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1.
Sci Transl Med ; 12(570)2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33139519

RESUMO

Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti-ß2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer's threshold and in 30% using a more stringent cutoff (≥40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.

2.
J Vet Intern Med ; 34(5): 1894-1902, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32881076

RESUMO

BACKGROUND: Neutrophil extracellular traps (NETs), webs of DNA and citrullinated histones extruded from activated neutrophils cause transfusion-related acute lung injury. Supernatants of stored red blood cell (RBC) units might promote NETosis in neutrophils from the units or from transfusion recipients. HYPOTHESES: (1) NETs form during storage of canine RBC, (2) leukoreduction (LR) before storage of RBC reduces NETosis, and (3) supernatant from stored, nonleukoreduced (NLR) RBC units induces NETosis in healthy canine neutrophils modeling transfusion recipients. ANIMALS: Six healthy purpose-bred research dogs were utilized for blood donation. METHODS: Prospective controlled study. RBC units were collected from each dog, aseptically divided into 2 equal subunits, 1 of which was leukoreduced, and stored for 42 days. Stored units were sampled biweekly for quantification of NET markers citrullinated histone H3 (Western blot) and cell-free DNA (cfDNA) (DNA dye binding). Unit supernatants were applied ex vivo to canine neutrophils and extracellular DNA release representing NETosis was assessed. RESULTS: Markers of NETs increased during RBC storage (cfDNA P < .0001 and citrullinated H3 P = .0002) and were higher in NLR than LR units (day 42 LR cfDNA 0.34 ± 0.82 ng/mL vs day 42 NLR 1361.07 ± 741.00 ng/mL, P < .0001; day 42 LR citrullinated H3 0.19 ± 0.13 AU vs NLR 0.57 ± 0.34 AU, P = .007). Isolated neutrophils did not form NETs when exposed to stored canine RBC supernatant. CONCLUSIONS AND CLINICAL IMPORTANCE: NETosis occurs in stored canine NLR RBC units, and is attenuated by LR before storage. NETs might be mediators of transfusion reactions.

3.
Nat Commun ; 11(1): 4035, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788578

RESUMO

Polyphosphates are linear polymers and ubiquitous metabolites. Bacterial polyphosphates are long chains of hundreds of phosphate units. Here, we report that mouse survival of peritoneal Escherichia coli sepsis is compromised by long-chain polyphosphates, and improves with bacterial polyphosphatekinase deficiency or neutralization using recombinant exopolyphosphatase. Polyphosphate activities are chain-length dependent, impair pathogen clearance, antagonize phagocyte recruitment, diminish phagocytosis and decrease production of iNOS and cytokines. Macrophages bind and internalize polyphosphates, in which their effects are independent of P2Y1 and RAGE receptors. The M1 polarization driven by E. coli derived LPS is misdirected by polyphosphates in favor of an M2 resembling phenotype. Long-chain polyphosphates modulate the expression of more than 1800 LPS/TLR4-regulated genes in macrophages. This interference includes suppression of hundreds of type I interferon-regulated genes due to lower interferon production and responsiveness, blunted STAT1 phosphorylation and reduced MHCII expression. In conclusion, prokaryotic polyphosphates disturb multiple macrophage functions for evading host immunity.


Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Polifosfatos/metabolismo , Animais , Apresentação do Antígeno/imunologia , Polaridade Celular , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon Tipo I/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Fenótipo , Sepse/imunologia , Análise de Sobrevida , Transcriptoma/genética
4.
PLoS One ; 15(8): e0237849, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822431

RESUMO

Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate. We now extend that approach by optimizing the reaction conditions and using readily available "bridging molecules" containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the terminal phosphates of polyphosphate. Additionally, we report three facile methods for purifying conjugated polyphosphate from excess reactants.


Assuntos
Aminas/química , Química Click/métodos , Peptídeos/química , Polifosfatos/química , Biotinilação , Ciclo-Octanos/química , Cistamina/química , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Polifosfatos/isolamento & purificação , Compostos de Sulfidrila/química , Temperatura , Fatores de Tempo
5.
J Thromb Haemost ; 18(11): 3043-3052, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32808449

RESUMO

BACKGROUND: Platelets secrete many pro-wound healing molecules such as growth factors and cytokines. We found that releasates from activated human platelets induced the differentiation of cultured murine and human fibroblasts into a myofibroblast phenotype. Surprisingly, most of this differentiation-inducing activity was heat-stable, suggesting it was not due to the protein component of the releasates. Inorganic polyphosphate is a major constituent of platelet-dense granules and promotes blood coagulation and inflammation. OBJECTIVES: We aim to investigate the contribution of polyphosphate on myofibroblast differentiating activity of platelet releasates. METHODS: Using NIH-3T3 cells and primary human fibroblasts, we examined the effect of human platelet releasates and chemically synthesized polyphosphate on fibroblast differentiation and migration. RESULTS: We found that the myofibroblast-inducing activity of platelet releasates was severely attenuated after incubation with a polyphosphate-degrading enzyme, and that fibroblasts responded to platelet-sized polyphosphate by increased levels of α-smooth muscle actin, stress fibers, and collagen. Furthermore, fibroblasts were chemotactic toward polyphosphate. CONCLUSIONS: These findings indicate that platelet-derived polyphosphate acts as a cell signaling molecule by inducing murine and human fibroblasts to differentiate into myofibroblasts, a cell type known to drive both wound healing and fibrosing diseases. Polyphosphate therefore not only promotes early wound responses through enhancing fibrin clot formation, but also may play roles in the later stages of wound healing, and, potentially, progression of fibrotic diseases, by recruiting fibroblasts and inducing their differentiation into myofibroblasts.

6.
Biotechnol Bioeng ; 117(7): 2089-2099, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32190899

RESUMO

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2-40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO3 , in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, 31 P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.

7.
J Thromb Haemost ; 17(12): 2131-2140, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31420909

RESUMO

BACKGROUND: Inorganic polyphosphate modulates the contact pathway of blood clotting, which is implicated in thrombosis and inflammation. Polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60-100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. We previously reported that optimal triggering of clotting via the contact pathway requires very long polyphosphates, although the impact of shorter polyphosphate polymers on individual proteolytic reactions of the contact pathway was not interrogated. OBJECTIVES AND METHODS: We conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high molecular weight kininogen and Zn2+ , to mediate four individual proteolytic reactions of the contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekallikrein activation by factor XIIa, and prekallikrein autoactivation. RESULTS: The individual contact pathway reactions were differentially dependent on polyphosphate length. Very long-chain polyphosphate was required to support factor XII autoactivation, whereas platelet-size polyphosphate significantly accelerated the activation of factor XII by kallikrein, and the activation of prekallikrein by factor XIIa. Intriguingly, polyphosphate did not support prekallikrein autoactivation. We also report that high molecular weight kininogen was required only when kallikrein was the enzyme (ie, FXII activation by kallikrein), whereas Zn2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn2+ nor high molecular weight kininogen. CONCLUSIONS: Platelet polyphosphate and Zn2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states.


Assuntos
Coagulação Sanguínea , Cininogênio de Alto Peso Molecular/sangue , Polifosfatos/sangue , Zinco/sangue , Ativação Enzimática , Fator Xa/metabolismo , Humanos , Cinética , Cininogênio de Alto Peso Molecular/química , Peso Molecular , Polifosfatos/química , Proteólise
8.
Res Pract Thromb Haemost ; 3(1): 18-25, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30656272

RESUMO

This illustrated review focuses on polyphosphate as a potent modulator of the plasma clotting cascade, with possible roles in hemostasis, thrombosis, and inflammation. Polyphosphates are highly anionic, linear polymers of inorganic phosphates that are widespread throughout biology. Infectious microorganisms accumulate polyphosphates with widely varying polymer lengths (from a few phosphates to over a thousand phosphates long), while activated human platelets secrete polyphosphate with a very narrow size distribution (about 60-100 phosphates long). Work from our lab and others has shown that long-chain polyphosphate is a potent trigger of clotting via the contact pathway, while polyphosphate of the size secreted by platelets accelerates factor V activation, blocks the anticoagulant activity of tissue factor pathway inhibitor, promotes factor XI activation by thrombin, and makes fibrin fibrils thicker and more resistant to fibrinolysis. Polyphosphate also modulates inflammation by triggering bradykinin release, inhibiting the complement system, and modulating endothelial function. Polyphosphate and nucleic acids have similar physical properties and both will trigger the contact pathway-although polyphosphate is orders of magnitude more procoagulant than either DNA or RNA. Important caveats in these studies include observations that nucleic acids and polyphosphate may co-purify, and that these preparations can be contaminated with highly procoagulant microparticles if silica-based purification methods are employed. Polyphosphate has received attention as a possible therapeutic, with some recent studies exploring the use of polyphosphate in a variety of formulations to control bleeding. Other studies are investigating treatments that block polyphosphate function as novel antithrombotics with the possibility of reduced bleeding side effects.

9.
Arterioscler Thromb Vasc Biol ; 38(8): 1748-1760, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30354195

RESUMO

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Fator XII/metabolismo , Fator XIIa/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Polifosfatos/toxicidade , Trombose/induzido quimicamente , Animais , Plaquetas/metabolismo , Modelos Animais de Doenças , Fator XII/genética , Fator XIIa/genética , Feminino , Fibrina/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Papio ursinus , Pré-Calicreína/genética , Pré-Calicreína/metabolismo , Embolia Pulmonar/sangue , Embolia Pulmonar/induzido quimicamente , Embolia Pulmonar/genética , Sepse/sangue , Sepse/microbiologia , Transdução de Sinais/efeitos dos fármacos , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/microbiologia , Trombose/sangue , Trombose/genética , Calicreínas Teciduais/genética , Calicreínas Teciduais/metabolismo
10.
Mol Microbiol ; 110(6): 973-994, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30230089

RESUMO

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.


Assuntos
Polifosfatos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/metabolismo , Hidrolases Anidrido Ácido/química , Proteínas de Bactérias/química , Núcleo Celular/metabolismo , Microcorpos/metabolismo
11.
Electrophoresis ; 39(19): 2454-2459, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30009536

RESUMO

PAGE is often used to resolve inorganic polyphosphates (polyP), but unfortunately polyP size ladders are not commercially available. Since several dyes that are commonly used to detect nucleic acids in gels also stain polyP, we examined the utility of commercially available DNA size ladders for estimating polyP polymer lengths by gel electrophoresis. Narrow size fractions of polyP were prepared and their polymer lengths were quantified using NMR. Commercially available DNA ladders and these polyP fractions were then subjected to PAGE to determine the relationship between migration of DNA vs polyP, which was found to be: log10 (dsDNA length in bp) = 1.66 × log10 (polyP length in phosphate units) - 1.97. This relationship between DNA and polyP size held for a variety of different polyacrylamide concentrations, indicating that DNA size ladders can readily be employed to estimate polyP polymer lengths by PAGE.


Assuntos
DNA/química , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese em Gel de Poliacrilamida/normas , Polifosfatos/análise , Polifosfatos/química , DNA/análise , Padrões de Referência , Reprodutibilidade dos Testes
12.
Front Med (Lausanne) ; 5: 107, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29719836

RESUMO

Polyphosphate plays several roles in coagulation and inflammation, while extracellular DNA and RNA are implicated in thrombosis and as disease biomarkers. We sought to compare the procoagulant activities of polyphosphate versus DNA or RNA isolated from mammalian cells. In a recent study, we found that much of the procoagulant activity of DNA isolated from mammalian cells using Qiagen kits resisted digestion with nuclease or polyphosphatase, and even resisted boiling in acid. These kits employ spin columns packed with silica, which is highly procoagulant. Indeed, much of the apparent procoagulant activity of cellular DNA isolated with such kits was attributable to silica particles shed by the spin columns. Therefore, silica-based methods for isolating nucleic acids or polyphosphate from mammalian cells are not suitable for studying their procoagulant activities. We now report that polyphosphate readily co-purified with DNA and RNA using several popular isolation methods, including phenol/chloroform extraction. Thus, cell-derived nucleic acids are also subject to contamination with traces of cellular polyphosphate, which can be eliminated by alkaline phosphatase digestion. We further report that long-chain polyphosphate was orders of magnitude more potent than cell-derived DNA (purified via phenol/chloroform extraction) or RNA at triggering clotting. Additional experiments using RNA homopolymers found that polyG and polyI have procoagulant activity similar to polyphosphate, while polyA and polyC are not procoagulant. Thus, the procoagulant activity of RNA is rather highly dependent on base composition.

14.
Vet Clin Pathol ; 46(2): 278-286, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28370080

RESUMO

BACKGROUND: Neutrophil extracellular traps (NET), consisting of a filamentous DNA/chromatin-histone scaffold originating from neutrophils are part of the innate immune response, may be released under a variety of inflammatory conditions and are associated with an increased risk for thrombosis. OBJECTIVES: The purpose of this study was to evaluate a SYTOX green fluorescence assay and a histone-DNA complex (hisDNA) ELISA for quantification of NET-related DNA in canine plasma. METHODS: The influence of variations in blood sample handling on assay results was tested. Accuracy of the SYTOX green fluorescence and the hisDNA ELISA was evaluated with dilutional linearity using serial dilutions. Interference was assessed by addition of purified bilirubin or hemoglobin. Precision was determined by calculating the intra- and inter-assay CV. RESULTS: Preanalytic sample handling did not influence DNA measurements by either assay. Citrate and EDTA plasma samples were equivalent. For the DNA fluorescence assay, dilutional linearity was poor due to autofluorescence, which was corrected by addition of canine plasma to the diluent. The presence of bilirubin and hemoglobin also increased autofluorescence, and resulted in falsely low concentrations of DNA. On the hisDNA ELISA, pigmentemia had no effect. CONCLUSIONS: Both assays as modified in this study are suitable for measuring DNA in canine EDTA or citrate plasma. However, performance of the fluorescence assay was impacted by pigmentemia, and it was less sensitive than the ELISA in detecting the presence of nucleosome material in the plasma.


Assuntos
DNA/sangue , Doenças do Cão/sangue , Armadilhas Extracelulares , Inflamação/veterinária , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Imunofluorescência/veterinária , Inflamação/sangue
16.
Astrobiology ; 17(3): 253-265, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28282220

RESUMO

Planetary protection is governed by the Outer Space Treaty and includes the practice of protecting planetary bodies from contamination by Earth life. Although studies are constantly expanding our knowledge about life in extreme environments, it is still unclear what the probability is for terrestrial organisms to survive and grow on Mars. Having this knowledge is paramount to addressing whether microorganisms transported from Earth could negatively impact future space exploration. The objectives of this study were to identify cultivable microorganisms collected from the surface of the Mars Science Laboratory, to distinguish which of the cultivable microorganisms can utilize energy sources potentially available on Mars, and to determine the survival of the cultivable microorganisms upon exposure to physiological stresses present on the martian surface. Approximately 66% (237) of the 358 microorganisms identified are related to members of the Bacillus genus, although surprisingly, 22% of all isolates belong to non-spore-forming genera. A small number could grow by reduction of potential growth substrates found on Mars, such as perchlorate and sulfate, and many were resistant to desiccation and ultraviolet radiation (UVC). While most isolates either grew in media containing ≥10% NaCl or at 4°C, many grew when multiple physiological stresses were applied. The study yields details about the microorganisms that inhabit the surfaces of spacecraft after microbial reduction measures, information that will help gauge whether microorganisms from Earth pose a forward contamination risk that could impact future planetary protection policy. Key Words: Planetary protection-Spore-Bioburden-MSL-Curiosity-Contamination-Mars. Astrobiology 17, 253-265.


Assuntos
Bactérias/metabolismo , Meio Ambiente Extraterreno , Laboratórios , Marte , Viabilidade Microbiana , Astronave , Aerobiose , Anaerobiose , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Bactérias/efeitos da radiação , Dessecação , Elétrons , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Peróxidos/toxicidade , Filogenia , Raios Ultravioleta
17.
Sci Rep ; 7: 42119, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28186112

RESUMO

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Nanopartículas/química , Polifosfatos/farmacologia , Trombina/farmacologia , Trombose/sangue , Velocidade do Fluxo Sanguíneo , Plaquetas/metabolismo , Células Cultivadas , Simulação por Computador , Humanos , Microfluídica/instrumentação , Modelos Cardiovasculares , Ativação Plaquetária , Polifosfatos/química , Propriedades de Superfície , Trombina/química , Trombose/induzido quimicamente , Tempo de Coagulação do Sangue Total
18.
Ann Biomed Eng ; 45(5): 1328-1340, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27933406

RESUMO

In the contact activation pathway of the coagulation, zymogen factor XII (FXII) is converted to FXIIa, which triggers activation of FXI leading to the activation of FIX and subsequent thrombin generation and fibrin formation. Feedback activation of FXI by thrombin has been shown to promote thrombin generation in a FXII-independent manner and FXIIa can bypass FXI to directly activate FX and prothrombin in the presence of highly negatively charged molecules, such as long-chain polyphosphates (LC polyP). We sought to determine whether activation of FXII or FXI differentially regulate the physical biology of fibrin formation. Fibrin formation was initiated with tissue factor, ellagic acid (EA), or LC polyP in the presence of inhibitors of FXI and FXII. Our data demonstrated that inhibition of FXI decreased the rate of fibrin formation and fiber network density, and increased the fibrin network strength and rate of fibrinolysis when gelation was initiated via the contact activation pathway with EA. FXII inhibition decreased the fibrin formation and fibrin density, and increased the fibrinolysis rate only when fibrin formation was initiated via the contact activation pathway with LC polyP. Overall, we demonstrate that inhibition of FXI and FXII distinctly alter the biophysical properties of fibrin.


Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/química , Fator XII , Fator XI , Fibrina/química , Fibrinólise , Fator XI/antagonistas & inibidores , Fator XI/química , Fator XII/antagonistas & inibidores , Fator XII/química , Humanos , Polifosfatos/química
19.
J Biol Chem ; 292(5): 1808-1814, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28007958

RESUMO

The contact pathway of the plasma clotting cascade is dispensable for normal hemostasis, but contributes to thrombosis and serves as a bridge between inflammation and coagulation. This pathway is triggered upon exposure of plasma to certain anionic polymers and artificial surfaces. Recently, extracellular nucleic acids and inorganic polyphosphate (polyP) have been implicated as being important (patho)physiologically relevant activators of this pathway. However, mechanistic details regarding how nucleic acids or polyP modulate the individual reactions of the contact pathway have been lacking. In this study, we investigate the ability of RNA homopolymers and polyP to bind the primary constituents of the contact pathway: factor XIa, factor XIIa, and plasma kallikrein, in the presence and absence of high molecular weight kininogen (HK), an important cofactor in this pathway. We examine seven proteolytic activation reactions within the contact pathway and report that polyP greatly enhances the rate of all seven, while RNA is effective in supporting only a subset of these reactions. HK both enhances and suppresses these proteolytic activation reactions, depending on the specific reaction evaluated. Overall, we find that polyP is a potent mediator of contact pathway activation reactions in general, that RNA secondary structure may be important to its procoagulant activity, and that nucleic acids versus polyP may differentially modulate specific enzyme activation events within the contact pathway.


Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Coagulação Sanguínea/fisiologia , Polifosfatos/metabolismo , Proteólise , RNA/metabolismo , Humanos
20.
Am J Vet Res ; 77(12): 1340-1345, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27901385

RESUMO

OBJECTIVE To determine the predominant thromboxane (TX) metabolite in urine of healthy cats, evaluate whether the method of sample collection would impact concentration of that metabolite, and propose a reference interval for that metabolite in urine of healthy cats. ANIMALS 17 cats (11 purpose-bred domestic shorthair cats, 5 client-owned domestic shorthair cats, and 1 client-owned Persian cat). PROCEDURES All cats were deemed healthy on the basis of results for physical examination, a CBC, serum biochemical analysis, urinalysis, and measurement of prothrombin time and activated partial thromboplastin time. Voided and cystocentesis urine samples (or both) were collected. Aliquots of urine were stored at -80°C until analysis. Concentrations of TXB2, 11-dehydroTXB2, and 2,3 dinorTXB2 were measured with commercially available ELISA kits. Urinary creatinine concentration was also measured. RESULTS 11-dehydroTXB2 was the most abundant compound, representing (mean ± SD) 59 ± 18% of the total amount of TX detected. In all samples, the concentration of 11-dehydroTXB2 was greater than that of 2,3 dinorTXB2 (mean, 4.2 ± 2.7-fold as high). Mean concentration of 11-dehydroTXB2 for the 17 cats was 0.57 ± 0.47 ng/mg of creatinine. A reference interval (based on the 5% to 95% confidence interval) of 0.10 to 2.1 ng of 11-dehydroTXB2/mg of creatinine was proposed for healthy cats. CONCLUSIONS AND CLINICAL RELEVANCE In this study, 11-dehydroTXB2 was the major TX metabolite in feline urine. Measurement of this metabolite may represent a noninvasive, convenient method for monitoring in vivo platelet activation in cats at risk for thromboembolism.


Assuntos
Gatos/urina , Creatinina/urina , Tromboxanos/urina , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Valores de Referência , Manejo de Espécimes , Urinálise/veterinária
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