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1.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639143

RESUMO

Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Hemostáticos/metabolismo , Trombina/metabolismo , Animais , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , Hemostáticos/química , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Especificidade por Substrato , Trombina/química
2.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638997

RESUMO

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3ß. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3ß (Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/ß phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/ß reduced thrombin-mediated platelet aggregation, integrin αIIbß3 activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3ß phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3ß resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/ß KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3ß KI. In conclusion, our data indicate that GSK3α and GSK3ß have differential roles in regulating platelet function.


Assuntos
Plaquetas/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Ativação Plaquetária/genética , Agregação Plaquetária/genética , Transdução de Sinais/genética , Trombose/metabolismo , Animais , Doadores de Sangue , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trombina/metabolismo , Trombose/genética
3.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500640

RESUMO

Recently, the direct thrombin (thr) inhibitor dabigatran has proven to be beneficial in animal models of Alzheimer's disease (AD). Aiming at discovering novel multimodal agents addressing thr and AD-related targets, a selection of previously and newly synthesized potent thr and factor Xa (fXa) inhibitors were virtually screened by the Multi-fingerprint Similarity Searching aLgorithm (MuSSeL) web server. The N-phenyl-1-(pyridin-4-yl)piperidine-4-carboxamide derivative 1, which has already been experimentally shown to inhibit thr with a Ki value of 6 nM, has been flagged by a new, upcoming release of MuSSeL as a binder of cholinesterase (ChE) isoforms (acetyl- and butyrylcholinesterase, AChE and BChE), as well as thr, fXa, and other enzymes and receptors. Interestingly, the inhibition potency of 1 was predicted by the MuSSeL platform to fall within the low-to-submicromolar range and this was confirmed by experimental Ki values, which were found equal to 0.058 and 6.95 µM for eeAChE and eqBChE, respectively. Thirty analogs of 1 were then assayed as inhibitors of thr, fXa, AChE, and BChE to increase our knowledge of their structure-activity relationships, while the molecular determinants responsible for the multiple activities towards the target enzymes were rationally investigated by molecular cross-docking screening.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/farmacologia , Trombina/metabolismo , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Animais , Butirilcolinesterase/metabolismo , Bovinos , Fator Xa/metabolismo , Inibidores do Fator Xa/farmacologia , Humanos , Simulação de Acoplamento Molecular , Piperidinas/farmacologia , Relação Estrutura-Atividade
4.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361773

RESUMO

The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5' end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (KD) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA-thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.


Assuntos
Anticoagulantes/síntese química , Aptâmeros de Nucleotídeos/síntese química , Quadruplex G , Oligonucleotídeos/síntese química , Trombina/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Sequência de Bases , Sítios de Ligação , Coagulação Sanguínea/efeitos dos fármacos , Dendrímeros/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Ligação Proteica , Termodinâmica , Trombina/antagonistas & inibidores , Trombina/metabolismo
5.
FASEB J ; 35(9): e21835, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34449927

RESUMO

Circulating neutrophil extracellular traps (NETs) resistant to t-PA have not been studied completely although NETs in thrombi may contribute to tissue plasminogen activator (t-PA) resistance. This research intended to elucidate whether circulating NETs are associated with t-PA resistance and the underlying mechanism. The levels of NETs were detected in the circulating neutrophils, ischemic brain tissue of acute ischemic stroke (AIS) patients, and transient middle cerebral artery occlusion (tMCAO) models. NET formation in blood, thrombi, and ischemic brain tissue of mice were analyzed by immunofluorescence. Exposed phosphatidylserine (PS) was assessed using flow cytometry and confocal microscopy. Procoagulant activity (PCA) was evaluated using fibrin formation assays, thrombin, and purified coagulation complex. The plasma levels of NETs in AIS patients were significantly higher than those in healthy individuals. After thrombolysis, a significant increase was noted in NET markers in no-improvement patients, while the changes in improvement patients were not significant. Importantly, NETs were decorated with von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) in the blood and thrombi, which could reverse the fibrinolytic effects. In addition, NETs activated platelets (PLTs) and endothelial cells (ECs), stimulating a procoagulant phenotype and facilitating vWF and PAI-1 release. DNase I, activated protein C (APC), and sivelestat markedly inhibited these effects. Furthermore, targeting NETs protected mice from tMCAO-induced cerebral ischemia, possibly by regulating vWF and PAI-1. In summary, NETs may contribute to t-PA resistance in AIS through activation of PLTs and ECs. Strategies against NETs may present a promising therapeutic approach to improve the thrombolysis efficiency of t-PA in AIS patients.


Assuntos
Isquemia Encefálica/metabolismo , Armadilhas Extracelulares/metabolismo , AVC Isquêmico/metabolismo , Neutrófilos/metabolismo , Acidente Vascular Cerebral/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Idoso , Animais , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fosfatidilserinas/metabolismo , Trombina/metabolismo , Trombose/metabolismo
6.
Nutrients ; 13(8)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34444740

RESUMO

The aim of this study was to evaluate the effects of vitamin K1 on various vitamin K-dependent proteins in critically ill patients with prolonged Owren PT. We included critically ill non-bleeding adult patients without liver failure or anticoagulation treatment, with Owren PT > 1.2, who were prescribed intravenous vitamin K1. Blood was drawn at baseline and at 20-28 h after vitamin K1 administration. At both time points, we measured various vitamin K-dependent proteins and coagulation assays. ClinicalTrials.gov; Identifier: NTC3782025. In total, 52 patients were included. Intravenous vitamin K1 reduced Owren PT, Quick PT, protein induced by vitamin K absence/antagonist-II and desphospho-uncarboxylated matrix Gla protein (dp-ucMGP), but not to normal levels. Concomitantly, there were increases in thrombin generation and the activity of coagulation factors II, VII, IX and X that was only counteracted with a small increase in Protein C activity. In conclusion, the results suggest that vitamin K1 strengthens coagulation as measured by PT decrease and increases in the activity of vitamin K-dependent clotting factors and thrombin generation. The decreased dp-ucMGP, and its potential positive short- and long-term non-coagulative effects, merits further research.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Estado Terminal , Tempo de Protrombina , Vitamina K 1/administração & dosagem , Idoso , Biomarcadores/sangue , Fatores de Coagulação Sanguínea/metabolismo , Proteínas de Ligação ao Cálcio/sangue , Proteínas da Matriz Extracelular/sangue , Feminino , Humanos , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteína C/metabolismo , Precursores de Proteínas/sangue , Protrombina , Trombina/metabolismo
7.
Sci Rep ; 11(1): 16170, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373558

RESUMO

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling.


Assuntos
Calicreínas/metabolismo , Receptor PAR-1/antagonistas & inibidores , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Desenho de Fármacos , Humanos , Calicreínas/química , Calicreínas/genética , Cinética , Células MCF-7 , Mutagênese Sítio-Dirigida , Invasividade Neoplásica/prevenção & controle , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteólise , Receptor PAR-1/química , Receptor PAR-1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Especificidade por Substrato , Trombina/metabolismo
8.
FEBS Lett ; 595(20): 2628-2637, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34455594

RESUMO

We recently reported a physical interaction between the angiotensin II (AngII) receptor (AT1R) and thrombin receptor (PAR1) in HEK293 cells using bioluminescence resonance energy transfer (BRET) technology. This was characterized by thrombin trans-activating AT1R and the synergistic responses of the AT1R-PAR1 complex. Here, we investigated the other face of the coin by examining the effect of AT1R on PAR1 activity using BRET. AngII/AT1R did not promote PAR1 activation in the absence of thrombin. However, the combination of thrombin and AngII resulted in their synergistic/allosteric action. Moreover, AngII/AT1R potentiated the maximal thrombin responses, suggesting specific conformational changes within the AT1R-PAR1 complex. Overall, our data confirm the functional AT1R-PAR1 interplay and further support the implication of both AT1R and PAR1 protomers in their synergistic interaction as previously reported.


Assuntos
Transferência de Energia , Receptor Tipo 1 de Angiotensina/metabolismo , Trombina/metabolismo , Células HEK293 , Humanos , Luminescência
9.
Carbohydr Polym ; 270: 118347, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34364596

RESUMO

Fucoidan is a sulfated polysaccharide with various bioactivities. The application of fucoidan in cancer treatment, wound healing, and food industry has been extensively studied. However, the therapeutic value of fucoidan in cardiovascular diseases has been less explored. Increasing number of investigations in the past years have demonstrated the effects of fucoidan on cardiovascular system. In this review, we will focus on the bioactivities related to cardiovascular applications, for example, the modulation functions of fucoidan on coagulation system, inflammation, and vascular cells. Factors mediating those activities will be discussed in detail. Current therapeutic strategies and future opportunities and challenges will be provided to inspire and guide further research.


Assuntos
Fármacos Cardiovasculares/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Polissacarídeos/uso terapêutico , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Fármacos Cardiovasculares/farmacologia , Células Endoteliais/metabolismo , Humanos , Inflamação/tratamento farmacológico , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Polissacarídeos/farmacologia , Ratos , Selectinas/metabolismo , Sulfatos/metabolismo , Trombina/metabolismo
10.
Nat Protoc ; 16(8): 3981-4003, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34215864

RESUMO

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers and regulatory agencies responsible for the safety testing and release of immune globulin products. In this protocol, we describe a robust and sensitive version of the TG assay for quantitative measurement of thrombogenic activity in immune globulin products. Compared with the version of the assay commonly used in clinical laboratories that compares individual patient plasma samples with normal donor samples, our TG assay is suitable for quick (170-260 min) semiautomated analysis of multiple drug samples against the World Health Organization international standard for factor XIa. Commercially available reagents can be used for the assay, and it does not require specialized equipment. The protocol can be easily adapted for the measurement of the procoagulant activity of other biopharmaceuticals, e.g., coagulation factors.


Assuntos
Anticoagulantes/farmacologia , Fator XIa/metabolismo , Trombina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos
11.
Mol Immunol ; 137: 228-237, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34293590

RESUMO

Although high level of circulating C-reactive protein (pCRP) is considered as a biomarker for disease activity, the significance of CRP in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) has not been clarified. We once reported in AAV, pentameric CRP (pCRP) could dissociate into monomeric CRP (mCRP) and activate platelets. Recent studies have demonstrated that the activated platelets can release mitochondrial DNA (mtDNA). The purpose of this study was to further study the relationship between mCRP and platelets in AAV. We found the plasma level of mCRP in AAV patients was significantly higher than that of normal control and positively correlated with the proportion of mCRP-positive platelets. Platelets isolated from one normal donor could be activated by plasma from 5 AAV patients and this effect could be attenuated when mCRP had been removed. Only 0.1 µg/mL of recombinant mCRP was needed for inducing platelets to release mtDNA via interaction with lipid raft and through p38 MAPK/NF-κB pathway. The mCRP binding on platelets depended on the C-terminal octapeptide (aa 199-206). The released mtDNA did not induce respiratory burst alone, but enhanced the ANCA-induced neutrophils respiratory burst after binding Toll-like receptor 9 (TLR9). The mtDNA released by mCRP-activated platelets also enhanced thrombin generation of plasma. In conclusion, our data demonstrate that mCRP can bind platelets via interaction with lipid raft and induce the release of mtDNA. The released mtDNA can enhance the pathogenicity of ANCA and promote activation of coagulation system in AAV.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Plaquetas/metabolismo , Proteína C-Reativa/metabolismo , DNA Mitocondrial/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Coagulação Sanguínea/fisiologia , Feminino , Humanos , Masculino , Microdomínios da Membrana/metabolismo , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Ativação Plaquetária/fisiologia , Explosão Respiratória/fisiologia , Trombina/metabolismo
12.
Sci Rep ; 11(1): 15572, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330995

RESUMO

Factor (F) VIII deficiency causes bleeding in haemophilia A patients because of the reduced formation of procoagulant enzyme thrombin, which is needed to make the blood clot. We measured the dynamics of coagulation in haemophilia A patients by measuring thrombin generation (TG). Additionally, we quantified the procoagulant process of prothrombin conversion and anticoagulant process of thrombin inhibitor complex formation. In haemophilia A, prothrombin conversion is severely reduced, causing TG to be low. Nevertheless, the thrombin inactivation capacity of these patients is comparable to that in healthy subjects, leading to a severe imbalance between procoagulant and anticoagulant processes and a subsequent increased bleeding risk. A novel therapy in haemophilia A is the targeting of anticoagulant pathway, e.g. thrombin inhibitor antithrombin (AT), to restore the haemostatic balance. We simulated the effect of AT reduction on TG in silico. Lowering AT levels restored TG dose-dependently and an AT reduction of 90-95% led to almost normal TG in most patients . However, the variation in response to AT reduction was large between patients, indicating that this approach should be tailored to each individual patients. Ideally, TG and thrombin dynamics simulation could in the future contribute to the management of patients undergoing AT targeting therapy.


Assuntos
Antitrombinas/farmacologia , Hemofilia A/tratamento farmacológico , Adulto , Coagulação Sanguínea/efeitos dos fármacos , Hemofilia A/metabolismo , Hemofilia B/tratamento farmacológico , Hemofilia B/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Protrombina/metabolismo , Trombina/metabolismo
13.
J Trauma Acute Care Surg ; 91(4): 681-691, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34225342

RESUMO

BACKGROUND: Trauma patients have high concentrations of circulating extracellular vesicles (EVs) following injury, but the functional role of EVs in this setting is only partly deciphered. We aimed to describe in detail EV-associated procoagulant activity in individual trauma patients during the first 12 hours after injury to explore their putative function and relate findings to relevant trauma characteristics and outcome. METHODS: In a prospective observational study of 33 convenience recruited trauma patients, citrated plasma samples were obtained at trauma center admission and 2, 4, 6, and 8 hours thereafter. We measured thrombin generation from isolated EVs and the procoagulant activity of phosphatidylserine (PS)-exposing EVs. Correlation and multivariable linear regression analyses were used to explore associations between EV-associated procoagulant activity and trauma characteristics as well as outcome measures. RESULTS: EV-associated procoagulant activity was highest in the first 3 hours after injury. EV-associated thrombin generation normalized within 7 to 12 hours of injury, whereas the procoagulant activity of PS-exposing EVs declined to a level right above that of healthy volunteers. Increased EV-associated procoagulant activity at admission was associated with higher New Injury Severity Score, lower admission base excess, higher admission international normalized ratio, prolonged admission activated partial thromboplastin time, higher Sequential Organ Failure Assessment score at day 0, and fewer ventilator-free days. CONCLUSION: Our data suggest that EVs have a transient hypercoagulable function and may play a role in the early phase of hemostasis after injury. The role of EVs in trauma-induced coagulopathy and posttraumatic thrombosis should be studied bearing in mind this novel temporal pattern. LEVEL OF EVIDENCE: Prognostic/epidemiologic, level V.


Assuntos
Vesículas Extracelulares/metabolismo , Hemostasia/fisiologia , Trombina/metabolismo , Trombose/sangue , Adolescente , Adulto , Idoso , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Tempo de Tromboplastina Parcial , Fosfatidilserinas/sangue , Fosfatidilserinas/metabolismo , Projetos Piloto , Estudos Prospectivos , Trombina/análise , Trombose/diagnóstico , Trombose/etiologia , Fatores de Tempo , Ferimentos e Lesões/sangue , Ferimentos e Lesões/diagnóstico , Adulto Jovem
14.
Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205566

RESUMO

Placental abruption is the separation of the placenta from the lining of the uterus before childbirth. It is an infrequent perinatal complication with serious after-effects and a marked risk of maternal and fetal mortality. Despite the fact that numerous placental abruption risk factors are known, the pathophysiology of this issue is multifactorial and not entirely clear. The aim of this review was to examine the current state of knowledge concerning the molecular changes on the maternal-fetal interface occurring in placental abruption. Only original research articles describing studies published in English until the 15 March 2021 were considered eligible. Reviews, book chapters, case studies, conference papers and opinions were excluded. The systematic literature search of PubMed/MEDLINE and Scopus databases identified 708 articles, 22 of which were analyzed. The available evidence indicates that the disruption of the immunological processes on the maternal-fetal interface plays a crucial role in the pathophysiology of placental abruption. The features of chronic non-infectious inflammation and augmented immunological cytotoxic response were found to be present in placental abruption samples in the reviewed studies. Various molecules participate in this process, with only a few being examined. More advanced research is needed to fully explain this complicated process.


Assuntos
Descolamento Prematuro da Placenta/metabolismo , Placenta/metabolismo , Trombina/metabolismo , Descolamento Prematuro da Placenta/imunologia , Feminino , Humanos , Gravidez
15.
J Adv Res ; 31: 127-136, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34194837

RESUMO

Introduction: Cisplatin (CDDP) nephrotoxicity is one of the most significant complications limiting its use in cancer therapy. Objectives: This study investigated the pivotal role played by thrombin in CDDP-mediated nephrotoxicity. This work also aimed to clarify the possible preventive effect of Dabigatran (Dab), a direct thrombin inhibitor, on CDDP nephrotoxicity. Methods: Animals were grouped as follow; normal control group, CDDP nephrotoxicity group, CDDP + Dab 15, and CDDP + Dab 25 groups. Four days following CDDP administration, blood and urine samples were collected to evaluate renal function. Moreover, tissue samples were collected from the kidney to determine apoptosis markers, oxidative stress and histopathological evaluation. An immunofluorescence analysis of tissue factor (TF), thrombin, protease-activated receptor-2 (PAR2), fibrin, pERK1/2 and P53 proteins expression was also performed. Results: Thrombin, pERK, cleaved caspase-3, and oxidative stress markers were significantly elevated in CDDP-treated group. However, pretreatment of animals with either low or high doses of Dab significantly improved kidney function and decreased oxidative stress and apoptotic markers. Conclusion: We conclude that thrombin is an important factor in the pathogenesis of CDDP kidney toxicity via activation of ERK1/2, P53 and caspase-3 pathway, which can be effectively blocked by Dab.


Assuntos
Antitrombinas/farmacologia , Cisplatino/efeitos adversos , Dabigatrana/farmacologia , Nefropatias/tratamento farmacológico , Trombina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Caspase 3/metabolismo , Cisplatino/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Rim/patologia , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Ratos , Proteína Supressora de Tumor p53/metabolismo
16.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069309

RESUMO

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.


Assuntos
Afibrinogenemia/genética , Fibrinogênios Anormais/genética , Fibrinogênios Anormais/metabolismo , Mutação , Adulto , Afibrinogenemia/sangue , Animais , Testes de Coagulação Sanguínea , Células CHO , Cricetulus , Fator XIIIa/química , Fator XIIIa/metabolismo , Feminino , Fibrina/metabolismo , Fibrinogênios Anormais/química , Fibrinolisina/metabolismo , Heterozigoto , Humanos , Immunoblotting , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/metabolismo
17.
Food Chem ; 362: 130237, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34091163

RESUMO

Thrombin is a key therapeutic target protein of thrombosis. To date, massive studies have focused on the exploration of antithrombotic compounds. Here we capitalize on molecular docking, molecular simulations and spectroscopic experiments for virtually screening natural products that can inhibit thrombin and elucidating their interaction mechanism. Six compounds are screened from a natural product database by a cross-analysis based on two semi-flexible molecular docking methods. We show that four compounds can effectively inhibit thrombin and Calceolarioside B is the most competitive one based on enzyme inhibition experiments. Moreover, the binding free energies of these compounds with thrombin exhibit a consistent rank trend with their enzyme inhibition assay results. In addition, the Van der Waals is the main force to drive the interaction between the ligands and the receptor, which can be deduced from the fluorescence spectral results. This work provides a new insight into the development of antithrombotic natural compounds.


Assuntos
Ingredientes de Alimentos/análise , Alimento Funcional/análise , Produtos Biológicos/química , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Trombina/metabolismo , Interface Usuário-Computador
18.
Int J Hematol ; 114(3): 325-333, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34117991

RESUMO

INTRODUCTION: We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder. MATERIALS AND METHODS: Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting. RESULTS: Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells. CONCLUSION: We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.


Assuntos
Afibrinogenemia/genética , Alelos , Fibrinogênio/genética , Hemorragia/sangue , Hemorragia/etiologia , Infertilidade/etiologia , Mutação , Afibrinogenemia/sangue , Afibrinogenemia/complicações , Substituição de Aminoácidos , Animais , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Células CHO , Catálise , Cricetulus , Fibrina/metabolismo , Hemorragia/diagnóstico , Humanos , Infertilidade/diagnóstico , Infertilidade/terapia , Polimerização , Trombina/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-34147951

RESUMO

This study aims to screen potential anticoagulant components from leeches, a representative animal-sourced traditional Chinese medicine using thrombin (THR)-targeted ultrafiltration combined with ultrahigh performance liquid chromatography and high-resolution Orbitrap mass spectrometry (UPLC-HR-Orbitrap-MS). As a result, five small molecules in leech extract were discovered to interact with THR for the first time. Among them, two new compounds were isolated and their structures were identified by IR, HR-MS and NMR data. Furthermore, their THR inhibitory activity was confirmed with IC50 values of 4.74 and 8.31 µM, respectively. In addition, molecular docking analysis showed that the active (catalytic) site of THR might be the possible binding site of the two hits. Finally, reverse screening analysis indicated that LTA4-H, ACE and ALOX5AP were potential anticoagulant targets of the two new compounds. This study will broaden our understanding of the medicinal substance basis in leeches and further contribute to the discovery and development of clinical anticoagulant drugs from leeches.


Assuntos
Anticoagulantes , Produtos Biológicos , Sanguessugas/química , Trombina/metabolismo , Ultrafiltração/métodos , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Simulação de Acoplamento Molecular
20.
Lab Invest ; 101(10): 1394-1402, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34145381

RESUMO

Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms.


Assuntos
Ácidos e Sais Biliares/metabolismo , Hepatócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Tromboplastina/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ácido Desoxicólico/farmacologia , Células Hep G2 , Humanos , Isoxazóis/farmacologia , Fígado/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo
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