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1.
Food Chem ; 334: 127507, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32688180

RESUMO

A novel food-derived anticoagulant heptapeptides (P-3-CG) was isolated and characterized from oyster (Crassostrea gigas) pepsin hydrolysate. P-3-CG competed with fibrinogen against thrombin active domain by a spontaneous and exothermic reaction which was entropically driven. The residue Lys7 of P-3-CG anchored thrombin S1 pocket strongly, which inhibited fibrinogen binding to the thrombin, then blocked the conversion of fibrinogen to fibrin. The fibrinogen clotting time was prolonged to 27.55 s, and the reciprocally authenticated results of dynamic light scattering and scanning electron microscope further explained for fibrinogen clotting time extension. Inhibition of amidolytic activity of thrombin was affected significantly by reaction time and P-3-CG concentration. Furthermore, P-3-CG prolonged activated partial thromboplastin time significantly in vitro/vivo, and decreased the mortality which was confirmed by pulmonary pathological slide results. The obtained results demonstrated that P-3-CG may potentially serve as an alternative food-derived anticoagulant peptide that could be utilized for thrombosis prevention.


Assuntos
Anticoagulantes/metabolismo , Crassostrea/metabolismo , Oligopeptídeos/metabolismo , Trombina/metabolismo , Animais , Anticoagulantes/química , Fibrinogênio/metabolismo , Humanos , Hidrólise , Oligopeptídeos/química , Pepsina A/metabolismo , Trombina/química
2.
Nat Commun ; 11(1): 5468, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122656

RESUMO

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10-50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.


Assuntos
Dissulfetos/química , Fibrinogênio/química , Trombose/metabolismo , Alquilação , Testes de Coagulação Sanguínea , Fibrina/biossíntese , Fibrinólise , Hepatócitos , Humanos , Espectrometria de Massas , Oxirredução , Polimerização , Trombina/metabolismo
3.
Nucleic Acids Res ; 48(19): 10680-10690, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33021630

RESUMO

Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem-loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA Circular/química , Trombina/metabolismo , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/metabolismo , DNA Circular/sangue , DNA Circular/metabolismo , Quadruplex G , Humanos , Ligação Proteica , Trombina/química
4.
J Clin Invest ; 130(11): 6151-6157, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32759504

RESUMO

Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 (COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.


Assuntos
Betacoronavirus , Complexo de Ataque à Membrana do Sistema Complemento , Infecções por Coronavirus , Armadilhas Extracelulares , Neutrófilos , Pandemias , Pneumonia Viral , Tromboplastina , Trombose , Idoso , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Ativação do Complemento/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peptídeos Cíclicos/farmacologia , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/sangue , Receptor da Anafilatoxina C5a/imunologia , Síndrome do Desconforto Respiratório do Adulto/sangue , Síndrome do Desconforto Respiratório do Adulto/imunologia , Síndrome do Desconforto Respiratório do Adulto/virologia , Trombina/imunologia , Trombina/metabolismo , Tromboplastina/imunologia , Tromboplastina/metabolismo , Trombose/sangue , Trombose/imunologia , Trombose/virologia
5.
PLoS One ; 15(7): e0235392, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726315

RESUMO

Platelets upregulate the generation of thrombin and reinforce the fibrin clot which increases the incidence risk of venous thromboembolism (VTE). However, the role of platelets in the pathogenesis of venous cardiovascular diseases remains hard to quantify. An experimentally validated model of thrombin generation dynamics is formulated. The model predicts that a high platelet count increases the peak value of generated thrombin as well as the endogenous thrombin potential (ETP) as reported in experimental data. To investigate the effects of platelets density, shear rate, and wound size on the initiation of blood coagulation, we calibrate a previously developed model of venous thrombus formation and implement it in 3D using a novel cell-centered finite-volume solver. We conduct numerical simulations to reproduce in vitro experiments of blood coagulation in microfluidic capillaries. Then, we derive a reduced one-equation model of thrombin distribution from the previous model under simplifying hypotheses and we use it to determine the conditions of clotting initiation on the platelet count, the shear rate, and the plasma composition. The initiation of clotting also exhibits a threshold response to the size of the wounded region in good agreement with the reported experimental findings.


Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/fisiologia , Modelos Teóricos , Contagem de Plaquetas/métodos , Testes de Coagulação Sanguínea , Plaquetas/metabolismo , Fibrina/metabolismo , Humanos , Agregação Plaquetária/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Resistência ao Cisalhamento/fisiologia , Trombina/metabolismo , Tromboplastina/metabolismo , Trombose/metabolismo , Trombose/fisiopatologia , Veias/metabolismo , Veias/fisiologia
6.
Paediatr Respir Rev ; 35: 20-24, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32653469

RESUMO

Since the initial description in 2019, the novel coronavirus SARS-Cov-2 infection (COVID-19) pandemic has swept the globe. The most severe form of the disease presents with fever and shortness of breath, which rapidly deteriorates to respiratory failure and acute lung injury (ALI). COVID-19 also presents with a severe coagulopathy with a high rate of venous thromboembiolism. In addition, autopsy studies have revealed co-localized thrombosis and inflammation, which is the signature of thromboinflammation, within the pulmonary capillary vasculature. While the majority of published data is on adult patients, there are parallels to pediatric patients. In our experience as a COVID-19 epicenter, children and young adults do develop both the coagulopathy and the ALI of COVID-19. This review will discuss COVID-19 ALI from a hematological perspective with discussion of the distinct aspects of coagulation that are apparent in COVID-19. Current and potential interventions targeting the multiple thromboinflammatory mechanisms will be discussed.


Assuntos
Lesão Pulmonar Aguda/sangue , Infecções por Coronavirus/sangue , Inflamação/sangue , Pneumonia Viral/sangue , Trombose/sangue , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/fisiopatologia , Anticoagulantes/uso terapêutico , Antitrombinas/uso terapêutico , Betacoronavirus , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/imunologia , Transtornos da Coagulação Sanguínea/fisiopatologia , Capilares/imunologia , Capilares/fisiopatologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/fisiopatologia , Endotélio Vascular/imunologia , Endotélio Vascular/fisiopatologia , Inibidores do Fator Xa/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/fisiopatologia , Pandemias , Ativação Plaquetária , Inibidores da Agregação de Plaquetas/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Pneumonia Viral/fisiopatologia , Embolia Pulmonar/sangue , Embolia Pulmonar/imunologia , Embolia Pulmonar/fisiopatologia , Trombina/imunologia , Trombina/metabolismo , Trombose/tratamento farmacológico , Trombose/imunologia , Trombose/fisiopatologia
7.
Arterioscler Thromb Vasc Biol ; 40(9): 2127-2142, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32698684

RESUMO

OBJECTIVE: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kß, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). CONCLUSIONS: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.


Assuntos
Afibrinogenemia/sangue , Plaquetas/efeitos dos fármacos , Fibrinogênio/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Trombose/tratamento farmacológico , Afibrinogenemia/diagnóstico , Afibrinogenemia/genética , Plaquetas/metabolismo , Simulação por Computador , Fibrinogênio/genética , Fibrinolíticos/farmacologia , Humanos , Cinética , Microscopia de Vídeo , Modelos Biológicos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais , Estresse Mecânico , Trombina/metabolismo , Trombose/sangue , Trombose/diagnóstico , Trombose/genética
8.
Molecules ; 25(11)2020 May 29.
Artigo em Inglês | MEDLINE | ID: covidwho-436971

RESUMO

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than -8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.


Assuntos
Anticoagulantes/química , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anticoagulantes/farmacologia , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Hepacivirus/química , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Trombina/antagonistas & inibidores , Trombina/química , Trombina/genética , Trombina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
9.
Molecules ; 25(11)2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32485894

RESUMO

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than -8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.


Assuntos
Anticoagulantes/química , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anticoagulantes/farmacologia , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Hepacivirus/química , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Trombina/antagonistas & inibidores , Trombina/química , Trombina/genética , Trombina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(20): 10958-10969, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32366656

RESUMO

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants and an orphan disease with no specific treatment. Most patients with confirmed NEC develop moderate-severe thrombocytopenia requiring one or more platelet transfusions. Here we used our neonatal murine model of NEC-related thrombocytopenia to investigate mechanisms of platelet depletion associated with this disease [K. Namachivayam, K. MohanKumar, L. Garg, B. A. Torres, A. Maheshwari, Pediatr. Res. 81, 817-824 (2017)]. In this model, enteral administration of immunogen trinitrobenzene sulfonate (TNBS) in 10-d-old mouse pups produces an acute necrotizing ileocolitis resembling human NEC within 24 h, and these mice developed thrombocytopenia at 12 to 15 h. We hypothesized that platelet activation and depletion occur during intestinal injury following exposure to bacterial products translocated across the damaged mucosa. Surprisingly, platelet activation began in our model 3 h after TNBS administration, antedating mucosal injury or endotoxinemia. Platelet activation was triggered by thrombin, which, in turn, was activated by tissue factor released from intestinal macrophages. Compared to adults, neonatal platelets showed enhanced sensitivity to thrombin due to higher expression of several downstream signaling mediators and the deficiency of endogenous thrombin antagonists. The expression of tissue factor in intestinal macrophages was also unique to the neonate. Targeted inhibition of thrombin by a nanomedicine-based approach was protective without increasing interstitial hemorrhages in the inflamed bowel or other organs. In support of these data, we detected increased circulating tissue factor and thrombin-antithrombin complexes in patients with NEC. Our findings show that platelet activation is an important pathophysiological event and a potential therapeutic target in NEC.


Assuntos
Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Doenças do Recém-Nascido/metabolismo , Trombina/metabolismo , Animais , Animais Recém-Nascidos , Plaquetas/metabolismo , Modelos Animais de Doenças , Humanos , Recém-Nascido , Inflamação/metabolismo , Enteropatias/patologia , Intestinos/lesões , Intestinos/patologia , Macrófagos/metabolismo , Camundongos , Trombocitopenia/metabolismo
11.
PLoS One ; 15(5): e0227932, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469873

RESUMO

BACKGROUND AND OBJECTIVE: For many pathological states, microparticles are supposed to be one of the causes of hypercoagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test. RESULTS: Monocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0-200·103/ul range of microparticles concentrations. However, at concentrations greater than 500·103/ul, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine. CONCLUSION: Microparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Trombina/metabolismo , Trombose/sangue , Plaquetas/efeitos dos fármacos , Calcimicina/farmacologia , Cálcio/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliais/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Humanos , Monócitos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Trombose/tratamento farmacológico , Trombose/patologia
12.
PLoS One ; 15(5): e0231944, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365105

RESUMO

Intrauterine bleeding during pregnancy is a major risk factor for preterm birth. Thrombin, the most abundant coagulation factor in blood, is associated with uterine myometrial contraction. Here, we investigated the molecular mechanism and signaling of thrombin-induced myometrial contraction. First, histologic studies of placental abruption, as a representative intrauterine bleeding, revealed that thrombin was expressed within the infiltrating hemorrhage and that thrombin receptor (protease-activated receptor 1, PAR1) was highly expressed in myometrial cells surrounding the hemorrhage. Treatment of human myometrial cells with thrombin resulted in augmented contraction via PAR1. Thrombin-induced signaling to myosin was then mediated by activation of myosin light chain kinase- and Rho-induced phosphorylation of myosin light chain-2. In addition, thrombin increased prostaglandin-endoperoxidase synthase-2 (PTGS2 or COX2) mRNA and prostaglandin E2 and F2α synthesis in human myometrial cells. Thrombin significantly increased the mRNA level of interleukine-1ß, whereas it decreased the expressions of prostaglandin EP3 and F2α receptors. Progesterone partially blocked thrombin-induced myometrial contractions, which was accompanied by suppression of the thrombin-induced increase of PTGS2 and IL1B mRNA expressions as well as suppression of PAR1 expression. Collectively, thrombin induces myometrial contractions by two mechanisms, including direct activation of myosin and indirect increases in prostaglandin synthesis. The results suggest a therapeutic potential of progesterone for preterm labor complicated by intrauterine bleeding.


Assuntos
Miométrio/efeitos dos fármacos , Trombina/farmacologia , Contração Uterina/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/metabolismo , Feminino , Humanos , Contração Muscular/efeitos dos fármacos , Miométrio/fisiologia , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Gravidez , Progesterona/metabolismo , Progesterona/farmacologia , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Imagem com Lapso de Tempo , Contração Uterina/fisiologia
13.
PLoS One ; 15(5): e0233866, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470085

RESUMO

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.


Assuntos
Vacinas contra a AIDS/imunologia , Edição de Genes , Proteólise , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Alelos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Células CHO , Sequência Consenso , Cricetinae , Cricetulus , Fator VIII/metabolismo , Glicosilação , Humanos , Polissacarídeos/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Trombina/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
14.
PLoS One ; 15(5): e0233640, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453766

RESUMO

Understanding the coagulation process is critical to developing treatments for trauma and coagulopathies. Clinical studies on tranexamic acid (TXA) have resulted in mixed reports on its efficacy in improving outcomes in trauma patients. The largest study, CRASH-2, reported that TXA improved outcomes in patients who received treatment prior to 3 hours after the injury, but worsened outcomes in patients who received treatment after 3 hours. No consensus has been reached about the mechanism behind the duality of these results. In this paper we use a computational model for coagulation and fibrinolysis to propose that deficiencies or depletions of key anti-fibrinolytic proteins, specifically antiplasmin, a1-antitrypsin and a2-macroglobulin, can lead to worsened outcomes through urokinase-mediated hyperfibrinolysis.


Assuntos
Transtornos da Coagulação Sanguínea/tratamento farmacológico , Ácido Tranexâmico/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/genética , Ferimentos e Lesões/tratamento farmacológico , Antifibrinolíticos/uso terapêutico , Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/patologia , Simulação por Computador , Fibrina/genética , Tempo de Lise do Coágulo de Fibrina , Fibrinolisina/genética , Fibrinólise/efeitos dos fármacos , Hemorragia/sangue , Hemorragia/tratamento farmacológico , Hemorragia/genética , Humanos , Proteínas de Membrana/genética , Mortalidade , alfa 2-Macroglobulinas Associadas à Gravidez/genética , Trombina/genética , Trombina/metabolismo , Ferimentos e Lesões/sangue , Ferimentos e Lesões/genética , Ferimentos e Lesões/patologia , alfa 1-Antitripsina/genética
15.
Pharmacol Res ; 157: 104820, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32360484

RESUMO

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Animais , Anti-Inflamatórios/análise , Anti-Inflamatórios/farmacologia , Células Cultivadas , Simulação por Computador , Infecções por Coronavirus/genética , Expressão Gênica/efeitos dos fármacos , Ácido Glicirrízico/farmacologia , Humanos , Interleucina-6/metabolismo , Lipopeptídeos/antagonistas & inibidores , Lipopeptídeos/farmacologia , Lipopolissacarídeos , Masculino , Pandemias , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia Viral/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Receptores Toll-Like/metabolismo
16.
J Vis Exp ; (159)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32421005

RESUMO

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.


Assuntos
Centrifugação/métodos , Células-Tronco Mesenquimais/citologia , Plasma Rico em Plaquetas/química , Medicina Regenerativa , Trombina/metabolismo , Adulto , Proliferação de Células , Células Cultivadas , Humanos , Masculino
18.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023815

RESUMO

Platelet cryopreservation has been investigated for several decades as an alternative to room temperature storage of platelet concentrates. The use of dimethylsulfoxide as a cryoprotectant has improved platelet storage and cryopreserved concentrates can be kept at -80 °C for two years. Cryopreserved platelets can serve as emergency backup to support stock crises or to disburden difficult logistic areas like rural or military regions. Cryopreservation significantly influences platelet morphology, decreases platelet activation and severely abrogates platelet aggregation. Recent data indicate that cryopreserved platelets have a procoagulant phenotype because thrombin and fibrin formation kicks in earlier compared to room temperature stored platelets. This happens both in static and hydrodynamic conditions. In a clinical setting, low 1-h post transfusion recoveries of cryopreserved platelets represent fast clearance from circulation which may be explained by changes to the platelet GPIbα receptor. Cryopreservation splits the concentrate in two platelet subpopulations depending on GPIbα expression levels. Further research is needed to unravel its physiological importance. Proving clinical efficacy of cryopreserved platelets is difficult because of the heterogeneity of indications and the ambiguity of outcome measures. The procoagulant character of cryopreserved platelets has increased interest for use in trauma stressing the need for double-blinded randomized clinical trials in actively bleeding patients.


Assuntos
Plaquetas/metabolismo , Criopreservação/métodos , Coleta de Amostras Sanguíneas , Fibrina/metabolismo , Humanos , Agregação Plaquetária , Trombina/metabolismo
19.
Clin Chem ; 66(2): 379-389, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32040579

RESUMO

BACKGROUND: Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS: To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. RESULTS: Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION: Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.


Assuntos
Protrombina/genética , Trombose/genética , Adulto , Animais , Testes de Coagulação Sanguínea , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Éxons/genética , Feminino , Hemostasia , Heterozigoto , Humanos , Masculino , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Protrombina/metabolismo , Estudos Retrospectivos , Fatores de Risco , Mutação Silenciosa/genética , Trombina/metabolismo , Trombofilia/genética , Trombofilia/metabolismo , Trombose/metabolismo , Tromboembolia Venosa/genética , Tromboembolia Venosa/metabolismo
20.
Nucleic Acids Res ; 48(7): 3975-3986, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32095808

RESUMO

Guanine-rich regions of the human genome can adopt non-canonical secondary structures. Their role in regulating gene expression has turned them into promising targets for therapeutic intervention. Ligands based on polyaromatic moieties are especially suitable for targeting G-quadruplexes utilizing their size complementarity to interact with the large exposed surface area of four guanine bases. A predictable way of (de)stabilizing specific G-quadruplex structures through efficient base stacking of polyaromatic functional groups could become a valuable tool in our therapeutic arsenal. We have investigated the effect of pyrene-modified uridine nucleotides incorporated at several positions of the thrombin binding aptamer (TBA) as a model system. Characterization using spectroscopic and biophysical methods provided important insights into modes of interaction between pyrene groups and the G-quadruplex core as well as (de)stabilization by enthalpic and entropic contributions. NMR data demonstrated that incorporation of pyrene group into G-rich oligonucleotide such as TBA may result in significant changes in 3D structure such as formation of novel dimeric topology. Site specific structural changes induced by stacking of the pyrene moiety on nearby nucleobases corelate with distinct thrombin binding affinities and increased resistance against nuclease degradation.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Pirenos/química , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/metabolismo , Desoxirribonucleases , Dimerização , Entropia , Humanos , Termodinâmica , Trombina/metabolismo , Nucleotídeos de Uracila/química
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