RESUMEN
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.
Asunto(s)
Plaquetas , Quimiocina CXCL12 , Megacariocitos , Proteínas Proto-Oncogénicas c-pim-1 , Receptores CXCR4 , Transducción de Señal , Receptores CXCR4/metabolismo , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Megacariocitos/metabolismo , Megacariocitos/efectos de los fármacos , Megacariocitos/citología , Humanos , Transducción de Señal/efectos de los fármacos , Animales , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Quimiocina CXCL12/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Movimiento Celular/efectos de los fármacos , Línea CelularRESUMEN
Cucurbitacins are dietary compounds that have been shown to elicit a range of anti-tumour, anti-inflammatory and anti-atherosclerotic activities. Originally identified as signal transducer and activator of transcription, STAT, inhibitors, a variety of mechanisms of action have since been described, including dysregulation of the actin cytoskeleton and disruption of integrin function. Integrin outside-in signalling and cytoskeletal rearrangements are critical for the propagation of stable thrombus formation and clot retraction following platelet adhesion at the site of vessel damage. The effects of cucurbitacins on platelet function and thrombus formation are unknown. We report for the first time anti-platelet and anti-thrombotic effects of cucurbitacins B, E and I in human platelets. Treatment of platelets with cucurbitacins resulted in attenuation of platelet aggregation, secretion and fibrinogen binding following stimulation by platelet agonists. Cucurbitacins were also found to potently inhibit other integrin- and cytoskeleton-mediated events, including adhesion, spreading and clot retraction. Further investigation of cytoskeletal dynamics found treatment with cucurbitacins altered cofilin phosphorylation, enhanced activation and increased F actin polymerisation and microtubule assembly. Disruption to cytoskeletal dynamics has been previously shown to impair integrin activation, platelet spreading and clot retraction. Anti-platelet properties of cucurbitacins were found to extend to a disruption of stable thrombus formation, with an increase in thrombi instability and de-aggregation under flow. Our research identifies novel, anti-platelet and anti-thrombotic actions of cucurbitacins that appear to be linked to dysregulation of cytoskeletal dynamics and integrin function.
Asunto(s)
Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Trombosis , Plaquetas/metabolismo , Cucurbitacinas/metabolismo , Cucurbitacinas/farmacología , Citoesqueleto/metabolismo , Humanos , Microtúbulos/metabolismo , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombosis/metabolismoRESUMEN
Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.
Asunto(s)
Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria , Animales , Plaquetas , Humanos , Lectinas Tipo C , Ratones , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton tyrosine kinase used in the treatment of B-cell malignancies. They bind irreversibly to cysteine 481 of Bruton tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of downstream substrates including phospholipase C-γ2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton tyrosine kinase activity, as shown by loss of phosphorylation at tyrosine 223 and phospholipase C-γ2, delay but do not block aggregation in response to a maximally-effective concentration of collagen-related peptide or collagen. In contrast, 10- to 20-fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation in response to glycoprotein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, showed a reduction of platelet aggregation in response to collagen-related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton tyrosine kinase blockade. Unexpectedly, low concentrations of ibrutinib inhibited aggregation in response to collagen-related peptide in patients deficient in Bruton tyrosine kinase. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavorable pharmacodynamics.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia/tratamiento farmacológico , Plaquetas/efectos de los fármacos , Enfermedades Genéticas Ligadas al Cromosoma X/tratamiento farmacológico , Glicoproteínas de Membrana Plaquetaria/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa/genética , Agammaglobulinemia Tirosina Quinasa/metabolismo , Agammaglobulinemia/sangre , Agammaglobulinemia/genética , Benzamidas/administración & dosificación , Benzamidas/metabolismo , Plaquetas/metabolismo , Proteínas Portadoras/administración & dosificación , Enfermedades Genéticas Ligadas al Cromosoma X/sangre , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Mutación , Péptidos/administración & dosificación , Piperidinas , Activación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria , Glicoproteínas de Membrana Plaquetaria/agonistas , Inhibidores de Proteínas Quinasas/metabolismo , Pirazinas/administración & dosificación , Pirazinas/metabolismo , Pirazoles/administración & dosificación , Pirazoles/metabolismo , Pirimidinas/administración & dosificación , Pirimidinas/metabolismoRESUMEN
The Bruton tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side effects of ibrutinib. The second-generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with non-Hodgkin lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide, and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, whereas size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited Src family kinases, which have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of von Willebrand factor (VWF) and factor VIII (FVIII) ex vivo by addition of intermediate purity FVIII (Haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma VWF and FVIII.