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1.
Hamostaseologie ; 41(5): 379-385, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34695854

RESUMO

In 2019 first reports about a new human coronavirus emerged, which causes common cold symptoms as well as acute respiratory distress syndrome. The virus was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and severe thrombotic events including deep vein thrombosis, pulmonary embolism, and microthrombi emerged as additional symptoms. Heart failure, myocardial infarction, myocarditis, and stroke have also been observed. As main mediator of thrombus formation, platelets became one of the key aspects in SARS-CoV-2 research. Platelets may also directly interact with SARS-CoV-2 and have been shown to carry the SARS-CoV-2 virus. Platelets can also facilitate the virus uptake by secretion of the subtilisin-like proprotein convertase furin. Cleavage of the SARS-CoV-2 spike protein by furin enhances binding capabilities and virus entry into various cell types. In COVID-19 patients, platelet count differs between mild and serious infections. Patients with mild symptoms have a slightly increased platelet count, whereas thrombocytopenia is a hallmark of severe COVID-19 infections. Low platelet count can be attributed to platelet apoptosis and the incorporation of platelets into microthrombi (peripheral consumption) and severe thrombotic events. The observed excessive formation of thrombi is due to hyperactivation of platelets caused by the infection. Various factors have been suggested in the activation of platelets in COVID-19, such as hypoxia, vessel damage, inflammatory factors, NETosis, SARS-CoV-2 interaction, autoimmune reactions, and autocrine activation. COVID-19 does alter chemokine and cytokine plasma concentrations. Platelet chemokine profiles are altered in COVID-19 and contribute to the described chemokine storms observed in severely ill COVID-19 patients.


Assuntos
Plaquetas/fisiologia , Plaquetas/virologia , COVID-19/sangue , Plaquetas/imunologia , COVID-19/complicações , COVID-19/imunologia , Quimiocinas/sangue , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/etiologia , Interações entre Hospedeiro e Microrganismos/imunologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Modelos Biológicos , Pandemias , Ativação Plaquetária/imunologia , Ativação Plaquetária/fisiologia , SARS-CoV-2/patogenicidade , Trombose/sangue , Trombose/etiologia
2.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638529

RESUMO

Bernard-Soulier syndrome (BSS) is an autosomal-recessive bleeding disorder caused by biallelic variants in the GP1BA, GP1BB, and GP9 genes encoding the subunits GPIbα, GPIbß, and GPIX of the GPIb-IX complex. Pathogenic variants usually affect the extracellular or transmembrane domains of the receptor subunits. We investigated a family with BSS caused by the homozygous c.528_550del (p.Arg177Serfs*124) variant in GP1BB, which is the first mutation ever identified that affects the cytoplasmic domain of GPIbß. The loss of the intracytoplasmic tail of GPIbß results in a mild form of BSS, characterized by only a moderate reduction of the GPIb-IX complex expression and mild or absent bleeding tendency. The variant induces a decrease of the total platelet expression of GPIbß; however, all of the mutant subunit expressed in platelets is correctly assembled into the GPIb-IX complex in the plasma membrane, indicating that the cytoplasmic domain of GPIbß is not involved in assembly and trafficking of the GPIb-IX receptor. Finally, the c.528_550del mutation exerts a dominant effect and causes mild macrothrombocytopenia in heterozygous individuals, as also demonstrated by the investigation of a second unrelated pedigree. The study of this novel GP1BB variant provides new information on pathophysiology of BSS and the assembly mechanisms of the GPIb-IX receptor.


Assuntos
Síndrome de Bernard-Soulier/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Trombocitopenia/genética , Adulto , Síndrome de Bernard-Soulier/sangue , Síndrome de Bernard-Soulier/patologia , Plaquetas/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Domínios Proteicos/genética , Trombocitopenia/patologia , Fator de von Willebrand/metabolismo
3.
Biomed Res Int ; 2021: 7086108, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34513996

RESUMO

The paper presents the results of studying the immunological parameters of 369 people who were practically healthy at the time of the survey, 298 women and 71 men, of which 216 people are living in the European North of the Russian Federation (173 women and 43 men) and 153 are residents of the Arctic (125 women and 28 men). The study was carried out in the morning (08:00-10:00 am). The study included the determination of the aggregation of erythrocytes, platelets, neutrophilic granulocytes, lymphocytes, hemogram study, hematological analysis, enzyme immunoassay, and flow cytometry. Statistical processing of the obtained data was carried out using the Statistica 7.0 software package (StatSoft, USA). It was found that the activity of aggregation of cells of peripheral venous blood in Arctic residents is 1.5-1.7 times higher than that in people living in more favourable climatic conditions. The frequency of registration of aggregation of erythrocytes and platelets is actually 2 times higher than the aggregation of leukocytes. Aggregation of erythrocytes is associated with an increase in the concentrations of transferrin and receptors for this transport protein. The frequency of detection of platelet aggregation is accompanied by an increase in transferrin concentrations; in cases of aggregation of nonnuclear blood cells, the content of NO2 in the blood serum is increased. Aggregation of neutrophilic granulocytes and lymphocytes is associated with an increase in the content of free adhesion molecules. Aggregation of erythrocytes and platelets is in evidence when it is necessary to trigger reactions of changes in the hemodynamics of microcirculation to increase the efficiency of oxygen and trophic supply of tissues. The adhesion of leukocytes to the endothelium determines the secretion of biologically active substances that contribute to a change in microcirculation and an increase in the migration of leukocytes into tissues for the implementation of phagocytic and cytolytic functions.


Assuntos
Células Sanguíneas/citologia , Agregação Celular/fisiologia , Adulto , Idoso , Altitude , Plaquetas/citologia , Plaquetas/fisiologia , Adesão Celular/fisiologia , Eritrócitos/citologia , Feminino , Citometria de Fluxo/métodos , Granulócitos/citologia , Voluntários Saudáveis , Humanos , Leucócitos/citologia , Linfócitos/citologia , Masculino , Microcirculação/fisiologia , Pessoa de Meia-Idade , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Federação Russa
4.
Rev Bras Ginecol Obstet ; 43(8): 595-599, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34547793

RESUMO

OBJECTIVE: To describe the hematological changes, the platelet indices in particular, in pregnant women with coronavirus disease 2019 (COVID-19) compared to healthy pregnant women. METHODS: A retrospective case-control study conducted at the Al Yarmouk Teaching Hospital, in Baghdad, Iraq, involving 100 pregnant women, 50 with positive viral DNA for COVID-19 (case group), and 50 with negative results (control group); both groups were subjected to a thorough hematological evaluation. RESULTS: Among the main hematological variables analyzed, the platelet indices, namely the mean platelet volume (MPV) and the platelet distribution width (PDW), showed statistically significant differences (MPV: 10.87 ± 66.92 fL for the case group versus 9.84 ± 1.2 fL for the control group; PDW: 14.82 ± 3.18 fL for the case group versus 13.3 ± 2.16 fL for the controls). The criterion value of the receiver operating characteristic (ROC) curve for PDW at a cutoff point of > 11.8 fL showed a weak diagnostic marker, while the MPV at a cutoff value of > 10.17 fL showed a good diagnostic marker. CONCLUSION: The MPV and PDW are significantly affected by the this viral infection, even in asymptomatic confirmed cases, and we recommend that both parameters be included in the diagnostic panel of this infection.


Assuntos
Plaquetas/virologia , COVID-19/sangue , Complicações Infecciosas na Gravidez/sangue , Adulto , Doenças Assintomáticas , Biomarcadores/sangue , Plaquetas/fisiologia , COVID-19/diagnóstico , Teste para COVID-19 , Estudos de Casos e Controles , Feminino , Humanos , Volume Plaquetário Médio , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Estudos Retrospectivos
5.
Int J Mol Sci ; 22(17)2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34502520

RESUMO

In our previous study, we introduced the platelet endothelial cell adhesion molecule 1 (PECAM-1)/thrombus ratio, which is a parameter indicating the proportion of PECAM-1 in laser-induced thrombi in mice. Because PECAM-1 is an antithrombotic molecule, the higher the PECAM-1/thrombus ratio, the less activated the platelets. In this study, we used an extracorporeal model of thrombosis (flow chamber model) to verify its usefulness in the assessment of the PECAM-1/thrombus ratio in animal and human studies. Using the lipopolysaccharide (LPS)-induced inflammation model, we also evaluated whether the PECAM-1/thrombus ratio determined in the flow chamber (without endothelium) differed from that calculated in laser-induced thrombosis (with endothelium). We observed that acetylsalicylic acid (ASA) decreased the area of the thrombus while increasing the PECAM-1/thrombus ratio in healthy mice and humans in a dose-dependent manner. In LPS-treated mice, the PECAM-1/thrombus ratio decreased as the dose of ASA increased in both thrombosis models, but the direction of change in the thrombus area was inconsistent. Our study demonstrates that the PECAM-1/thrombus ratio can more accurately describe the platelet activation status than commonly used parameters such as the thrombus area, and, hence, it can be used in both human and animal studies.


Assuntos
Ativação Plaquetária/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Animais , Aspirina/análise , Plaquetas/metabolismo , Plaquetas/fisiologia , Adesão Celular , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Feminino , Voluntários Saudáveis , Humanos , Inflamação , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Trombose/metabolismo
6.
Int J Mol Sci ; 22(17)2021 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-34502272

RESUMO

The actin cytoskeleton plays a central role in platelet formation and function. Alpha-actinins (actinins) are actin filament crosslinking proteins that are prominently expressed in platelets and have been studied in relation to their role in platelet activation since the 1970s. However, within the past decade, several groups have described mutations in ACTN1/actinin-1 that cause congenital macrothrombocytopenia (CMTP)-accounting for approximately 5% of all cases of this condition. These findings are suggestive of potentially novel functions for actinins in platelet formation from megakaryocytes in the bone marrow and/or platelet maturation in circulation. Here, we review some recent insights into the well-known functions of actinins in platelet activation before considering possible roles for actinins in platelet formation that could explain their association with CMTP. We describe what is known about the consequences of CMTP-linked mutations on actinin-1 function at a molecular and cellular level and speculate how these changes might lead to the alterations in platelet count and morphology observed in CMTP patients. Finally, we outline some unanswered questions in this area and how they might be addressed in future studies.


Assuntos
Actinina/metabolismo , Plaquetas/fisiologia , Trombocitopenia/etiologia , Actinina/genética , Humanos , Integrinas , Megacariócitos/patologia , Megacariócitos/fisiologia , Mutação , Adesividade Plaquetária , Trombocitopenia/sangue
7.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502548

RESUMO

Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A2 and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood.


Assuntos
Transtornos da Coagulação Sanguínea/fisiopatologia , Venenos de Crotalídeos/metabolismo , Hemorragia/fisiopatologia , Hemostasia/fisiologia , Trombose/fisiopatologia , Animais , Antivenenos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Venenos de Crotalídeos/antagonistas & inibidores , Humanos
8.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502240

RESUMO

Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.


Assuntos
Anticoagulantes/farmacologia , Plaquetas/fisiologia , Respiração Celular/fisiologia , Meios de Cultura/farmacologia , Mitocôndrias/fisiologia , Ativação Plaquetária , Plaquetas/efeitos dos fármacos , Citometria de Fluxo , Humanos , Mitocôndrias/efeitos dos fármacos
9.
Biomolecules ; 11(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34356647

RESUMO

During surgical procedures, cotton abdominal swabs with their high absorptive capacity and malleability are used to retain organs and absorb blood or other body fluids. Such properties of the natural material cotton are advantageous for most operations, but in cardiopulmonary bypass (CPB) surgery, a high blood volume can accumulate in the thoracic cavity that is quickly retransfused via the heart-lung machine (HLM). This common practice is supposed to be safe due to the high anticoagulation. However, in vitro analyses showed that blood cells and plasma proteins were activated despite a high anticoagulation, which can propagate especially an inflammatory response in the patient. Thus, we investigated patients' blood during CPB surgery for inflammatory and coagulation-associated activation after contact to the HLM and either cotton or synthetic abdominal swabs. Contact with cotton significantly increased thrombocyte and neutrophil activation measured as ß-thromboglobulin and PMN-elastase secretion, respectively, compared to synthetic abdominal swabs. Both inflammatory cytokines, interleukin (IL) 1ß and IL6, were also significantly increased in the cotton over the synthetic patient group, while SDF-1α was significantly lower in the synthetic group. Our data show for the first time that cotton materials can activate platelets and leukocytes despite a high anticoagulation and that this activation is lower with synthetic materials. This additional activation due to the material on top of the activation exerted by the tissue contact that blood is exposed to during CPB surgery can propagate further reactions in patients after surgery, which poses a risk for this already vulnerable patient group.


Assuntos
Procedimentos Cirúrgicos Cardíacos/instrumentação , Ativação Plaquetária , Tampões Cirúrgicos , Têxteis , Idoso , Plaquetas/fisiologia , Procedimentos Cirúrgicos Cardíacos/métodos , Fibra de Algodão , Citocinas/sangue , Feminino , Máquina Coração-Pulmão , Humanos , Inflamação/sangue , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Tampões de Gaze Cirúrgicos
10.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445311

RESUMO

BACKGROUND: Today there are many devices that can be used to study blood clotting disorders by identifying abnormalities in blood platelets. The Total Thrombus Formation Analysis System is an automated microchip flow chamber system that is used for the quantitative analysis of clot formation under blood flow conditions. For several years, researchers have been using a tool to analyse various clinical situations of patients to identify the properties and biochemical processes occurring within platelets and their microenvironment. METHODS: An investigation of recent published literature was conducted based on PRISMA. This review includes 52 science papers directly related to the use of the Total Clot Formation Analysis System in relation to bleeding, surgery, platelet function assessment, anticoagulation monitoring, von Willebrand factor and others. CONCLUSION: Most available studies indicate that The Total Thrombus Formation Analysis System may be useful in diagnostic issues, with devices used to monitor therapy or as a significant tool for predicting bleeding events. However, T-TAS not that has the potential for diagnostic indications, but allows the direct observation of the flow and the interactions between blood cells, including the intensity and dynamics of clot formation. The device is expected to be of significant value for basic research to observe the interactions and changes within platelets and their microenvironment.


Assuntos
Coagulação Sanguínea , Plaquetas/fisiologia , Dispositivos Lab-On-A-Chip/normas , Microfluídica/métodos , Trombose/sangue , Plaquetas/metabolismo , Humanos , Microfluídica/instrumentação , Trombose/diagnóstico
12.
Cells ; 10(8)2021 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-34440857

RESUMO

Human blood cells may offer a minimally invasive strategy to study systemic alterations of mitochondrial function. Here we tested the reliability of a protocol designed to study mitochondrial respiratory control in human platelets (PLTs) in field studies, using high-resolution respirometry (HRR). Several factors may trigger PLT aggregation during the assay, altering the homogeneity of the cell suspension and distorting the number of cells added to the two chambers (A, B) of the Oroboros Oxygraph-2k (O2k). Thus, inter-chamber variability (∆ab) was calculated by normalizing oxygen consumption to chamber volume (JO2) or to a specific respiratory control state (flux control ratio, FCR) as a reliable parameter of experimental quality. The method's reliability was tested by comparing the ∆ab of laboratory-performed experiments (LAB, N = 9) to those of an ultramarathon field study (three sampling time-points: before competition (PRE, N = 7), immediately after (POST, N = 10) and 24 h after competition (REC; N = 10)). Our results show that ∆ab JO2 changed PRE-POST, but also for LAB-POST and LAB-REC, while all ∆ab FCR remained unchanged. Thus, we conclude that our method is reliable for assessing PLT mitochondrial function in LAB and field studies and after systemic stress conditions.


Assuntos
Plaquetas/fisiologia , Mitocôndrias/fisiologia , Plaquetas/citologia , Plaquetas/metabolismo , Respiração Celular/fisiologia , Exercício Físico/fisiologia , Humanos , Mitocôndrias/metabolismo , Consumo de Oxigênio , Reprodutibilidade dos Testes
13.
Int Immunopharmacol ; 99: 107995, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34304001

RESUMO

Despite endorsed and exponential research to improve diagnostic and therapeutic strategies, efforts have not yet converted into a better prospect for patients infected with the novel coronavirus (2019nCoV), and still, the name of SARS-CoV-2 is coupled with numerous unanswered questions. One of these questions is concerning how this respiratory virus reduces the number of platelets (PLTs)? The results of laboratory examinations showed that about a quarter of COVID-19 cases experience thrombocytopenia, and more remarkably, about half of these patients succumb to the infection due to coagulopathy. These findings have positioned PLTs as a pillar in the management as well as stratifying COVID-19 patients; however, not all the physicians came into a consensus about the prognostic value of these cells. The current review aims to unravel the contributory role of PLTs s in COVID-19; and alsoto summarize the original data obtained from international research laboratories on the association between COVID-19 and PLT production, activation, and clearance. In addition, we provide a special focus on the prognostic value of PLTs and their related parameters in COVID-19. Questions on how SARS-CoV-2 induces thrombocytopenia are also responded to. The last section provides a general overview of the most recent PLT- or thrombocytopenia-related therapeutic approaches. In conclusion, since SARS-CoV-2 reduces the number of PLTs by eliciting different mechanisms, treatment of thrombocytopenia in COVID-19 patients is not as simple as it appears and serious cautions should be considered to deal with the problem through scrutiny awareness of the causal mechanisms.


Assuntos
Plaquetas/fisiologia , COVID-19/diagnóstico , COVID-19/fisiopatologia , Trombocitopenia/fisiopatologia , Humanos
14.
Clin Pharmacol Ther ; 110(3): 702-713, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34255863

RESUMO

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.


Assuntos
Plaquetas/fisiologia , Grupos de Populações Continentais/genética , RNA Mensageiro/genética , Adolescente , Afro-Americanos/genética , Biomarcadores/sangue , Plaquetas/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Citocinas/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Masculino , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária/métodos
15.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298979

RESUMO

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.


Assuntos
Plaquetas/fisiologia , Antígenos CD18/fisiologia , Degranulação Celular , Córnea/irrigação sanguínea , Eritrócitos/fisiologia , Hiperemia/fisiopatologia , Mastócitos/fisiologia , Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Vasculite/imunologia , Vênulas/metabolismo , Animais , Antígenos CD18/deficiência , Movimento Celular , Quimiotaxia de Leucócito , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Epitélio Corneano/fisiologia , Feminino , Hiperemia/sangue , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Microscopia Eletrônica , Modelos Animais , Fagocitose , Regeneração/fisiologia , Vasculite/sangue , Vênulas/patologia , Cicatrização/fisiologia
16.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208643

RESUMO

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.


Assuntos
Plaquetas/fisiologia , Sistemas Microeletromecânicos/métodos , Testes de Função Plaquetária/métodos , Algoritmos , Técnicas Biossensoriais , Plaquetas/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Sistemas Microeletromecânicos/instrumentação , Modelos Teóricos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Testes de Função Plaquetária/instrumentação
17.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209789

RESUMO

Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.


Assuntos
Materiais Biocompatíveis/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Meios de Cultura/farmacologia , Adulto , Materiais Biocompatíveis/química , Plaquetas/citologia , Meios de Cultura/química , Endotélio/citologia , Feminino , Humanos , Masculino , Teste de Materiais , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Polímeros/farmacologia , Tecidos Suporte/química
18.
Sci Rep ; 11(1): 12201, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108583

RESUMO

Acute normovolemic hemodilution (ANH) is a potential transfusion method for platelets, as well as for red blood cells. However, previous studies have shown that whole blood storage in ANH decreases platelet aggregability by 14.7-76.3% and that this decrease is not recovered by reinfusion. We investigated whether a new whole blood storage method for 6 h using a polyolefin bag, based on the platelet concentrates storage method, would maintain platelet function better than the conventional method using a polyvinyl chloride bag. We demonstrated that storage of whole blood in a polyolefin bag maintained ADP-induced aggregation rates at more than twofold higher than those in a polyvinyl chloride bag, and also significantly suppressed P-selectin expression, a platelet activation marker (ADP-induced aggregation rates: 24.6 ± 5.1% vs. 51.7 ± 11.5%, p = 0.002; P-selectin expression; 50.3 ± 8.4MFI vs. 31.6 ± 9.3MFI, p = 0.018). These results could be attributed to the high gas permeability of polyolefin, which lowered PCO2 and maintained a high pH with or without agitation. There were no significant changes in platelet count and red blood cell parameters due to the storage methods. Our results suggest that ANH using polyolefin bags is advantageous in improving hemostatic function compared to the conventional method.


Assuntos
Plaquetas/fisiologia , Preservação de Sangue/instrumentação , Preservação de Sangue/métodos , Determinação do Volume Sanguíneo/métodos , Hemodiluição/métodos , Hemostasia , Polienos/química , Hemodiluição/estatística & dados numéricos , Humanos , Ativação Plaquetária , Agregação Plaquetária , Testes de Função Plaquetária
19.
Life Sci ; 277: 119612, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33991548

RESUMO

AIM: Hepatocellular carcinoma (HCC) is a primary liver cancer that usually develops in a background of chronic liver disease and prolonged inflammation. A major contributor in the complex molecular pathogenesis of HCC is the highly intertwined cross-talk between the tumor and the surrounding stromal cells, such as hepatic stellate cells, endothelial cells, macrophages and other immune cells. These tumor-stroma interactions actively fuel tumor growth and modulate the hepatic microenvironment to benefit tumor invasion and disease progression. Platelets have been reported to interact with different cell types in the tumor microenvironment, including tumor cells, stellate cells and macrophages. MATERIALS AND METHODS: Mice were treated with hepatocarcinogenic compound diethylnitrosamine for 25 weeks to induce HCC in the background of fibrosis and inflammation. From week 10, anti-platelet drug Clopidogrel was added to the drinking water and mice were given ad libitum access. KEY FINDINGS: In this study, we show that activated platelets promote tumor cell proliferation and contribute to the adverse tumor-stroma cross-talk that fuels tumor progression. We also show that inhibiting platelet activation with the P2Y12-inhibitor Clopidogrel decreases the number of tumors in a chemically induced mouse model for HCC. SIGNIFICANCE: These results suggest an important role for platelets in the pathogenesis of HCC and that the use of anti-platelet drugs may be therapeutically relevant for patients with liver cancer.


Assuntos
Plaquetas/metabolismo , Carcinoma Hepatocelular/metabolismo , Animais , Plaquetas/fisiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Clopidogrel/farmacologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Células Estreladas do Fígado/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia
20.
Indian J Pathol Microbiol ; 64(2): 347-350, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33851632

RESUMO

Immature platelet fraction (IPF) is a quantification of immature platelets in the circulation reflecting the state of thrombopoiesis in the marrow. Normal reference range for IPF has been established in adults. Reference intervals in neonates are highly dependent on gestational age of the neonate. Complete blood counts (CBC) with IPF of all neonates admitted in neonatal intensive care unit (NICU) were analyzed using Mindray BC-6800 Auto Hematology analyzer. Platelet count of less than 150 × 10^9/L was assigned as thrombocytopenia. Neonates were divided into four groups as per the corrected gestational age (CGA) on the day of CBC analysis: 28-32 weeks, 32-34 weeks, 34-37 weeks, and >37 weeks according to World Health Organization (WHO) classification. Mean, standard deviation, and 95% confidence interval for IPF was calculated in each group and reference range for IPF was derived. Mean IPF in neonates with normal platelet count was term--3.58 (95% CI 3.29 to 3.87), late preterm Neonates (34-37 weeks)--4.14 (95% CI 3.82 to 5.0), moderate preterm neonates (32-34 weeks)--4.14 (95% CI 3.46 to 4.82), and in Very Preterm neonates (28-32 weeks)--IPF of 5.51 (95% CI 3.95 to 7.07). We aimed to establish a reference range for IPF in neonates of different gestational age groups. The IPF values in neonates were comparable between hematology analyzers in neonates with normal platelet counts.


Assuntos
Plaquetas/citologia , Plaquetas/fisiologia , Trombocitopenia/diagnóstico , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Contagem de Plaquetas , Padrões de Referência , Valores de Referência , Trombopoese/fisiologia
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