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1.
Biochem Biophys Res Commun ; 537: 7-14, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33383564

RESUMO

Hydrogen sulfide (H2S) prevents platelet activation and neutrophils extracellular traps (NETs) formation. However, the mechanism of sodium hydrosulfide (NaHS, a donor that produces H2S) inhibits the formation of NETs in hyperhomocysteinemia (HHcy) rats has not been previously investigated. In the experiment, the expressions of HMGB1 of platelets, the expressions of TLR4, PAD4 and the phosphor-p38 of neutrophils were measured. The NETs formations, the concentration of DNA in the serum and the culture solution of cultured neutrophils which was stimulated by platelet-rich plasma (PRP) were tested. Additionally, the cellular ROS level and SOD activity were detected. The platelets were activated and the expression of HMGB1 of platelets and NETs formation, the concentration of DNA, and the expressions of TLR4, phosphor-p38 and PAD4, the ROS level were all increased while the activity of SOD decreased in the HHcy group compared to the control group. NaHS significantly inhibited the activation of platelets, the production of ROS and the formation of NETs in neutrophils, reversed the expressions of HMGB1, TLR4, phosphor-p38, PAD4 and decreased concentration of DNA which was caused by high homocysteine. Our results demonstrate that the donor of H2S inhibits NETs formation of neutrophils via the HMGB1/TLR4/p38 MAPK/ROS pathway in hyperhomocysteinemia.


Assuntos
Armadilhas Extracelulares/metabolismo , Proteína HMGB1/metabolismo , Sulfeto de Hidrogênio/farmacologia , Hiper-Homocisteinemia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Modelos Animais de Doenças , Armadilhas Extracelulares/efeitos dos fármacos , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosforilação/efeitos dos fármacos , Plasma Rico em Plaquetas/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
3.
Life Sci ; 260: 118295, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32822720

RESUMO

Advanced chronic kidney disease is associated with high rates of cardiovascular disease. Considering the crucial role of capillaries in renal function, our study aimed to evaluate microparticles related to vascular physiology examining the link between stages of chronic kidney disease with circulating endothelial (EMP), platelet (PMP) and monocytic (MMP) microparticles. Cross-sectional study with blinded endpoints included subjects of both sexes, aged 40-75 years (n = 247), with established cardiovascular disease (coronary heart disease, ischemic stroke, or peripheral artery disease). They were stratified 1:1 by the presence or absence of decreased glomerular filtration rate (GFR < 60 mL/min/1.73 m2) estimated by the CKD-EPI criteria, and according to the stages of CKD. Microparticles were quantified by flow-cytometry using specific antibodies to identify endothelial, platelet, and monocytic derived microparticles. Higher percentages of circulating MMP (p = 0.036), but not for EMP or PMP, were observed in subjects with reduced GFR. Circulating MMP were also related to the stages of chronic kidney disease (trend analysis across renal stages, p = 0.038). Higher percentages of circulating MMP were found in subjects with reduced GFR, and their percentages were progressively higher according to the stage of chronic renal function.


Assuntos
Micropartículas Derivadas de Células , Monócitos/ultraestrutura , Insuficiência Renal Crônica/sangue , Adulto , Idoso , Plaquetas/ultraestrutura , Doenças Cardiovasculares/sangue , Estudos Transversais , Progressão da Doença , Células Endoteliais/ultraestrutura , Feminino , Taxa de Filtração Glomerular , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/fisiopatologia
4.
Rev Esp Patol ; 53(3): 182-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32650969
6.
Platelets ; 31(4): 544-547, 2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32436471

RESUMO

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles. Clinical manifestations include a bleeding diathesis due to a platelet delta storage pool deficiency, oculocutaneous albinism, inflammatory bowel disease, neutropenia, and pulmonary fibrosis. Ten genes associated with HPS are identified to date, and each gene encodes a protein subunit of either Biogenesis of Lysosome-related Organelles Complex (BLOC)-1, BLOC-2, BLOC-3, or the Adaptor Protein-3 complex. Several genetic variants and phenotypic heterogeneities are reported in individuals with HPS, who generally exhibit easy bruisability and increased bleeding. Desmopressin, pro-coagulants, or platelet transfusion may be used as prophylaxis or treatment for excessive bleeding in patients with HPS. However, response to desmopressin can be variable. Platelets are effective in preventing or treating bleeding in individuals with HPS, but platelets should be transfused judiciously to limit alloimmunization in patients with HPS who are at risk of developing pulmonary fibrosis and may be potential candidates for lung transplantation. The discovery of new genes associated with HPS in people with excessive bleeding and hypopigmentation of unknown etiology may be facilitated by the use of next-generation sequencing or panel-based genetic testing.


Assuntos
Plaquetas/metabolismo , Síndrome de Hermanski-Pudlak/genética , Lisossomos/genética , Ácido Aminocaproico/farmacologia , Antifibrinolíticos/farmacologia , Plaquetas/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Contusões/genética , Desamino Arginina Vasopressina/uso terapêutico , Hemorragia/genética , Síndrome de Hermanski-Pudlak/tratamento farmacológico , Síndrome de Hermanski-Pudlak/fisiopatologia , Humanos , Hipopigmentação/genética , Lisossomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas/genética , Proteínas/metabolismo , Ácido Tranexâmico/farmacologia
7.
Platelets ; 31(5): 627-632, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32397915

RESUMO

Coronavirus disease 2019 (COVID-19) is a new infectious disease that currently lacks standardized and established laboratory markers to evaluate its severity. In COVID-19 patients, the number of platelets (PLTs) and dynamic changes of PLT-related parameters are currently a concern. The present paper discusses the potential link between PLT parameters and COVID-19. Several studies have identified a link between severe COVID-19 patients and specific coagulation index, in particular, high D-dimer level, prolonged prothrombin time, and low PLT count. These alterations reflect the hypercoagulable state present in severe COVID-19 patients, which could promote microthrombosis in the lungs, as well as in other organs. Further information and more advanced hematological parameters related to PLTs are needed to better estimate this link, also considering COVID-19 patients at different disease stages and stratified in different cohorts based on preexisting co-morbidity, age, and gender. Increasing the understanding of PLT functions in COVID-19 will undoubtedly improve our knowledge on disease pathogenesis, clinical management, and therapeutic options, but could also lead to the development of more precise therapeutic strategies for COVID-19 patients.


Assuntos
Betacoronavirus , Plaquetas/fisiologia , Infecções por Coronavirus/sangue , Pandemias , Pneumonia Viral/sangue , Trombofilia/etiologia , Anticoagulantes/administração & dosagem , Anticoagulantes/uso terapêutico , Biomarcadores/sangue , Plaquetas/ultraestrutura , Moléculas de Adesão Celular/metabolismo , Infecções por Coronavirus/complicações , Infecções por Coronavirus/patologia , Citocinas/metabolismo , Coagulação Intravascular Disseminada/etiologia , Interações Medicamentosas , Células Endoteliais/patologia , Endotélio Vascular/patologia , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Inflamação , Pulmão/patologia , Peptidil Dipeptidase A/fisiologia , Contagem de Plaquetas , Testes de Função Plaquetária , Pneumonia Viral/complicações , Pneumonia Viral/patologia , Tempo de Protrombina , Receptores Virais/fisiologia , /prevenção & controle , Síndrome Respiratória Aguda Grave/sangue , Síndrome Respiratória Aguda Grave/patologia , Trombofilia/sangue , Trombofilia/tratamento farmacológico , Trombose Venosa/epidemiologia , Trombose Venosa/etiologia , Trombose Venosa/patologia , Trombose Venosa/prevenção & controle
8.
Arterioscler Thromb Vasc Biol ; 40(6): 1441-1453, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375545

RESUMO

Megakaryocyte-derived platelets and endothelial cells store their hemostatic cargo in α- and δ-granules and Weibel-Palade bodies, respectively. These storage granules belong to the lysosome-related organelles (LROs), a heterogeneous group of organelles that are rapidly released following agonist-induced triggering of intracellular signaling pathways. Following vascular injury, endothelial Weibel-Palade bodies release their content into the vascular lumen and promote the formation of long VWF (von Willebrand factor) strings that form an adhesive platform for platelets. Binding to VWF strings as well as exposed subendothelial collagen activates platelets resulting in the release of α- and δ-granules, which are crucial events in formation of a primary hemostatic plug. Biogenesis and secretion of these LROs are pivotal for the maintenance of proper hemostasis. Several bleeding disorders have been linked to abnormal generation of LROs in megakaryocytes and endothelial cells. Recent reviews have emphasized common pathways in the biogenesis and biological properties of LROs, focusing mainly on melanosomes. Despite many similarities, LROs in platelet and endothelial cells clearly possess distinct properties that allow them to provide a highly coordinated and synergistic contribution to primary hemostasis by sequentially releasing hemostatic cargo. In this brief review, we discuss in depth the known regulators of α- and δ-granules in megakaryocytes/platelets and Weibel-Palade bodies in endothelial cells, starting from transcription factors that have been associated with granule formation to protein complexes that promote granule maturation. In addition, we provide a detailed view on the interplay between platelet and endothelial LROs in controlling hemostasis as well as their dysfunction in LRO related bleeding disorders.


Assuntos
Plaquetas/ultraestrutura , Grânulos Citoplasmáticos/fisiologia , Células Endoteliais/ultraestrutura , Hemostasia/fisiologia , Lisossomos/fisiologia , Transtornos da Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/fisiopatologia , Colágeno/fisiologia , Grânulos Citoplasmáticos/ultraestrutura , Humanos , Lisossomos/ultraestrutura , Corpos de Weibel-Palade/fisiologia , Corpos de Weibel-Palade/ultraestrutura , Fator de von Willebrand/metabolismo
9.
Sci Rep ; 10(1): 4621, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165642

RESUMO

Histones are typically located within the intracellular compartment, and more specifically, within the nucleus. When histones are located within the extracellular compartment, they change roles and become damage-associated molecular patterns (DAMPs), promoting inflammation and coagulation. Patients with sepsis have increased levels of extracellular histones, which have been shown to correlate with poor prognosis and the development of sepsis-related sequelae, such as end-organ damage. Until now, neutrophils were assumed to be the primary source of circulating histones during sepsis. In this paper, we show that megakaryocytes contain extranuclear histones and transfer histones to their platelet progeny. Upon examination of isolated platelets from patients with sepsis, we identified that patients with sepsis have increased amounts of platelet-associated histones (PAHs), which appear to be correlated with the type of infection. Taken together, these results suggest that megakaryocytes and platelets may be a source of circulating histones during sepsis and should be further explored.


Assuntos
Plaquetas/metabolismo , Citoplasma/metabolismo , Histonas/metabolismo , Megacariócitos/metabolismo , Sepse/metabolismo , Biomarcadores , Coagulação Sanguínea , Plaquetas/ultraestrutura , Citoplasma/ultraestrutura , Imunofluorescência , Humanos , Megacariócitos/ultraestrutura , Modelos Biológicos , Sepse/sangue , Sepse/etiologia
10.
Toxicol Appl Pharmacol ; 391: 114912, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32014540

RESUMO

Arsenic, an environmental contaminant in drinking water worldwide is well-established to increase cardiovascular diseases (CVDs) in humans. Of these, thrombotic events represent a major adverse effect associated with arsenic exposure, for which an abundance of epidemiological evidence exists. Platelet aggregation constitutes a pivotal step in thrombosis but arsenic alone doesn't induce aggregation and the mechanism underlying arsenic-induced thrombosis still remains unclear. Here we demonstrated that arsenic induces morphological changes of platelets, i.e., contraction and pseudopod projection, the primal events of platelet activation, which can increase platelet reactivity. Arsenite induced prominent platelet shape changes in a dose-dependent manner in freshly isolated human platelets. Of note, arsenite suppressed focal adhesion kinase (FAK) activity, which in turn activated RhoA, leading to altered actin assembly through LIMK activation, and subsequent cofilin inactivation. Arsenic-induced platelet shape change appeared to increase the sensitivity to thrombin and ADP-induced aggregation. Supporting this, latrunculin A, an inhibitor of actin-dynamics abolished it. Taken together, we demonstrated that arsenic induces cytoskeletal changes and shape changes of platelets through FAK-mediated alteration of actin dynamics, which renders platelets reactive to activating stimuli, ultimately contributing to increased thrombosis.


Assuntos
Actinas/metabolismo , Arsenitos/toxicidade , Plaquetas/patologia , Plaquetas/ultraestrutura , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Compostos de Sódio/toxicidade , Difosfato de Adenosina/farmacologia , Adolescente , Adulto , Humanos , Técnicas In Vitro , Quinases Lim/antagonistas & inibidores , Masculino , Selectina-P/biossíntese , Agregação Plaquetária/efeitos dos fármacos , Adulto Jovem , Proteína rhoA de Ligação ao GTP
11.
J Dermatol ; 47(2): 185-189, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31820501

RESUMO

Hermansky-Pudlak syndrome type 2 (HPS2) is an extremely rare autosomal recessive inherited disease characterized by partial oculocutaneous albinism (OCA), bleeding diathesis due to a storage pool deficiency and immunodeficiency. The disorder is caused by disruption of the adapter protein 3 complex, which is involved in impaired intracellular vesicle transport. Here, we report the first case of a 1-year-old girl with HPS2 in Asia. She had no specific symptoms other than OCA and neutropenia. We analyzed her platelet function using transmission electron microscopy and a platelet aggregation test, cytotoxic degranulation assay of her natural killer (NK) cells and bleeding time, the results of which led to the diagnosis of HPS2. Although her NK-cell cytotoxic degranulation was impaired, she had not developed signs of hemophagocytic lymphohistiocytosis (HLH) or fibrosing lung disease. Molecular genetic analyses showed novel heterozygous mutations (c.188T>A [p.M63K] and c.2546>A [p.L849X]) in AP3B1. When examining patients with OCA, blood tests should be performed to confirm neutrophil count, bleeding time and platelet agglutination. When HPS2 is suspected, detailed immunological tests should be considered, and attention should be paid to HLH and pulmonary lesions immediately and over the long term.


Assuntos
Complexo 3 de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Síndrome de Hermanski-Pudlak/genética , Plaquetas/patologia , Plaquetas/ultraestrutura , Análise Mutacional de DNA , Feminino , Cabelo/patologia , Cabelo/ultraestrutura , Síndrome de Hermanski-Pudlak/sangue , Síndrome de Hermanski-Pudlak/diagnóstico , Síndrome de Hermanski-Pudlak/patologia , Heterozigoto , Humanos , Lactente , Japão , Microscopia Eletrônica de Transmissão , Mutação
12.
Platelets ; 31(1): 33-42, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30721642

RESUMO

Exposure to hypoxia, through ascension to high altitudes (HAs), air travel, or human disease, is associated with an increased incidence of thrombosis in some settings. Mechanisms underpinning this increased thrombosis risk remain incompletely understood, and the effects of more sustained hypoxia on the human platelet molecular signature and associated functional responses have never been examined. We examined the effects of prolonged (≥2 months continuously) hypobaric hypoxia on platelets isolated from subjects residing at HA (3,700 meters) and, for comparison, matched subjects residing under normoxia conditions at sea level (50 meters). Using complementary transcriptomic, proteomic, and functional methods, we identified that the human platelet transcriptome is markedly altered under prolonged exposure to hypobaric hypoxia at HA. Among the significantly, differentially expressed genes (mRNA and protein), were those having canonical roles in platelet activation and thrombosis, including membrane glycoproteins (e.g. GP4, GP6, GP9), integrin subunits (e.g. ITGA2B), and alpha-granule chemokines (e.g. SELP, PF4V1). Platelets from subjects residing at HA were hyperactive, as demonstrated by increased engagement and adhesion to fibrinogen, fewer alpha granules by transmission electron microscopy, increased circulating PF4 and ADP, and significantly enhanced clot retraction. In conclusion, we identify that prolonged hypobaric hypoxia exposure due to HA alters the platelet transcriptome and proteome, triggering increased functional activation responses that may contribute to thrombosis. Our findings may also have relevance across a range of human diseases where chronic hypoxia, platelet activation, and thrombosis are increased.


Assuntos
Altitude , Plaquetas/metabolismo , Hipóxia/metabolismo , Proteoma , Transcriptoma , Adulto , Biomarcadores , Plaquetas/ultraestrutura , Biologia Computacional/métodos , Citoesqueleto/metabolismo , Exposição Ambiental , Perfilação da Expressão Gênica , Humanos , Masculino , Ativação Plaquetária , Adesividade Plaquetária , Proteômica/métodos , Trombose/etiologia , Trombose/metabolismo
13.
Platelets ; 31(4): 536-540, 2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-31502501

RESUMO

Gray platelet syndrome (GPS) is an inherited disorder. Patients harboring GPS have thrombocytopenia with large platelets lacking α-granules. A long-term complication is myelofibrosis with pancytopenia. Hematopoietic stem cell transplant (HSCT) could be a curative treatment. We report a male GPS patient with severe pancytopenia, splenomegaly and a secondary myelofibrosis needing red blood cells transfusion. He received an HSCT from a 10/10 matched HLA-unrelated donor after a myeloablative conditioning regimen. Transfusion independence occurred at day+21, with a documented neutrophil engraftment. At day+ 180, we added ruxolitinib to cyclosporine and steroids for a moderate chronic graft versus host disease (GVHD) and persistent splenomegaly. At day+240 GVHD was controlled and splenomegaly reduced. Complete donor chimesrism was documented in blood and marrow and platelets functions and morphology normalized. At day+ 720, the spleen size normalized and there was no evidence of marrow fibrosis on the biopsy. In GPS, HSCT may be a curative treatment in selected patients with pancytopenia and myelofibrosis.


Assuntos
Plaquetas/patologia , Síndrome da Plaqueta Cinza/terapia , Transplante de Células-Tronco Hematopoéticas , Mielofibrose Primária/terapia , Adulto , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Ciclosporina/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Síndrome da Plaqueta Cinza/tratamento farmacológico , Síndrome da Plaqueta Cinza/fisiopatologia , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pirazóis/uso terapêutico , Esplenomegalia/tratamento farmacológico , Esplenomegalia/etiologia , Fatores de Tempo , Condicionamento Pré-Transplante
14.
Platelets ; 31(2): 226-235, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30977703

RESUMO

Platelet concentrates are used in clinic for therapy and prophylaxis of conditions associated with platelet deficiency or malfunction. The characteristics of platelet concentrates gradually change during pretransfusion storage, affecting their clinical effectiveness and the risk of adverse transfusion reactions. The presence of platelet-derived membrane vesicles is an important characteristic of platelet concentrates. Due to their functionality, changes in the number and molecular compositions of platelet-derived vesicles have major effects on the clinical properties of platelet preparations. The existence of different subpopulations of membrane vesicles requires analytical methods capable of providing information at the individual vesicle level. Such methods include flow cytometry and electron microscopy. However, conventional flow cytometry has certain limitations, since the diameters of many platelet-derived membrane vesicles are smaller than its detection limit. The use of classical scanning electron microscopy is also limited due to the requirement for coating with a layer of conductive material, which impedes the detection of small extracellular vesicles. Here, a combination of high-sensitivity flow cytometry and low-voltage scanning electron microscopy was used to increase sensitivity and resolution in the detection of nanosized objects present in platelet concentrates during storage. Apheresis platelet concentrates from eight healthy adult donors were investigated on days 2 and 7 of storage. Fractions of nanosized objects were obtained by differential centrifugation. Fluorophore-conjugated antibodies were used to detect marker-positive vesicles derived from platelets (CD41), red blood cells (CD235a), leukocytes (CD45), and endothelial cells (VEGFR2). Near-spherical objects with diameters ranging from 25 to 700 nm were observed by low-voltage scanning electron microscopy in platelet concentrates and its fractions. On day 7 of storage, objects with diameters of less than 100 nm were attached to and clustered near the terminal ends of pseudopod-like projections. High-sensitivity flow cytometry showed that during storage numbers of CD41(pos) vesicles elevated more than fivefold and numbers of marker-negative nanosized objects, which did not carry any of the investigated cell type-specific markers elevated more than twofold. Major changes in both CD41(pos) vesicles and marker-negative nanosized objects abundances were observed for objects with diameters around 100 nm bead equivalents. Overall, these results emphasized the importance of application of high-sensitivity methods for monitoring the characteristics of cell-derived nanosized objects during platelet concentrate storage.


Assuntos
Plaquetas/ultraestrutura , Preservação de Sangue , Citometria de Fluxo , Vesículas Extracelulares/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Varredura , Nanoestruturas/ultraestrutura , Plasma/citologia , Plasma/metabolismo , Transfusão de Plaquetas , Plasma Rico em Plaquetas/citologia , Plasma Rico em Plaquetas/metabolismo , Plaquetoferese , Fatores de Tempo
15.
Exp Cell Res ; 385(2): 111692, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31689412

RESUMO

Arterial hypertension (HTN) can lead to serious organ damage. Several mechanisms have been implicated in the pathogenesis of HTN including constitutive activation of platelets, which increases the risk of aggregation and clot formation. We recently demonstrated the plasma membranes of platelets from patients with HTN exhibit modified structural and physicochemical properties; Raman and Fourier transform infrared by attenuated total reflectance (FTIR-ATR) spectroscopy also indicated lipid content and protein structure alterations. This study aimed to precisely quantify the constituents of the main structural phospholipids and cholesterol in the plasma membranes of platelets from patients with HTN and normotensive individuals. We also assessed the consequence of these alterations on platelet structure and function. Liquid chromatography coupled to triple quadrupole mass spectrometry revealed the plasma membranes of HTN platelets contained less cholesterol and phosphatidylcholine, more phosphatidylserine and phosphatidylethanolamine and had similar sphingosine contents. Atomic force microscopy revealed HTN platelets exhibited increased surface roughness and more pleats. Transmission electron microscopy revealed diminution of the internal membranous structures in HTN platelets. Our findings strongly suggest plasma membrane lipid content alterations-including cholesterol depletion-occur in HTN, and these alterations may induce morphological and physiological abnormalities that participate in the functional changes associated with hypertension.


Assuntos
Plaquetas/metabolismo , Membrana Celular/ultraestrutura , Hipertensão/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Idoso , Plaquetas/ultraestrutura , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Fluidez de Membrana , Pessoa de Meia-Idade
16.
Transfusion ; 59(12): 3783-3793, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31642072

RESUMO

Developments during the past few years have resulted in multiple kinds of platelet products for transfusion. This involves different collection methods, containers, preservative solutions, modifications of storage temperatures and durations, and additional treatments such as pathogen reduction. Much experience has been obtained testing these processes in vitro to seek indications of in vivo effectiveness. Availability of an in vitro method that correlated with in vivo effectiveness would be extremely valuable for these different kinds of platelet products and as more innovation in platelet preparation occurs in the future. This report reviews the methods for in vitro platelet testing with a view to their in vivo implications and whether such testing could be helpful in projecting the clinical effectiveness of different platelet products.


Assuntos
Plaquetas/metabolismo , Preservação de Sangue/métodos , Hemostáticos/metabolismo , Transfusão de Plaquetas/métodos , Plaquetas/ultraestrutura , Citometria de Fluxo , Humanos , Microscopia Eletrônica
17.
ACS Nano ; 13(9): 10576-10586, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31483602

RESUMO

Advances in cardiovascular materials have brought us improved artificial vessels with larger diameters for reducing adverse responses that drive acute thrombosis and the associated complications. Nonetheless, the challenge is still considerable when applying these materials in small-diameter blood vessels. Here we report the biomimetic design of an acellular small-diameter vascular graft with specifically lamellar nanotopography on the luminal surface via a modified freeze-cast technique. The experimental findings verify that the well-designed nanolamellar structure is able to inhibit the adherence and activation of platelets, induce oriented growth of endothelial cells, and eventually remodel a neovessel to maintain long-term patency in vivo. Furthermore, the results of numerical simulations in physically mimetic conditions reveal that the regularly lamellar nanopattern can manipulate blood flow to reduce the flow disturbance compared with random topography. Our current work not only creates a freeze-cast small-diameter vascular graft that employs topographic architecture to direct the vascular cell fates for revasculature but also rekindles confidence in biophysical cues for modulating in situ tissue regeneration.


Assuntos
Prótese Vascular , Nanopartículas/química , Túnica Íntima/cirurgia , Animais , Velocidade do Fluxo Sanguíneo , Plaquetas/ultraestrutura , Sistema Livre de Células , Simulação por Computador , Matriz Extracelular/metabolismo , Masculino , Coelhos , Ratos , Ultrassonografia
18.
Int Wound J ; 16(6): 1457-1463, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31486290

RESUMO

In the present study, the age- and sex-related differences in platelet ultrastructure were investigated using transmission electron microscopy (TEM). A total of 15 healthy volunteers were grouped according to age, with 5 people in each of the following groups: young group (25-45 years), middle-aged group (46-65 years), and old-aged group (> 65 years). In the TEM micrographs, the internal components, specifically the α-granules, dense granules, and lysosomal granules, of 20 platelets were counted for each group. Two-way analysis of variance of age and sex variance was used to compare the results. The ultrastructure of the platelets in the old-aged group was observed to be quite different from those of the young and middle-aged groups. Specifically, with ageing, the platelet membrane becomes more irregular in shape and non-smooth, and multiple platelet membrane ruptures are observed. Furthermore, the pseudopodia and protuberances become more numerous and slender, and the number of α-granules is significantly reduced. These morphological changes indicate that ageing may affect the function of platelets, which in turn affects the efficacy of platelet concentrates. Thus, the effects of age should be considered when using platelet concentrates prepared from elderly autologous blood.


Assuntos
Envelhecimento , Plaquetas/ultraestrutura , Adulto , Idoso , Membrana Celular/ultraestrutura , Extensões da Superfície Celular/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Voluntários Saudáveis , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade
19.
Blood Adv ; 3(17): 2617-2626, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501156

RESUMO

Platelet α-granules play important roles in platelet function. They contain hundreds of proteins that are synthesized by the megakaryocyte or taken up by endocytosis. The trafficking pathways that mediate platelet α-granule biogenesis are incompletely understood, especially with regard to cargo synthesized by the megakaryocyte. Vacuolar-protein sorting 33B (VPS33B) and VPS16B are essential proteins for α-granule biogenesis, but they are largely uncharacterized. Here, we adapted a powerful method to directly map the pathway followed by newly synthesized cargo proteins to reach α-granules. Using this method, we revealed the recycling endosome as a key intermediate compartment in α-granule biogenesis. We then used CRISPR/Cas9 gene editing to knock out VPS33B in pluripotent stem cell-derived immortalized megakaryocyte cells (imMKCLs). Consistent with the observations in platelets from patients with VPS33B mutation, VPS33B-knockout (KO) imMKCLs have drastically reduced levels of α-granule proteins platelet factor 4, von Willebrand factor, and P-selectin. VPS33B and VPS16B form a distinct and small complex in imMKCLs with the same hydrodynamic radius as the recombinant VPS33B-VPS16B heterodimer purified from bacteria. Mechanistically, the VPS33B-VPS16B complex ensures the correct trafficking of α-granule proteins. VPS33B deficiency results in α-granule cargo degradation in lysosomes. VPS16B steady-state levels are significantly lower in VPS33B-KO imMKCLs, suggesting that VPS16B is destabilized in the absence of its partner. Exogenous expression of green fluorescent protein-VPS33B in VPS33B-KO imMKCLs reconstitutes the complex, which localizes to the recycling endosome, further defining this compartment as a key intermediate in α-granule biogenesis. These results advance our understanding of platelet α-granule biogenesis and open new avenues for the study of these organelles.


Assuntos
Plaquetas/ultraestrutura , Grânulos Citoplasmáticos/química , Vesículas Citoplasmáticas/química , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Plaquetas/metabolismo , Linhagem Celular , Endossomos/metabolismo , Humanos , Megacariócitos/citologia , Transporte Proteico , Vesículas Transportadoras/química
20.
Blood Adv ; 3(15): 2342-2354, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31391167

RESUMO

Platelet activation requires fully functional mitochondria, which provide a vital energy source and control the life span of platelets. Previous reports have shown that both general autophagy and selective mitophagy are critical for platelet function. However, the underlying mechanisms remain incompletely understood. Here, we show that Nix, a previously characterized mitophagy receptor that plays a role in red blood cell maturation, also mediates mitophagy in platelets. Genetic ablation of Nix impairs mitochondrial quality, platelet activation, and FeCl3-induced carotid arterial thrombosis without affecting the expression of platelet glycoproteins (GPs) such as GPIb, GPVI, and αIIbß3 Metabolic analysis revealed decreased mitochondrial membrane potential, enhanced mitochondrial reactive oxygen species level, diminished oxygen consumption rate, and compromised adenosine triphosphate production in Nix -/- platelets. Transplantation of wild-type (WT) bone marrow cells or transfusion of WT platelets into Nix-deficient mice rescued defects in platelet function and thrombosis, suggesting a platelet-autonomous role (acting on platelets, but not other cells) of Nix in platelet activation. Interestingly, loss of Nix increases the life span of platelets in vivo, likely through preventing autophagic degradation of the mitochondrial protein Bcl-xL. Collectively, our findings reveal a novel mechanistic link between Nix-mediated mitophagy, platelet life span, and platelet physiopathology. Our work suggests that targeting platelet mitophagy Nix might provide new antithrombotic strategies.


Assuntos
Plaquetas/metabolismo , Proteínas de Membrana/metabolismo , Mitofagia , Ativação Plaquetária , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Biomarcadores , Tempo de Sangramento , Plaquetas/ultraestrutura , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/metabolismo , Trombose das Artérias Carótidas/patologia , Sobrevivência Celular/genética , Humanos , Imunofenotipagem , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fenótipo , Ativação Plaquetária/genética , Testes de Função Plaquetária , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética
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