboembolism/blood , Venous Thromboembolism/epidemiology
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