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1.
Biosens Bioelectron ; 253: 116184, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38452569

RESUMO

Direct electron transfer (DET) between an electrode and redox labels is feasible in electrochemical biosensors using small aptamer-aptamer sandwiches; however, its application is limited in biosensors that rely on larger antibody-antibody sandwiches. The development of sandwich-type biosensors utilizing DET is challenged by the scarcity of aptamer-aptamer sandwich pairs with high affinity in complex biological samples. Here, we introduce an electrochemical biosensor using an antibody-aptamer hybrid sandwich for detecting thrombin in human serum. The biosensor enables rapid DET through an antibody-aptamer hybrid configuration comprising (i) an antibody capture probe that provides high and specific affinity to the target in human serum, (ii) the target thrombin, and (iii) an aptamer detection probe that facilitates convenient terminal conjugation with long flexible spacer DNA and polylinker peptide containing multiple amine-reactive phenazine ethosulfate (arPES) redox labels, allowing the conjugated labels to easily approach the electrode. Rapid repeated DET using arPES-catalyzed NADH oxidation strongly enhanced the electrochemical signals. Properly sized spacer and polylinker provided low nonspecific adsorption of the aptamer probe conjugated with multiple arPESs and low interference with the binding of the aptamer probe. Methods for immobilizing thiol-terminated antibodies on Au electrodes were compared and optimized. The developed biosensor using the antibody-aptamer hybrid sandwich exhibited high sensitivity and selectivity in detecting thrombin, surpassing the limitations of an aptamer-aptamer sandwich owing to the low affinity of thrombin aptamers in human serum. The calculated detection limit of the biosensor was ∼1.5 pM in buffer and ∼2.7 nM in human serum.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Humanos , Técnicas Biossensoriais/métodos , Trombina/metabolismo , Elétrons , Anticorpos/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Oxirredução , Eletrodos , Limite de Detecção , Ouro
2.
Sci Rep ; 14(1): 6229, 2024 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-38486006

RESUMO

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.


Assuntos
Cálcio , Fenotiazinas , Inibidores da Agregação Plaquetária , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Cálcio/metabolismo , Trombina/metabolismo , Sinalização do Cálcio , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptor PAR-1/metabolismo , Plaquetas/metabolismo , Ativação Plaquetária , Cálcio da Dieta/farmacologia , Agregação Plaquetária
3.
Int J Mol Sci ; 25(5)2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38474244

RESUMO

Adrenaline has recently been found to trigger phosphatidylserine (PS) exposure on blood platelets, resulting in amplification of the coagulation process, but the mechanism is only fragmentarily established. Using a panel of platelet receptors' antagonists and modulators of signaling pathways, we evaluated the importance of these in adrenaline-evoked PS exposure by flow cytometry. Calcium and sodium ion influx into platelet cytosol, after adrenaline treatment, was examined by fluorimetric measurements. We found a strong reduction in PS exposure after blocking of sodium and calcium ion influx via Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (NCX), respectively. ADP receptor antagonists produced a moderate inhibitory effect. Substantial limitation of PS exposure was observed in the presence of GPIIb/IIIa antagonist, phosphoinositide-3 kinase (PI3-K) inhibitors, or prostaglandin E1, a cyclic adenosine monophosphate (cAMP)-elevating agent. We demonstrated that adrenaline may develop a procoagulant response in human platelets with the substantial role of ion exchangers (NHE and NCX), secreted ADP, GPIIb/IIIa-dependent outside-in signaling, and PI3-K. Inhibition of the above mechanisms and increasing cytosolic cAMP seem to be the most efficient procedures to control adrenaline-evoked PS exposure in human platelets.


Assuntos
Plaquetas , Ativação Plaquetária , Humanos , Plaquetas/metabolismo , Cálcio/metabolismo , Epinefrina/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Sódio/metabolismo , Trombina/metabolismo
4.
FASEB J ; 38(4): e23468, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38334433

RESUMO

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.


Assuntos
Glicoproteínas da Membrana de Plaquetas , Trombina , Humanos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombina/metabolismo , Proteínas Quinases/metabolismo , Óxido Nítrico/metabolismo , Células Endoteliais/metabolismo , Ativação Plaquetária/fisiologia , Plaquetas/metabolismo , Endotélio/metabolismo , Prostaglandinas I
5.
Int J Mol Sci ; 25(3)2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38339082

RESUMO

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.


Assuntos
Serpinas , Serpinas/metabolismo , Heparina/química , Serina Proteases , Inibidores de Serino Proteinase/metabolismo , Anticoagulantes , Trombina/metabolismo
6.
Acta Physiol (Oxf) ; 240(4): e14116, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38400621

RESUMO

AIM: Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. METHODS: Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed in vivo with magnetic resonance imaging and ex vivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. RESULTS: The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine in vivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated ex vivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. CONCLUSION: In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.


Assuntos
Angiotensina II , Doenças Vasculares , Camundongos , Masculino , Animais , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/farmacologia , Análise de Onda de Pulso , Trombina/metabolismo , Trombina/farmacologia , Camundongos Endogâmicos C57BL , Doenças Vasculares/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Endotélio Vascular , Óxido Nítrico/metabolismo
7.
Biomolecules ; 14(2)2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38397467

RESUMO

Altered properties of fibrin clots have been associated with bleeding and thrombotic disorders, including hemophilia or trauma and heart attack or stroke. Clotting factors, such as thrombin and tissue factor, or blood plasma proteins, such as fibrinogen, play critical roles in fibrin network polymerization. The concentrations and combinations of these proteins affect the structure and stability of clots, which can lead to downstream complications. The present work includes clots made from plasma and purified fibrinogen and shows how varying fibrinogen and activation factor concentrations affect the fibrin properties under both conditions. We used a combination of scanning electron microscopy, confocal microscopy, and turbidimetry to analyze clot/fiber structure and polymerization. We quantified the structural and polymerization features and found similar trends with increasing/decreasing fibrinogen and thrombin concentrations for both purified fibrinogen and plasma clots. Using our compiled results, we were able to generate multiple linear regressions that predict structural and polymerization features using various fibrinogen and clotting agent concentrations. This study provides an analysis of structural and polymerization features of clots made with purified fibrinogen or plasma at various fibrinogen and clotting agent concentrations. Our results could be utilized to aid in interpreting results, designing future experiments, or developing relevant mathematical models.


Assuntos
Fibrinogênio , Trombose , Humanos , Fibrinogênio/metabolismo , Trombina/metabolismo , Coagulação Sanguínea , Plasma/metabolismo , Fibrina/química
8.
Anesth Analg ; 138(4): 696-711, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38324297

RESUMO

Physiological hemostasis is a balance between pro- and anticoagulant pathways, and in sepsis, this equilibrium is disturbed, resulting in systemic thrombin generation, impaired anticoagulant activity, and suppression of fibrinolysis, a condition termed sepsis-induced coagulopathy (SIC). SIC is a common complication, being present in 24% of patients with sepsis and 66% of patients with septic shock, and is often associated with poor clinical outcomes and high mortality. 1 , 2 Recent preclinical and clinical studies have generated new insights into the molecular pathogenesis of SIC. In this article, we analyze the complex pathophysiology of SIC with a focus on the role of procoagulant innate immune signaling in hemostatic activation--tissue factor production, thrombin generation, endotheliopathy, and impaired antithrombotic functions. We also review clinical presentations of SIC, the diagnostic scoring system and laboratory tests, the current standard of care, and clinical trials evaluating the efficacies of anticoagulant therapies.


Assuntos
Transtornos da Coagulação Sanguínea , Sepse , Humanos , Trombina/metabolismo , Transtornos da Coagulação Sanguínea/diagnóstico , Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/terapia , Hemostasia , Sepse/complicações , Sepse/diagnóstico , Sepse/terapia , Anticoagulantes/uso terapêutico
10.
Int Immunopharmacol ; 128: 111459, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38181675

RESUMO

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition play a central role in refractory hypoxemia in acute respiratory distress syndrome (ARDS), but it lacks effective drugs for prevention and treatment of this pathophysiology. Our previous experiment confirmed that RUNX1 promoted alveolar hypercoagulation and fibrinolytic inhibition through NF-κB pathway. Other studies demonstrated that 6-gingerol regulated inflammation and metabolism by inhibiting the NF-κB signaling pathway. We assume that 6-gingerol would ameliorate alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/ NF-κB pathway in LPS-induced ARDS. METHODS: Rat ARDS model was replicated through LPS inhalation. Before LPS inhalation, the rats were intraperitoneally treated with different doses of 6-gingerol or the same volume of normal saline (NS) for 12 h, and then intratracheal inhalation of LPS for 24 h. In cell experiment, alveolar epithelial cell type II (AECII) was treated with 6-gingerol for 6 h and then with LPS for another 24 h. RUNX1 gene was down-regulated both in pulmonary tissue and in cells. Tissue factor (TF), plasminogen Activator Inhibitor 1(PAI-1) and thrombin were determined by Wester-blot (WB), qPCR or by enzyme-linked immunosorbent (ELISA). Lung injury score, pulmonary edema and pulmonary collagen III in rat were assessed. NF-κB pathway were also observed in vivo and in vitro. The direct binding capability of 6-gingerol to RUNX1 was confirmed by using Drug Affinity Responsive Target Stability test (DARTS). RESULTS: 6-gingerol dose-dependently attenuated LPS-induced lung injury and pulmonary edema. LPS administration caused excessive TF and PAI-1 expression both in pulmonary tissue and in AECII cell and a large amount of TF, PAI-1 and thrombin in bronchial alveolar lavage fluid (BALF), which all were effectively decreased by 6-gingerol treatment in a dose-dependent manner. The high collagen Ⅲ level in lung tissue provoked by LPS was significantly abated by 6-gingerol. 6-gingerol was seen to dramatically inhibit the LPS-stimulated activation of NF-κB pathway, indicated by decreases of p-p65/total p65, p-IKKß/total IKKß, and also to suppress the RUNX1 expression. RUNX1 gene knock down or RUNX1 inhibitor Ro5-3335 significantly enhanced the efficacies of 6-gingerol in vivo and in vitro, but RUNX1 over expression remarkably impaired the effects of 6-gingerol on TF, PAI-1 and on NF-κB pathway. DARTS result showed that 6-gingerol directly bond to RUNX1 molecules. CONCLUSIONS: Our experimental data demonstrated that 6-gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/NF-κB pathway in LPS-induced ARDS. 6-gingerol is expected to be an effective drug in ARDS.


Assuntos
Catecóis , Álcoois Graxos , Lesão Pulmonar , Edema Pulmonar , Síndrome do Desconforto Respiratório , Ratos , Animais , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio , Subunidade alfa 2 de Fator de Ligação ao Core , Quinase I-kappa B/metabolismo , Trombina/metabolismo , Trombina/farmacologia , Trombina/uso terapêutico , Transdução de Sinais , Síndrome do Desconforto Respiratório/tratamento farmacológico , Colágeno/farmacologia
11.
Stem Cells Dev ; 33(3-4): 89-103, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38164089

RESUMO

Mesenchymal stem cells (MSCs) directly differentiate into neurons and endothelial cells after transplantation, and their secretome has considerable potential for treating brain injuries. Previous studies have suggested that the effects of MSCs priming with exposure to hypoxia, cytokines, growth factors, or chemical agents could optimize the paracrine potency and therapeutic potential of MSCs. Studies have suggested that thrombin-primed Wharton's Jelly-derived mesenchymal stem cells (Th.WJ-MSCs) significantly enhance the neuroprotective beneficial effects of naive MSCs in brain injury such as hypoxic-ischemic brain injury (HIE) and intraventricular hemorrhage (IVH). This study aimed to characterize WJ-MSCs in terms of stem cell markers, differentiation, cell proliferation, and paracrine factors by comparing naive and Th.WJ-MSCs. We demonstrated that compared with naive MSCs, Th.MSCs significantly enhanced the neuroprotective effects in vitro. Moreover, we identified differentially expressed proteins in the conditioned media of naive and Th.WJ-MSCs by liquid chromatography-tandem mass spectrometry analysis. Secretome analysis of the conditioned medium of WJ-MSCs revealed that such neuroprotective effects were mediated by paracrine effects with secretomes of Th.WJ-MSCs, and hepatocyte growth factor was identified as a key paracrine mediator. These results can be applied further in the preclinical and clinical development of effective and safe cell therapeutics for brain injuries such as HIE and IVH.


Assuntos
Lesões Encefálicas , Células-Tronco Mesenquimais , Fármacos Neuroprotetores , Fator de Transcrição STAT3 , Geleia de Wharton , Humanos , Fator de Crescimento de Hepatócito/metabolismo , Fármacos Neuroprotetores/farmacologia , Trombina/farmacologia , Trombina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Endoteliais/metabolismo , Células Cultivadas , Transdução de Sinais , Diferenciação Celular , Fatores Imunológicos/metabolismo , Lesões Encefálicas/metabolismo , Proliferação de Células
12.
Biochem Biophys Res Commun ; 696: 149473, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38241814

RESUMO

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.


Assuntos
Anticoagulantes , Hirudinas , Anticoagulantes/farmacologia , Hirudinas/farmacologia , Hirudinas/genética , Hirudinas/metabolismo , Trombina/metabolismo , Fator Xa , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo
13.
Int J Mol Sci ; 25(2)2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38279219

RESUMO

Serine proteases regulate cell functions through G protein-coupled protease-activated receptors (PARs). Cleavage of one peptide bond of the receptor amino terminus results in the formation of a new N-terminus ("tethered ligand") that can specifically interact with the second extracellular loop of the PAR receptor and activate it. Activation of PAR1 by thrombin (canonical agonist) and activated protein C (APC, noncanonical agonist) was described as a biased agonism. Here, we have supposed that synthetic peptide analogs to the PAR1 tethered ligand liberated by APC could have neuroprotective effects like APC. To verify this hypothesis, a model of the ischemic brain impairment based on glutamate (Glu) excitotoxicity in primary neuronal cultures of neonatal rats has been used. It was shown that the nanopeptide NPNDKYEPF-NH2 (AP9) effectively reduced the neuronal death induced by Glu. The influence of AP9 on cell survival was comparable to that of APC. Both APC and AP9 reduced the dysregulation of intracellular calcium homeostasis in cultured neurons induced by excitotoxic Glu (100 µM) or NMDA (200 µM) concentrations. PAR1 agonist synthetic peptides might be noncanonical PAR1 agonists and a basis for novel neuroprotective drugs for disorders related to Glu excitotoxicity such as brain ischemia, trauma and some neurodegenerative diseases.


Assuntos
Fármacos Neuroprotetores , Receptor PAR-1 , Ratos , Animais , Receptor PAR-1/metabolismo , Fármacos Neuroprotetores/farmacologia , Ligantes , Trombina/metabolismo , Peptídeos/farmacologia , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Células Cultivadas
14.
Medicine (Baltimore) ; 103(2): e36849, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38215083

RESUMO

Unusual blood clots can cause serious health problems, such as lung embolism, stroke, and heart attack. Inhibiting thrombin activity was adopted as an effective strategy for preventing blood clots. In this study, we explored computational-based method for designing peptide inhibitors of human thrombin therapeutic peptides to prevent platelet aggregation. The random peptides and their 3-dimentional structures were generated to build a virtual peptide library. The generated peptides were docked into the binding pocket of human thrombin. The designed strong binding peptides were aligned with the native binder by comparative study, and we showed the top 5 peptide binders display strong binding affinity against human thrombin. The 5 peptides were synthesized and validated their inhibitory activity. Our result showed the 5-mer peptide AEGYA, EVVNQ, and FASRW with inhibitory activity against thrombin, range from 0.53 to 4.35 µM. In vitro anti-platelet aggregation assay was carried out, suggesting the 3 peptides can inhibit the platelet aggregation induced by thrombin. This study showed computer-aided peptide inhibitor design can be a robust method for finding potential binders for thrombin, which provided solutions for anticoagulation.


Assuntos
Antitrombinas , Trombina , Trombose , Humanos , Anticoagulantes/farmacologia , Plaquetas/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Trombina/metabolismo , Antitrombinas/química , Antitrombinas/farmacologia
15.
Biophys J ; 123(3): 334-348, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38169215

RESUMO

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin cytoskeleton and substrate to study their role in permeability. Our studies show that mechanochemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tricellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force due to disturbed flow that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer extracellular matrix. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechanochemical model to investigate the variation in the permeability of endothelial monolayer due to multiple loads acting simultaneously.


Assuntos
Células Endoteliais , Endotélio Vascular , Humanos , Caderinas/metabolismo , Citoesqueleto de Actina/metabolismo , Trombina/metabolismo , Permeabilidade , Permeabilidade Capilar/fisiologia , Células Cultivadas
16.
J Thromb Haemost ; 22(4): 905-914, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38266676

RESUMO

Thrombomodulin (TM) is a type 1 receptor best known for its function as an anticoagulant cofactor for thrombin activation of protein C on the surface of vascular endothelial cells. In addition to its anticoagulant cofactor function, TM also regulates fibrinolysis, complement, and inflammatory pathways. TM is a multidomain receptor protein with a lectin-like domain at its N-terminus that has been shown to exhibit direct anti-inflammatory functions. This domain is followed by 6 epidermal growth factor-like domains that support the interaction of TM with thrombin. The interaction inhibits the procoagulant function of thrombin and enables the protease to regulate the anticoagulant and fibrinolytic pathways by activating protein C and thrombin-activatable fibrinolysis inhibitor. TM has a Thr/Ser-rich region immediately above the membrane surface that harbors chondroitin sulfate glycosaminoglycans, and this region is followed by a single-spanning transmembrane and a C-terminal cytoplasmic domain. The structure and physiological function of the extracellular domains of TM have been extensively studied, and numerous excellent review articles have been published. However, the physiological function of the cytoplasmic domain of TM has remained poorly understood. Recent data from our laboratory suggest that intracellular signaling by the cytoplasmic domain of TM plays key roles in maintaining quiescence by modulating phosphatase and tensin homolog signaling in endothelial cells. This article briefly reviews the structure and function of extracellular domains of TM and focuses on the mechanism and possible physiological importance of the cytoplasmic domain of TM in modulating phosphatase and tensin homolog signaling in endothelial cells.


Assuntos
Trombina , Trombomodulina , Humanos , Trombomodulina/metabolismo , Trombina/metabolismo , Proteína C/metabolismo , Células Endoteliais/metabolismo , Tensinas , Anticoagulantes , Monoéster Fosfórico Hidrolases
17.
Mol Cell Proteomics ; 23(2): 100717, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38237698

RESUMO

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.


Assuntos
Proteoma , Trombina , Proteoma/metabolismo , Trombina/farmacologia , Trombina/metabolismo , Glicosilação , Plaquetas/metabolismo , Ativação Plaquetária
19.
Int J Hematol ; 119(2): 196-204, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38228939

RESUMO

The protein C (PC) pathway involves physiological anticoagulant factors (PC, protein S [PS], and factor V) and performs major anticoagulant functions in adults. Variations in overall PC pathway function due to dynamic changes in PC and PS in early childhood are poorly understood. We aimed to evaluate the contributions of PC pathway function during early childhood by measuring changes in plasma thrombin generation (TG) after administration of the PC activator protac. We evaluated correlations between anticoagulant factors and percentage of protac-induced coagulation inhibition (PiCi%). Before protac addition, TG in newborns (n = 35), infants (n = 42), young children (n = 35), and adults (n = 20) were 525 ± 74, 720 ± 96, 785 ± 53, and 802 ± 64 mOD/min, and PiCi% were 42.1 ± 9.9, 69.8 ± 11.0, 82.9 ± 4.4, and 86.9 ± 3.4%, respectively. The distribution of PiCi% on the two axes of TG (with or without protac) changed continuously with age and differed from that of warfarin-treated plasma and adult PC- or PS-deficient plasma. PiCi% increased dynamically during infancy and correlated with PS levels in newborns and PC levels in young children. Addition of PC or fresh frozen plasma equivalent to approximately 25% PC to PC-deficient plasma improved PiCi%. This automatic measurement requires only a small sample volume and is useful for analysis of developmental hemostasis.


Assuntos
Proteína C , Quimera de Direcionamento de Proteólise , Adulto , Criança , Pré-Escolar , Humanos , Recém-Nascido , Anticoagulantes/farmacologia , Antitrombinas/farmacologia , Coagulação Sanguínea , Proteína C/análise , Proteína C/metabolismo , Proteína C/farmacologia , Proteína S/metabolismo , Trombina/metabolismo , Lactente
20.
Blood Adv ; 8(6): 1550-1566, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38163324

RESUMO

ABSTRACT: Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.


Assuntos
Agregação Plaquetária , Trombose , Camundongos , Humanos , Animais , Trombina/metabolismo , Tromboxanos , Puromicina/efeitos adversos
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