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ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.
Assuntos
Megacariócitos , Complexo Glicoproteico GPIb-IX de Plaquetas , Trombocitopenia , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Citoplasma/metabolismo , Filaminas/genética , Filaminas/metabolismo , Megacariócitos/metabolismo , Morfogênese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismoRESUMO
Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.
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Proteoma , Trombina , Proteoma/metabolismo , Trombina/farmacologia , Trombina/metabolismo , Glicosilação , Plaquetas/metabolismo , Ativação PlaquetáriaRESUMO
BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.
Assuntos
Molécula 1 de Adesão Intercelular , Antígeno de Macrófago 1 , Humanos , Antígeno de Macrófago 1/fisiologia , Adesão Celular/fisiologia , Células Endoteliais , Inflamação , Movimento Celular/fisiologia , NeutrófilosRESUMO
Little is known about the physiological role of beta-2-glycoprotein I (ß2GPI) despite it being the major auto-antigen in the antiphospholipid syndrome. A systematic study of the role of ß2GPI in thrombus formation in vivo has not been performed to date. Herein, we report that ß2GPI deficient (-/-) mice have enhanced thrombus formation compared to wild type (WT) mice in a laser-induced arteriole and venule model of thrombosis. Furthermore, neutrophil accumulation and elastase activity was enhanced in thrombi of ß2GPI -/- compared with WT mice. The antithrombotic function of ß2GPI is dependent on its fifth domain (domain V); intravenous administration of the ß2GPI domain deletion mutant lacking domain V (human recombinant domain I-IV) had no effect on platelet and fibrin thrombus size in ß2GPI -/- or WT mice. On the contrary, intravenous administration of human recombinant domain V significantly inhibited platelet and fibrin thrombus size in both ß2GPI -/- mice and WT mice. These findings reveal a major role for ß2GPI as a natural anticoagulant and implicate domain V of ß2GPI as a potential antithrombotic therapy.
Assuntos
Síndrome Antifosfolipídica , Trombose , beta 2-Glicoproteína I , Animais , Anticoagulantes , Síndrome Antifosfolipídica/tratamento farmacológico , Síndrome Antifosfolipídica/genética , Fibrinolíticos , Camundongos , Camundongos Knockout , beta 2-Glicoproteína I/fisiologiaRESUMO
Protein disulfide isomerase (PDI) and endoplasmic reticulum protein 57 (ERp57) are emerging as important regulators of thrombus formation. Another thiol isomerase, endoplasmic reticulum protein 5 (ERp5), is involved in platelet activation. We show here the involvement of ERp5 in thrombus formation using the mouse laser-injury model of thrombosis and a specific antibody raised against recombinant ERp5. Anti-ERp5 antibody inhibited ERp5-dependent platelet and endothelial cell disulfide reductase activity in vitro. ERp5 release at the thrombus site was detected after infusion of Alexa Fluor 488-labeled anti-ERp5 antibody at 0.05 µg/g body weight, a dose that does not inhibit thrombus formation. Anti-ERp5 at 3 µg/g body weight inhibited laser-induced thrombus formation in vivo by causing a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (P < .01). ERp5 binds to ß3 integrin with an equilibrium dissociation constant (KD) of 21 µM, measured by surface plasmon resonance. The cysteine residues in the ERp5 active sites are not required for binding to ß3 integrin. These results provide evidence for a novel role of ERp5 in thrombus formation, a function that may be mediated through its association with αIIbß3.
Assuntos
Modelos Animais de Doenças , Integrina beta3/metabolismo , Lasers/efeitos adversos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Trombose/patologia , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Western Blotting , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Fibrina/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação Plaquetária , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Trombose/enzimologia , Trombose/etiologiaRESUMO
Significance: The primary role of platelets is to generate a thrombus by platelet activation. Platelet activation relies on calcium mobilization from the endoplasmic reticulum (ER). ER resident proteins, which are externalized upon platelet activation, are essential for the function of platelet surface receptors and intercellular interactions. Recent Advances: The platelet ER is a conduit for changes in cellular function in response to the extracellular milieu. ER homeostasis is maintained by an appropriate redox balance, regulated calcium stores and normal protein folding. Alterations in ER function and ER stress results in ER proteins externalizing to the cell surface, including members of the protein disulfide isomerase family (PDIs) and chaperones. Critical Issues: The platelet ER is central to platelet function, but our understanding of its regulation is incomplete. Previous studies have focused on the function of PDIs in the extracellular space, and much less on their intracellular role. How platelets maintain ER homeostasis and how they direct ER chaperone proteins to facilitate intercellular signalling is unknown. Future Directions: An understanding of ER functions in the platelet is essential as these may determine critical platelet activities such as secretion and adhesion. Studies are necessary to understand the redox reactions of PDIs in the intracellular versus extracellular space, as these differentially affect platelet function. An unresolved question is how platelet ER proteins control calcium release. Regulation of protein folding in the platelet and downstream pathways of ER stress require further evaluation. Targeting the platelet ER may have therapeutic application in metabolic and neoplastic disease.
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A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.
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Here, we present a series of illustrated capsules from the State of the Art (SOA) speakers at the 2024 International Society on Thrombosis and Haemostasis Congress in Bangkok, Thailand. This year's Congress marks the first time that the International Society on Thrombosis and Haemostasis has held its flagship scientific meeting in Southeast Asia and is the first to be organized by an international Planning Committee. The Bangkok program will feature innovative science and clinical updates from around the world, reflecting the diversity and multidisciplinary growth of our field. In these illustrated SOA capsules, you will find an exploration of novel models of thrombosis and bleeding and biomaterial discoveries that can trigger or block coagulation. Thromboinflammation is now understood to drive many disease states, and the SOA speakers cover cellular and coagulation responses to COVID-19 and other infections. The theme of crosstalk between coagulation and inflammation expands with capsules on protein S signaling, complement, and fibrinolytic inhibitors. Novel agents for hemophilia and thrombosis prevention are introduced. Challenging clinical conditions are also covered, such as inherited platelet disorders and antiphospholipid antibody syndrome. The scientific program in Bangkok will also showcase the work of clinicians and scientists from all parts of the world and chronicle real-world challenges. For example, 2 SOA capsules address the diagnosis and management of von Willebrand disease in low-income settings. Take some time to browse through these short illustrated reviews; we're sure that you'll be entertained, educated, and inspired to further explore the world of thrombosis and hemostasis.
RESUMO
Extracellular protein disulfide isomerases (PDIs), including PDI, endoplasmic reticulum protein 57 (ERp57), ERp72, ERp46, and ERp5, are required for in vivo thrombus formation in mice. Platelets secrete PDIs upon activation, which regulate platelet aggregation. However, platelets secrete only â¼10% of their PDI content extracellularly. The intracellular role of PDIs in platelet function is unknown. Here, we aim to characterize the role of ERp5 (gene Pdia6) using platelet conditional knockout mice, platelet factor 4 (Pf4) Cre+/ERp5floxed (fl)/fl. Pf4Cre+/ERp5fl/fl mice developed mild macrothrombocytopenia. Platelets deficient in ERp5 showed marked dysregulation of their ER, indicated by a twofold upregulation of ER proteins, including PDI, ERp57, ERp72, ERp46, 78 kilodalton glucose-regulated protein (GRP78), and calreticulin. ERp5-deficient platelets showed an enhanced ER stress response to ex vivo and in vivo ER stress inducers, with enhanced phosphorylation of eukaryotic translation initiation factor 2A and inositol-requiring enzyme 1 (IRE1). ERp5 deficiency was associated with increased secretion of PDIs, an enhanced response to thromboxane A2 receptor activation, and increased thrombus formation in vivo. Our results support that ERp5 acts as a negative regulator of ER stress responses in platelets and highlight the importance of a disulfide isomerase in platelet ER homeostasis. The results also indicate a previously unanticipated role of platelet ER stress in platelet secretion and thrombosis. This may have important implications for the therapeutic applications of ER stress inhibitors in thrombosis.
Assuntos
Plaquetas , Trombose , Animais , Camundongos , Plaquetas/metabolismo , Agregação Plaquetária , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Hemostasia , Trombose/metabolismoRESUMO
ß2-Glycoprotein I (ß2GPI) is an evolutionary conserved, abundant circulating protein. Although its function remains uncertain, accumulated evidence points toward interactions with endothelial cells and components of the coagulation system, suggesting a regulatory role in vascular biology. Our group has shown that thioredoxin 1 (TRX-1) generates free thiols in ß2GPI, a process that may have a regulatory role in platelet adhesion. This report extends these studies and shows for the first time evidence of ß2GPI with free thiols in vivo in both multiple human and murine serum samples. To explore how the vascular surface may modulate the redox status of ß2GPI, unstimulated human endothelial cells and EAhy926 cells are shown to be capable of amplifying the effect of free thiol generation within ß2GPI. Multiple oxidoreductase enzymes, such as endoplasmic reticulum protein 46 (ERp 46) and TRX-1 reductase, in addition to protein disulfide isomerase are secreted on the surface of endothelial cells. Furthermore, one or more of these generated free thiols within ß2GPI are also shown to be nitrosylated. Finally, the functional significance of these findings is explored, by showing that free thiol-containing ß2GPI has a powerful effect in protecting endothelial cells and EAhy926 cells from oxidative stress-induced cell death.
Assuntos
Células Endoteliais/metabolismo , Estresse Oxidativo , Compostos de Sulfidrila/metabolismo , beta 2-Glicoproteína I/metabolismo , Adolescente , Adulto , Idoso , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Compostos de Sulfidrila/sangue , Tiorredoxinas/farmacologia , Adulto Jovem , beta 2-Glicoproteína I/sangue , beta 2-Glicoproteína I/genéticaRESUMO
Significance: How mechanical forces and biochemical cues are coupled remains a miracle for many biological processes. Integrins, well-known adhesion receptors, sense changes in mechanical forces and reduction-oxidation reactions (redox) in their environment to mediate their adhesive function. The coupling of mechanical and redox function is a new area of investigation. Disturbance of normal mechanical forces and the redox balance occurs in thromboinflammatory conditions; atherosclerotic plaques create changes to the mechanical forces in the circulation. Diabetes induces redox changes in the circulation by the production of reactive oxygen species and vascular inflammation. Recent Advances: Integrins sense changes in the blood flow shear stress at the level of focal adhesions and respond to flow and traction forces by increased signaling. Talin, the integrin-actin linker, is a traction force sensor and adaptor. Oxidation and reduction of integrin disulfide bonds regulate their adhesion. A conserved disulfide bond in integrin αlpha IIb beta 3 (αIIbß3) is directly reduced by the thiol oxidoreductase endoplasmic reticulum protein 5 (ERp5) under shear stress. Critical Issues: The coordination of mechano-redox events between the extracellular and intracellular compartments is an active area of investigation. Another fundamental issue is to determine the spatiotemporal arrangement of key regulators of integrins' mechanical and redox interactions. How thromboinflammatory conditions lead to mechanoredox uncoupling is relatively unexplored. Future Directions: Integrated approaches, involving disulfide bond biochemistry, microfluidic assays, and dynamic force spectroscopy, will aid in showing that cell adhesion constitutes a crossroad of mechano- and redox biology, within the same molecule, the integrin. Antioxid. Redox Signal. 37, 1072-1093.
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Integrinas , Trombose , Humanos , Integrinas/metabolismo , Tromboinflamação , Inflamação , Talina/metabolismo , Adesão Celular , DissulfetosRESUMO
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.
Assuntos
Púrpura Trombocitopênica Idiopática , Trombocitopenia , Trombose , ChAdOx1 nCoV-19 , Citometria de Fluxo , Heparina/uso terapêutico , Humanos , Imunoglobulinas Intravenosas/efeitos adversos , Fator Plaquetário 4 , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptores Ativados por Proteinase/uso terapêutico , Trombocitopenia/diagnóstico , Trombose/tratamento farmacológicoRESUMO
Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils' importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.
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Angiogenesis is a prerequisite for solid tumor growth, but there is relatively limited data regarding Hodgkin lymphoma. The purpose of this study was to examine the immunohistochemical expression of angiogenic and proliferation markers in Hodgkin biopsies in relation to clinical parameters. Immunostaining was performed on 65 Hodgkin biopsies with vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1alpha), platelet-derived growth factor receptor alpha (PDGFRalpha), Ki-67, and p53. Microvessel density (MVD) was determined by CD31 staining. In all cases, neoplastic cells and reactive background cells were evaluated. The neoplastic population expressed VEGF in 48% of the cases, HIF-1alpha in 54% of the cases, and PDGFRalpha in 95% of the cases. Both Ki-67 and p53 were positive in neoplastic cells in over 60% of the cases. The MVD had a median of 2.6/0.0625mm(2) which was not different from normal lymph nodes. VEGF in the non-neoplastic compartment showed increased staining in Ann Arbor stage I-II versus III-IV. In conclusion, VEGF, HIF-1alpha, and predominantly PDGFRalpha are expressed in neoplastic cells in the majority of Hodgkin lymphomas. As microvessel formation is not increased in Hodgkin, additional functions of these angiogenic molecules should be investigated.
Assuntos
Doença de Hodgkin/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/análise , Fator A de Crescimento do Endotélio Vascular/análise , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Feminino , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/patologia , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Masculino , Microvasos/patologia , Estadiamento de Neoplasias , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Resultado do Tratamento , Proteína Supressora de Tumor p53/análiseRESUMO
Microfluidic devices have become an integral method of cardiovascular research as they enable the study of shear force in biological processes, such as platelet function and thrombus formation. Furthermore, microfluidic chips offer the benefits of ex vivo testing of platelet adhesion using small amounts of blood or purified platelets. Microfluidic chips comprise flow channels of varying dimensions and geometries which are connected to a syringe pump. The pump draws blood or platelet suspensions through the channel(s) allowing for imaging of platelet adhesion and thrombus formation by fluorescence microscopy. The chips can be fabricated from various blood-compatible materials. The current protocol uses commercial plastic or in-house polydimethylsiloxane (PDMS) chips. Commercial biochips offer the advantage of standardization whereas in-house chips offer the advantage of decreased cost and flexibility in design. Microfluidic devices are a powerful tool to study the biorheology of platelets and other cell types with the potential of a diagnostic and monitoring tool for cardiovascular diseases.
RESUMO
Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.
Assuntos
Plaquetas/imunologia , Armadilhas Extracelulares/imunologia , Heparina/efeitos adversos , Neutrófilos/imunologia , Receptores de IgG/genética , Trombocitopenia/imunologia , Trombose/imunologia , Animais , Complexo Antígeno-Anticorpo/biossíntese , Plaquetas/efeitos dos fármacos , Citrulinação , Inibidores Enzimáticos/farmacologia , Armadilhas Extracelulares/química , Armadilhas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica , Histonas/genética , Histonas/imunologia , Humanos , Imunoglobulina G/biossíntese , Camundongos , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/imunologia , Fator Plaquetário 4/genética , Fator Plaquetário 4/imunologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/imunologia , Receptores de IgG/imunologia , Transdução de Sinais , Trombocitopenia/induzido quimicamente , Trombocitopenia/patologia , Trombose/induzido quimicamente , Trombose/patologia , Trombose/prevenção & controleAssuntos
Oxirredutases/metabolismo , Compostos de Sulfidrila/metabolismo , beta 2-Glicoproteína I/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Especificidade por Substrato , Compostos de Sulfidrila/sangue , beta 2-Glicoproteína I/sangueRESUMO
Angiogenesis is implicated in the progression of myelodysplastic syndromes (MDS). Bone marrow microvascular density (MVD), serum angiogenin (ANG) and interleukin 6 (IL-6) were measured in 67 patients with untreated MDS. MVD, ANG and IL-6 were significantly higher in the patient group as a whole when compared to controls (P < 0.01). MVD and ANG were significantly higher in subtypes with a high-risk for leukemic transformation (RAEB, RAEB-t and CMML) than in low-risk subtypes (RA and RARS) (P < 0.01). In the MDS group, a positive correlation was found between ANG and IL-6 (P < 0.001) and also between MVD and IL-6 (P < 0.05). Using multivariate analysis, only IL-6 displayed independent prognostic value and was inversely related to MDS survival.
Assuntos
Medula Óssea/irrigação sanguínea , Interleucina-6/sangue , Síndromes Mielodisplásicas/sangue , Neovascularização Patológica , Ribonuclease Pancreático/sangue , Idoso , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/mortalidade , Prognóstico , Risco , Análise de SobrevidaRESUMO
OBJECTIVE: To summarize current knowledge regarding the diagnosis and management of gastrointestinal vasculitis. METHODS: Three cases of gastrointestinal vasculitis with acute abdominal ischemia as their first manifestation are presented. Underlying diseases were microscopic polyangiitis, systemic lupus erythematosus (SLE), and polyarteritis nodosa (PAN). Relevant English-language articles collected from the PubMed database were reviewed. RESULTS: Among the angiitides, PAN, SLE, and Henoch-Schönlein are those most commonly accompanied by gastrointestinal complications. Intestinal vasculitis usually occurs when there is evidence of generalized disease activity. Abdominal computerized tomography is a valuable tool for diagnosing intestinal ischemia and suspected vasculitis. CONCLUSIONS: In young patients presenting with intestinal ischemia, it is essential to assess the possibility of an underlying systemic disease. With prompt initiation of immunosuppressive treatment, surgery may be avoided. Prognosis is improved when there is minimal delay in surgical intervention.
Assuntos
Intestinos/irrigação sanguínea , Isquemia/diagnóstico , Vasculite/diagnóstico , Abdome Agudo/diagnóstico , Abdome Agudo/etiologia , Dor Abdominal/diagnóstico , Dor Abdominal/etiologia , Adulto , Diagnóstico Diferencial , Evolução Fatal , Feminino , Seguimentos , Humanos , Laparotomia , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Pessoa de Meia-Idade , Poliarterite Nodosa/diagnóstico , Medição de RiscoRESUMO
Nearly all hematologic malignancies can occasionally present with or develop pleural effusions during the clinical course of disease. Among the most common disorders are Hodgkin and non-Hodgkin lymphomas, with a frequency of 20 to 30%, especially if mediastinal involvement is present. Acute and chronic leukemias, myelodysplastic syndromes, are rarely accompanied by pleural involvement. Furthermore, 10 to 30% of patients receiving bone marrow transplantation develop pleural effusions. In cases of hematologic pleural effusions, drug toxicity, underlying infectious, secondary malignant or rarely autoimmune causes should be carefully sought. In most cases, the pleural fluid responds to treatment of the primary disease, whereas resistant or relapsing cases may necessitate pleurodesis.