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1.
J Thromb Haemost ; 21(9): 2596-2610, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37331519

RESUMO

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.


Assuntos
Agregação Plaquetária , Ristocetina , Humanos , Ácido Araquidônico/farmacologia , Reprodutibilidade dos Testes , Difosfato de Adenosina/farmacologia , Testes de Função Plaquetária/métodos , Inibidores da Agregação Plaquetária/farmacologia , Epinefrina/farmacologia , Comunicação , Plaquetas
2.
Biomedicines ; 11(6)2023 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-37371764

RESUMO

The study of endothelial dysfunction (ED) is crucial to identify the pathogenetic mechanism(s) and provide indications for patient management in cardiovascular diseases. It is currently hindered by the limited availability of patient-specific primary endothelial cells (ECs). Endothelial colony-forming cells (ECFCs) represent an optimal non-invasive tool to overcome this issue. Therefore, we investigated the use of ECFCs as a substrate in thrombogenesis and thrombin generation assay (TGA) to assess ED. Both assays were set up on human umbilical vein endothelial cells (HUVECs) and then tested on ECFCs obtained from healthy donors. To prove the ability of the assays to detect endothelial activation, ECs stimulated with TNFα were compared with unstimulated ECs. EC activation was confirmed by the upregulation of VCAM-1 and Tissue Factor expression. Both assays discriminated between unstimulated and activated HUVECs and ECFCs, as significantly higher platelet deposition and fibrin formation in thrombogenesis assay, and thrombin generation in TGA, were observed when TNFα-activated ECs were used as a substrate. The amount of fibrin and thrombin measured in the two assays were directly correlated. Our results support the combined use of a thrombogenesis assay and TGA performed on patient-derived ECFCs to provide a personalized global assessment of ED relevant to the patient's hemostatic profile.

3.
Blood Transfus ; 20(5): 420-432, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-34369869

RESUMO

Acquired platelet function disorders (PFD) are rare bleeding diseases that should be suspected in all patients with unexplained mucocutaneous bleedings of recent onset, with no previous history of haemorrhages, and with normal coagulation test and platelet count. Drug-induced platelet function bleeding disorders are the most frequent PFDs and can easily be identified on the basis of recent administration of platelet-inhibiting drugs. Apart from these, the most challenging acquired PFDs are those caused by autoimmune mechanisms. In fact, demonstration of autoantibodies inhibiting platelet function may be difficult in most non-specialised centres. Among autoimmune PFDs (aPFDs), acquired Glanzmann thrombasthenia (aGT), which is caused by autoantibodies that bind to platelet αIIbß3 integrin, inhibiting its function, is the most frequent. aGT can be associated with underlying haematological malignancies or autoimmune diseases but can also be idiopathic. More rarely, other immune-mediated PFDs can occur, such as acquired delta storage pool disease (aδSPD). Treatment of aPFDs must rely on the control of acute and chronic bleedings, treatment of the underlying disease in secondary forms, and immunosuppressive treatment for autoantibody reduction or eradication. aPFDs may completely resolve upon treatment of any underlying disease that may be present. In primary aPFDs, and in the majority of secondary forms, treatment relies on immunosuppressive therapies.Here we present a systematic review of previously described immune-mediated aGT and aδSPD cases. Clinical and laboratory characteristics, treatments for the control of bleedings and for the eradication of autoantibodies, and responses to treatments are also discussed. Although no guidelines are available for the management of these very rare conditions, presentation of all cases reported so far can help clinicians in the diagnosis and treatment of these life-threatening diseases.


Assuntos
Doenças Autoimunes , Trombastenia , Albinismo , Autoanticorpos , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/etiologia , Transtornos Hemorrágicos , Síndrome de Hermanski-Pudlak , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/uso terapêutico , Trombastenia/terapia
5.
Nat Immunol ; 22(1): 19-24, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33208929

RESUMO

Long pentraxin 3 (PTX3) is an essential component of humoral innate immunity, involved in resistance to selected pathogens and in the regulation of inflammation1-3. The present study was designed to assess the presence and significance of PTX3 in Coronavirus Disease 2019 (COVID-19)4-7. RNA-sequencing analysis of peripheral blood mononuclear cells, single-cell bioinformatics analysis and immunohistochemistry of lung autopsy samples revealed that myelomonocytic cells and endothelial cells express high levels of PTX3 in patients with COVID-19. Increased plasma concentrations of PTX3 were detected in 96 patients with COVID-19. PTX3 emerged as a strong independent predictor of 28-d mortality in multivariable analysis, better than conventional markers of inflammation, in hospitalized patients with COVID-19. The prognostic significance of PTX3 abundance for mortality was confirmed in a second independent cohort (54 patients). Thus, circulating and lung myelomonocytic cells and endothelial cells are a major source of PTX3, and PTX3 plasma concentration can serve as an independent strong prognostic indicator of short-term mortality in COVID-19.


Assuntos
Proteína C-Reativa/genética , COVID-19/genética , Perfilação da Expressão Gênica/métodos , Macrófagos/metabolismo , SARS-CoV-2/isolamento & purificação , Componente Amiloide P Sérico/genética , Células A549 , Adulto , Proteína C-Reativa/metabolismo , COVID-19/epidemiologia , COVID-19/virologia , Linhagem Celular Tumoral , Células Cultivadas , Estudos de Coortes , Células Endoteliais/metabolismo , Epidemias , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Neutrófilos/metabolismo , Prognóstico , SARS-CoV-2/fisiologia , Componente Amiloide P Sérico/metabolismo
6.
Cardiovasc Res ; 116(3): 698-707, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31135876

RESUMO

AIMS: The pathogenetic mechanisms underlying unprovoked venous thromboembolism (uVTE) are largely unknown. In this study, we investigated the molecular mechanisms involved in uVTE pathogenesis by using ex vivo expanded endothelial colony-forming cells (ECFCs), which represent a valuable non-invasive tool for the assessment of endothelial function. METHODS AND RESULTS: We isolated and expanded ECFCs from the peripheral blood of uVTE patients and observed that these cells underwent earlier senescence and showed lower growth rate compared with ECFCs obtained from healthy donors. Through microarray expression profiling, we demonstrated that 2905 genes were differentially expressed between patients and controls. Among them, the anti-angiogenic cytokine TNF superfamily member 15 (TNFSF15) and its death-receptor TNFRSF25 were up-regulated in uVTE ECFCs, and this finding was validated by RT-qPCR. TNFSF15 up-regulation was confirmed at the protein level in ECFC supernatants, and the in vivo relevance of these findings was further corroborated by demonstrating that also the plasmatic levels of TNFSF15 are increased in uVTE patients. After proving that exogenous TNFSF15 exerts pro-apoptotic and anti-proliferative activity on control ECFCs, we demonstrated through blocking experiments that TNFSF15 up-regulation contributes to impaired survival and proliferation of uVTE ECFCs. CONCLUSION: By providing evidence that TNFSF15 impairs ECFC functions crucial to endothelial repair, and that uVTE patients have increased TNFSF15 levels both ex vivo and in vivo, the results of this study suggest that pathologic up-regulation of TNFSF15-TNFRSF25 axis may contribute to uVTE pathogenesis, and may represent the target for novel therapeutic strategies aimed at preventing recurrences in uVTE patients.


Assuntos
Células Progenitoras Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Tromboembolia Venosa/metabolismo , Adulto , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Senescência Celular , Células Progenitoras Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Transdução de Sinais , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Tromboembolia Venosa/patologia , Tromboembolia Venosa/fisiopatologia
8.
Cardiovasc Res ; 111(4): 373-84, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27297888

RESUMO

AIMS: Platelets express functional interleukin-1 receptor-1 (IL-1R1) as well as a repertoire of toll-like receptors (TLRs) involved in platelet activation, platelet-leucocyte reciprocal activation, and immunopathology. IL-1R8, also known as single Ig IL-1-related receptor (SIGIRR) or TIR8, is a member of the IL-1R family that negatively regulates responses to IL-1R family members and TLRs. In the present study, we addressed the expression of IL-1R8 in platelets and megakaryocytes and its role in the control of platelet activation during inflammatory conditions and thromboembolism. METHODS AND RESULTS: Here, we show by flow cytometry analysis, western blot, confocal microscopy, and quantitative real-time polymerase chain reaction that IL-1R8 is expressed on human and mouse platelets at high levels and on megakaryocytes. IL-1R8-deficient mice show normal levels of circulating platelets. Homotypic and heterotypic (platelet-neutrophil) aggregation triggered by Adenosine DiPhosphate (ADP) and IL-1 or lipopolysaccharide (LPS) was increased in IL-1R8-deficient platelets. IL-1R8-deficient mice showed increased soluble P-selectin levels and increased platelet-neutrophil aggregates after systemic LPS administration. Commensal flora depletion and IL-1R1 deficiency abated platelet hyperactivity and the increased platelet/neutrophil aggregation observed in Il1r8(-/-) mice in vitro and in vivo, suggesting a key role of IL-1R8 in regulating platelet TLR and IL-1R1 function. In a mouse model of platelet-dependent pulmonary thromboembolism induced by ADP administration, IL-1R8-deficient mice showed an increased frequency of blood vessel complete obstruction. CONCLUSION: These results show that platelets, which have a large repertoire of TLRs and IL-1 receptors, express high levels of IL-1R8, which plays a non-redundant function as a regulator of thrombocyte activity in vitro and in vivo.


Assuntos
Plaquetas/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/metabolismo , Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos Transgênicos , Ativação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo
10.
Am J Pathol ; 175(2): 547-56, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19574425

RESUMO

The mechanisms that sustain endometrial tissues at ectopic sites in patients with endometriosis are poorly understood. Various leukocytes, including macrophages, infiltrate endometriotic lesions. In this study, we depleted mouse macrophages by means of either clodronate liposomes or monoclonal antibodies before the injection of syngeneic endometrial tissue. In the absence of macrophages, tissue fragments adhered and implanted into the peritoneal wall, but endometriotic lesions failed to organize and develop. When we depleted macrophages after the establishment of endometriotic lesions, blood vessels failed to reach the inner layers of the lesions, which stopped growing. Macrophages from patients with endometriosis and experimental mice, but not nonendometriotic patients who underwent surgery for uterine leiomyomas or control mice, expressed markers of alternative activation. These markers included high levels of scavenger receptors, CD163 and CD206, which are involved in both the scavenging of hemoglobin with iron transfer into macrophages and the silent clearance of inflammatory molecules. Macrophages in both inflammatory liquid and ectopic lesions were equally polarized, suggesting a critical role of environmental cues in the peritoneal cavity. Adoptively transferred, alternatively activated macrophages dramatically enhanced endometriotic lesion growth in mice. Inflammatory macrophages effectively protected mice from endometriosis. Therefore, endogenous macrophages involved in tissue remodeling appear as players in the natural history of endometriosis, required for effective vascularization and ectopic lesion growth.


Assuntos
Endometriose/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Neovascularização Patológica/imunologia , Animais , Modelos Animais de Doenças , Endometriose/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/patologia
11.
Int J Gen Med ; 2: 141-4, 2009 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20360897

RESUMO

BACKGROUND: Atherosclerosis is a well known progressive disease that recognizes risk factors such as diabetes, hypertension, smoking, dyslipidemia, and inflammation. Mechanisms underlying atherosclerotic processes during inflammation are not completely understood, but cytokines are also involved, in particular tumor necrosis factor-alpha (TNF-alpha). Chronic inflammatory diseases such as rheumatoid arthritis (RA) are commonly associated with atherosclerotic complication. Little is known about the role of treatment of chronic inflammatory disease on the evolution of atherosclerosis in this kind of disease. Usually, evolution of atherosclerosis is monitored by intima-media thickness and the presence of plaques on several arteries such as common carotid. AIM: The aim of the study was to monitor atherosclerosis evolution in seven RA patients on common treatment with infliximab (an anti-TNF-alpha drug) compared with seven RA patients during common treatment but not treated with infliximab. PATIENTS AND METHODS: We selected 14 patients with RA according to the American College of Rheumatology classification criteria. Seven patients were selected before and after common treatment for RA based on nonsteroidal anti-inflammatory drugs (NSAIDs), methotrexate, and steroids (12 months), and seven patients before and after treatment based on infliximab associated with NSAIDs, methotrexate, and steroids (12 months). Ultrasound vascular imaging was performed to screen intima-media thickness and the presence of atherosclerotic plaques on common carotid artery and identify evolution of atherosclerosis. RESULTS: After 12 months, patients that were treated with infliximab showed significant worsening of atherosclerosis with an increase of intima-media thickness and the presence of further atherosclerotic plaques compared to patients that were treated traditionally and showed a nonsignificant increase of the same parameters. DISCUSSION: Treatment based on anti-TNF-alpha such as infliximab shows a worsening evolution of atherosclerosis based on our data. If these data are associated with a poor clinical outcome such as atherothrombosis of cerebral vessels and/or coronary vessels, this should be evaluated by further studies.

12.
J Leukoc Biol ; 81(1): 84-91, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17035340

RESUMO

Chemokines regulate the migration and the maturation of dendritic cells (DC) licensed by microbial constituents. We have recently found that the function of DC, including their ability to activate naïve, allogeneic CD4+ T cells, requires the autocrine/paracrine release of the nuclear protein high mobility group box 1 (HMGB1). We show here that human myeloid DC, which rapidly secrete upon maturation induction their own HMGB1, remodel their actin-based cytoskeleton, up-regulate the CCR7 and the CXCR4 chemokine receptors, and acquire the ability to migrate in response to chemokine receptor ligands. The events are apparently causally related: DC challenged with LPS in the presence of HMGB1-specific antibodies fail to up-regulate the expression of the CCR7 and CXCR4 receptors and to rearrange actin-rich structures. Moreover, DC matured in the presence of anti-HMGB1 antibodies fail to migrate in response to the CCR7 ligand CCL19 and to the CXCR4 ligand CXCL12. The blockade of receptor for advanced glycation end products (RAGE), the best-characterized membrane receptor for HMGB1, impinges as well on the up-regulation of chemokine receptors and on responsiveness to CCL19 and CXCL12. Our data suggest that the autocrine/paracrine release of HMGB1 and the integrity of the HMGB1/RAGE pathway are required for the migratory function of DC.


Assuntos
Quimiotaxia , Células Dendríticas/fisiologia , Proteína HMGB1/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Células Cultivadas , Quimiocina CCL19 , Quimiocina CXCL12 , Quimiocinas/metabolismo , Quimiocinas CC/metabolismo , Quimiocinas CXC/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Humanos , Linfonodos/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores CCR7 , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , Regulação para Cima
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