RESUMO
Proteoglycans are differentially expressed in different atherosclerotic plaque phenotypes, with biglycan and decorin characteristic of ruptured plaques and versican and hyaluronan more prominent in eroded plaques. Following plaque disruption, the exposure of extracellular matrix (ECM) proteins triggers platelet adhesion and thrombus formation. In this study, the impact of differential plaque composition on platelet function and thrombus formation was investigated. Platelet adhesion, activation and thrombus formation under different shear stress conditions were assessed in response to individual proteoglycans and composites representing different plaque phenotypes. The results demonstrated that all the proteoglycans tested mediated platelet adhesion but not platelet activation, and the extent of adhesion observed was significantly lower than that observed with type I and type III collagens. Thrombus formation upon the rupture and erosion ECM composites was significantly reduced (p < 0.05) compared to relevant collagen alone, indicating that proteoglycans negatively regulate platelet collagen responses. This was supported by results demonstrating that the addition of soluble biglycan or decorin to whole blood markedly reduced thrombus formation on type I collagen (p < 0.05). Interestingly, thrombus formation upon the erosion composite displayed aspirin sensitivity, whereas the rupture composite was intensive to aspirin, having implications for current antiplatelet therapy regimes. In conclusion, differential platelet responses and antiplatelet efficacy are observed on ECM composites phenotypic of plaque rupture and erosion. Proteoglycans inhibit thrombus formation and may offer a novel plaque-specific approach to limit arterial thrombosis.
Assuntos
Aterosclerose , Placa Aterosclerótica , Trombose , Humanos , Biglicano , Decorina , Proteínas da Matriz Extracelular , Aspirina , Colágeno Tipo IRESUMO
A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.
Assuntos
Plaquetas , Quimiocina CXCL12 , Megacariócitos , Proteínas Proto-Oncogênicas c-pim-1 , Receptores CXCR4 , Transdução de Sinais , Receptores CXCR4/metabolismo , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Megacariócitos/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/citologia , Humanos , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Quimiocina CXCL12/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Movimento Celular/efeitos dos fármacos , Linhagem CelularRESUMO
Connexins oligomerise to form hexameric hemichannels in the plasma membrane that can further dock together on adjacent cells to form gap junctions and facilitate intercellular trafficking of molecules. In this study, we report the expression and function of an orphan connexin, connexin-62 (Cx62), in human and mouse (Cx57, mouse homolog) platelets. A novel mimetic peptide (62Gap27) was developed to target the second extracellular loop of Cx62, and 3-dimensional structural models predicted its interference with gap junction and hemichannel function. The ability of 62Gap27 to regulate both gap junction and hemichannel-mediated intercellular communication was observed using fluorescence recovery after photobleaching analysis and flow cytometry. Cx62 inhibition by 62Gap27 suppressed a range of agonist-stimulated platelet functions and impaired thrombosis and hemostasis. This was associated with elevated protein kinase A-dependent signaling in a cyclic adenosine monophosphate-independent manner and was not observed in Cx57-deficient mouse platelets (in which the selectivity of 62Gap27 for this connexin was also confirmed). Notably, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Together, our data reveal a fundamental role for a hitherto uncharacterized connexin in regulating the function of circulating cells.
Assuntos
Plaquetas/metabolismo , Conexinas/fisiologia , Animais , Comunicação Celular/fisiologia , Linhagem Celular , Conexinas/sangue , Conexinas/química , Conexinas/deficiência , Conexinas/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Junções Comunicantes/fisiologia , Hemostasia/fisiologia , Humanos , Integrinas/sangue , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Adesividade Plaquetária , Agregação Plaquetária , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade , Trombose/sangueRESUMO
Multiple myeloma (MM) and its precursor states, smoldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS) are associated with increased incidence of thrombosis, however the cause of this is unknown. Lenalidomide treatment of MM substantially improves patient survival, although significantly increases thrombotic risk by an unknown mechanism. This pilot study aimed to establish the impact of MM and its treatment with Lenalidomide on platelet function. We analyzed platelet function in MGUS, SM and MM compared to healthy controls. We report an increase in platelet reactivity in MGUS, SM, and MM where increases in fibrinogen binding, P-selectin exposure, altered receptor expression, elevated levels of aggregation and enhanced sensitivity to agonist stimulation were observed. We also demonstrate an increase in patient platelet reactivity post Lenalidomide treatment compared to pre-treatment. We show Lenalidomide treatment of platelets ex vivo increased reactivity that was associated with formation of larger thrombi at arterial shear rates but not venous shear rates. This study demonstrates a clear increase in platelet reactivity and prothrombotic potential in patients with MGUS, SM and MM which is elevated further upon treatment with Lenalidomide. Our observations suggest that more detailed studies are warranted to determine mechanisms of thrombotic complications to enable the development of new preventative strategies that specifically target platelets.
What is the context?Multiple myeloma is associated with increased risk of thrombosis, although the potential role of platelets in this has not been evaluated.What is new?We show in this pilot study that multiple myeloma and its precursor states of smoldering myeloma and monoclonal gammopathy of undetermined significance are associated with increased levels of platelet responses. This is further exacerbated by treatment with the immunomodulatory drug lenalidomide.What is the impact?This study suggests that more detailed studies are warranted to explore the mechanisms that cause these effects in a larger population of patients, since this may reveal new approaches to prevent myeloma-associated thrombotic complications.
Assuntos
Gamopatia Monoclonal de Significância Indeterminada , Mieloma Múltiplo , Trombose , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/complicações , Lenalidomida/farmacologia , Lenalidomida/uso terapêutico , Projetos Piloto , Trombose/complicações , Gamopatia Monoclonal de Significância Indeterminada/complicaçõesRESUMO
Pim Kinases; Pim-1, Pim-2, and Pim-3, are a family of constitutively active serine/threonine kinases, widely associated with cell survival, proliferation, and migration. Historically considered to be functionally redundant, independent roles for the individual isoforms have been described. Whilst most established for their role in cancer progression, there is increasing evidence for wider pathological roles of Pim kinases within the context of cardiovascular disease, including inflammation, thrombosis, and cardiac injury. The Pim kinase isoforms have widespread expression in cardiovascular tissues, including the heart, coronary artery, aorta, and blood, and have been demonstrated to be upregulated in several co-morbidities/risk factors for cardiovascular disease. Pim kinase inhibition may thus be a desirable therapeutic for a multi-targeted approach to treat cardiovascular disease and some of the associated risk factors. In this review, we discuss what is known about Pim kinase expression and activity in cells of the cardiovascular system, identify areas where the role of Pim kinase has yet to be fully explored and characterised and review the suitability of targeting Pim kinase for the prevention and treatment of cardiovascular events in high-risk individuals.
Assuntos
Doenças Cardiovasculares , Humanos , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-pim-1 , Proteínas Serina-Treonina Quinases/metabolismo , Isoformas de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The vascular extracellular matrix (ECM) produced by endothelial and smooth muscle cells is composed of collagens and glycoproteins and plays an integral role in regulating the structure and function of the vascular wall. Alteration in the expression of these proteins is associated with endothelial dysfunction and has been implicated in the development and progression of atherosclerosis. The ECM composition of atherosclerotic plaques varies depending on plaque phenotype and vulnerability, with distinct differences observed between ruptured and erodes plaques. Moreover, the thrombi on the exposed ECM are diverse in structure and composition, suggesting that the best antithrombotic approach may differ depending on plaque phenotype. This review provides a comprehensive overview of the role of proteoglycans in atherogenesis and thrombosis. It discusses the differential expression of the proteoglycans in different plaque phenotypes and the potential impact on platelet function and thrombosis. Finally, the review highlights the importance of this concept in developing a targeted approach to antithrombotic treatments to improve clinical outcomes in cardiovascular disease.
Assuntos
Aterosclerose , Placa Aterosclerótica , Trombose , Humanos , Proteoglicanas/metabolismo , Fibrinolíticos/uso terapêutico , Placa Aterosclerótica/metabolismoRESUMO
Zinc (Zn2+) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn2+]o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn2+ influx are unknown. Fluctuations in intracellular zinc ([Zn2+]i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn2+]o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn2+ influx. [Zn2+]o stimulation resulted in the phosphorylation of PKC substates, MLC, and ß3 integrin. Platelet activation via GPVI or Zn2+ resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn2+]i that were sensitive to the inhibition of Orai1, ZIP7, or IP3R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn2+]o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn2+ influx. Increases in [Zn2+]i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn2+ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn2+ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis.
Assuntos
Proteínas de Transporte de Cátions , Proteínas de Transporte de Cátions/metabolismo , Zinco/farmacologia , Zinco/metabolismo , Retículo Endoplasmático/metabolismo , Ativação Plaquetária , Plaquetas/metabolismo , Cátions/metabolismo , Cálcio/metabolismoRESUMO
Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX-1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding.
Assuntos
Receptores de Tromboxano A2 e Prostaglandina H2 , Trombose , Plaquetas , Humanos , Agregação Plaquetária , Proteínas Proto-Oncogênicas c-pim-1/genética , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Trombose/tratamento farmacológicoRESUMO
The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction. We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro. Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azetidinas/uso terapêutico , Plaquetas/efeitos dos fármacos , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azetidinas/farmacologia , Humanos , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologiaRESUMO
OBJECTIVE: Platelets have been found to express intracellular nuclear receptors including the retinoid X receptors (RXRα and RXRß). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2; however, the effects on responses to other platelet agonists and the underlying mechanism have not been fully characterized. APPROACH AND RESULTS: The effect of 9-cis-retinoic acid, docosahexaenoic acid and methoprene acid on collagen receptor (glycoprotein VI [GPVI]) agonists and thrombin-stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilization, integrin αIIbß3 outside-in signaling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses after stimulation by GPVI agonists or thrombin and inhibition of integrin αIIbß3 outside-in signaling. Treatment with 9-cis-retinoic acid caused inhibition of thrombus formation in vitro and an impairment of thrombosis and hemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and nuclear factor κ-light-chain-enhancer of activated B cell activity. CONCLUSIONS: This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of protein kinase A, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo after treatment with RXR ligands.
Assuntos
Plaquetas/efeitos dos fármacos , Fibrinolíticos/farmacologia , Hemostasia/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores X de Retinoides/agonistas , Trombose/prevenção & controle , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Ligantes , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , NF-kappa B/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores X de Retinoides/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Trombina/farmacologia , Trombose/sangue , Fatores de TempoRESUMO
OBJECTIVES: The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. APPROACH AND RESULTS: We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbß3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. CONCLUSIONS: We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbß3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo.
Assuntos
Benzoatos/farmacologia , Benzilaminas/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Isoxazóis/farmacologia , Receptores X do Fígado/agonistas , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Ciclofilinas/sangue , Relação Dose-Resposta a Droga , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Ligantes , Receptores X do Fígado/sangue , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Selectina-P/sangue , Fosfatidilserinas/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Espécies Reativas de Oxigênio/sangue , Receptores Citoplasmáticos e Nucleares/sangueRESUMO
The Eph kinases, EphA4 and EphB1, and their ligand, ephrinB1, have been previously reported to be present in platelets where they contribute to thrombus stability. Although thrombus formation allows for Eph-ephrin engagement and bidirectional signaling, the importance specifically of Eph kinase or ephrin signaling in regulating platelet function remained unidentified. In the present study, a genetic approach was used in mice to establish the contribution of signaling orchestrated by the cytoplasmic domain of EphB2 (a newly discovered Eph kinase in platelets) in platelet activation and thrombus formation. We conclude that EphB2 signaling is involved in the regulation of thrombus formation and clot retraction. Furthermore, the cytoplasmic tail of this Eph kinase regulates initial platelet activation in a contact-independent manner in the absence of Eph-ephrin ligation between platelets. Together, these data demonstrate that EphB2 signaling not only modulates platelet function within a thrombus but is also involved in the regulation of the function of isolated platelets in a contact-independent manner.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/enzimologia , Ativação Plaquetária/fisiologia , Receptor EphB2/metabolismo , Transdução de Sinais/fisiologia , Animais , Plaquetas/citologia , Camundongos , Camundongos Transgênicos , Receptor EphB2/genéticaRESUMO
OBJECTIVE: Although initially seemingly paradoxical because of the lack of nucleus, platelets possess many transcription factors that regulate their function through DNA-independent mechanisms. These include the farnesoid X receptor (FXR), a member of the superfamily of ligand-activated transcription factors, that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6α-ethyl-chenodeoxycholic acid, modulate platelet activation nongenomically. APPROACH AND RESULTS: FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization, secretion, fibrinogen binding, and aggregation. Exposure to FXR ligands also reduced integrin αIIbß3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cyclic guanosine monophosphate levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the nongenomic actions of these ligands to the FXR. CONCLUSIONS: This study provides support for the ability of FXR ligands to modulate platelet activation. The atheroprotective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for the prevention of atherothrombotic disease.
Assuntos
Plaquetas/efeitos dos fármacos , Ácido Quenodesoxicólico/análogos & derivados , Hemostasia/efeitos dos fármacos , Isoxazóis/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Trombose/prevenção & controle , Animais , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ácido Quenodesoxicólico/farmacologia , GMP Cíclico/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Genótipo , Humanos , Ligantes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores Citoplasmáticos e Nucleares/sangue , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Trombose/sangue , Fatores de TempoRESUMO
OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbß3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbß3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.
Assuntos
Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Colágeno/metabolismo , Hemorragia/induzido quimicamente , Hemostasia/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Inibidores de Proteínas Quinases/toxicidade , Pirazóis/toxicidade , Pirimidinas/toxicidade , Adenina/análogos & derivados , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Tirosina Quinase da Agamaglobulinemia , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Hemorragia/sangue , Humanos , Piperidinas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/sangue , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Fatores de Risco , Fatores de TempoRESUMO
As part of the Biomedical Sciences undergraduate degree course students are required to apply biological principles to the interpretation of clinical case studies and the diagnosis of patients. Case study-based learning, i.e., application of knowledge to patient diagnosis, is new to most students as case studies do not form part of non-applied A level courses in biological sciences. This approach is an example of Problem Based Learning (PBL) which has been shown to support higher levels of student learning, encouraging critical thinking and analysis. PBL approaches have also been shown to increase academic satisfaction and student engagement. In recent years we have observed a downwards trend in student engagement and historically student performance in applied case study-based assessments to be lower than that observed for assessments based on detailing fundamental biological principles. We hypothesised that PBL teaching delivery would support students in preparing for case study-based assessments, helping them to demonstrate their critical evaluation and problem-solving skills, and hence, improve student performance. We also hypothesised that the student learning experience would be enhanced by a PBL teaching delivery approach which would improve overall engagement. We therefore redesigned a second year Biomedical Sciences degree haematology and clinical biochemistry unit: "Blood Science," with a stronger focus on PBL, including case study focussed activities throughout the unit. We subsequently analysed whether this PBL-focussed unit design improved student experience and feedback, student engagement and student confidence for biomedical science undergraduate students. We present here, our teaching strategy and the impact our changes had on student feedback for the 21/22 and 22/23 academic years. Our findings demonstrate that case study-based activities and tutorial PBL exercises, when incorporated into the curriculum design, can improve student experience in the Biomedical Sciences and other biological science undergraduate degree courses.
Assuntos
Currículo , Aprendizagem Baseada em Problemas , Humanos , EstudantesRESUMO
BACKGROUND: The response of platelets to activating stimuli and pharmaceutical agents varies greatly within the normal population. Current platelet function tests are used to measure end-point levels of platelet activation without taking the speed at which platelets activate into account, potentially missing vital metrics to characterize platelet reactivity. OBJECTIVES: To identify variability, to agonists and among individuals, in platelet activation kinetics and assess the impact of this on thrombus formation. METHODS: We have developed a bespoke real-time flow cytometry assay and analysis package to measure the rate of platelet activation over time using 2 parameters of platelet activation, fibrinogen binding and P-selectin exposure. RESULTS: The rate of platelet activation varied considerably within the normal population but did not correlate with maximal platelet activation, demonstrating that platelet activation rate is a separate and novel metric to describe platelet reactivity. The relative rate of platelet response between agonists was strongly correlated, suggesting that a central control mechanism regulates the rate of platelet response to all agonists. CONCLUSION: For the first time, we have shown that platelet response rate corresponds to thrombus size and structure, wherein faster responders form larger, more densely packed thrombi at arterial, but crucially not venous, shear. We have demonstrated that the rate of platelet activation is an important metric in stratifying individual platelet responses and will provide a novel focus for the design and development of antiplatelet therapy, targeting high-shear thrombosis without exacerbating bleeding at low shear.
Assuntos
Ativação Plaquetária , Trombose , Humanos , Trombose/metabolismo , Plaquetas/metabolismo , Testes de Função Plaquetária , Artérias , Agregação PlaquetáriaRESUMO
Background: Factor XIII (FXIII) is an important proenzyme in the hemostatic system. The plasma-derived enzyme activated FXIII cross-links fibrin fibers within thrombi to increase their mechanical strength and cross-links fibrin to fibrinolytic inhibitors, specifically α2-antiplasmin, to increase resistance to fibrinolysis. We have previously shown that cellular FXIII (factor XIII-A [FXIII-A]), which is abundant in the platelet cytoplasm, is externalized onto the activated membrane and cross-links extracellular substrates. The contribution of cellular FXIII-A to platelet activation and platelet function has not been extensively studied. Objectives: This study aims to identify the role of platelet FXIII-A in platelet function. Methods: We used normal healthy platelets with a cell permeable FXIII inhibitor and platelets from FXIII-deficient patients as a FXIII-free platelet model in a range of platelet function and clotting tests. Results: Our data demonstrate that platelet FXIII-A enhances fibrinogen binding to the platelet surface upon agonist stimulation and improves the binding of platelets to fibrinogen and aggregation under flow in a whole-blood thrombus formation assay. In the absence of FXIII-A, platelets show reduced sensitivity to agonist stimulation, including decreased P-selectin exposure and fibrinogen binding. We show that FXIII-A is involved in platelet spreading where a lack of FXIII-A reduces the ability of platelets to fully spread on fibrinogen and collagen. Our data demonstrate that platelet FXIII-A is important for clot retraction where clots formed in its absence retracted to a lesser extent. Conclusion: Overall, this study shows that platelet FXIII-A functions during thrombus formation by aiding platelet activation and thrombus retraction in addition to its antifibrinolytic roles.
RESUMO
Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca(2+). Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCß but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCε but not PKC. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca(2+). This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca(2+) concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.
Assuntos
Difosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Cálcio/metabolismo , Proteína Quinase C/antagonistas & inibidores , Animais , Anticoagulantes/farmacologia , Heterozigoto , Humanos , Indóis/farmacologia , Camundongos , Ativação Plaquetária , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Isoformas de Proteínas , Proteína Quinase C/metabolismoRESUMO
Platelets are well known for their roles in hemostasis and thrombosis, and are increasingly recognized for their abilities to interact with white blood cells during inflammatory diseases, via secreted soluble factors as well as cell-cell contact. This interaction has been investigated in animal models and patient samples and has shown to be implicated in patient outcomes in several diseases. Platelet-leukocyte co-cultures are widely used to study platelet-leukocyte interactions ex vivo. However, there is a paucity with regard to the systematic characterization of cell activation and functional behaviors of platelets and leukocytes in these co-cultures. Hence we aimed to characterize a model of platelet-leukocyte co-culture ex vivo. Human peripheral blood mononuclear cell (PBMC) and platelets were isolated and co-cultured for 5 days at 37 °C in the presence or absence of anti-CD3/CD28 antibodies or PHA. We evaluated PF-4 secretion and p-selectin expression in platelets as markers of platelet activation. Lymphocyte activation was assessed by cell proliferation and cell population phenotyping, in addition to platelet-lymphocyte aggregation. Platelet secretion and p-selectin expression is maintained throughout the co-culture, indicating that platelets were viable and reactive over the 5 days. Similarly PBMCs were viable and maintained proliferative capacity. Finally, dynamic heterotypic conjugation between platelets and T lymphocytes was also observed throughout co-culture (with a peak at days 3 and 4) upon T lymphocyte activation. In conclusion, this in vitro model can successfully mimic the in vivo interaction between platelets and T lymphocytes, and can be used to confirm and/or support in vivo results.
RESUMO
We describe the use of HTML5P (H5P) content collaboration framework to deliver an interactive, online alternative to an assessed laboratory practical on the Biomedical Cell Biology unit at the Manchester Metropolitan University, U.K. H5P is free, open-source technology to deliver bespoke interactive, self-paced online sessions. To determine if the use of H5P affected learning and student attainment, we compared the student grades among three cohorts: the 18/19 cohort who had 'wet' laboratory classes, the 19/20 cohort who had 'wet' laboratory classes with additional video support and the 20/21 cohort who had the H5P alternative. Our analysis shows that students using the H5P were not at a disadvantage to students who had 'wet' laboratory classes with regard to assessment outcomes. Student feedback, mean grade attained and an upward trend in the number of students achieving first-class marks (≥70%), indicate H5P may enhance students' learning experience and be a valuable learning source augmenting traditional practical classes in the future.