RESUMEN
PURPOSE: Growing evidence suggests different systemic exposure of anti-vascular endothelial growth factor (anti-VEGF) agents with repeated intravitreal application. Since the penetration of anti-VEGF agents through vascular barrier was reported, the interaction of anti-VEGF with nonresident platelets has become a topic of interest. The purpose of this study was to evaluate, with the help of visualization techniques, whether platelets take up the anti-VEGF agents ranibizumab, aflibercept, and bevacizumab. METHODS: The uptake of anti-VEGF agents with or without VEGF treatment was investigated using immunofluorescence and immunogold staining in human platelets. The role of actin filaments and clathrin-coated vesicles in the transport of ranibizumab, aflibercept, and bevacizumab was evaluated by two pharmacologic inhibitors: staurosporine (protein kinase C inhibitor) and cytochalasin D. RESULTS: All three anti-VEGF agents were taken up by platelets and colocalized with VEGF. Ranibizumab and aflibercept were mainly detected in alpha-granules; however, bevacizumab was equally localized in alpha-granules and in platelet vesicles. Both staurosporine and cytochalasin D completely inhibited the uptake of aflibercept into platelets. Both pharmacological inhibitors also decreased the transport of ranibizumab and bevacizumab into platelets. Bevacizumab was significantly more frequently colocalized within clathrin-coated vesicles than ranibizumab and aflibercept. CONCLUSION: All three anti-VEGF agents are taken up by platelets and internalized in alpha-granules, which may result in a higher local exposure of anti-VEGF after the activation of platelets, potentially contributing to arterial thromboembolic events. Clathrin-coated vesicles seem to be more prominent in the transport of bevacizumab than ranibizumab and aflibercept. Nevertheless, whether the different localization and transport of bevacizumab are truly related to specific differences of receptor-mediated endocytosis has to be revealed by further research.
RESUMEN
Essentials Phosphoinositide 3-kinase and MAPK pathways crosstalk via PDK1. PDK1 is required for adenosine diphosphate-induced platelet activation and thromboxane generation. PDK1 regulates RAF proto-oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway. Genetic ablation of PDK1 protects against platelet-dependent thrombosis in vivo. SUMMARY: Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3ß. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1-/- mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.
Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Plaquetas/enzimología , Proteínas Quinasas Activadas por Mitógenos/sangre , Agregación Plaquetaria/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/sangre , Embolia Pulmonar/enzimología , Trombosis/enzimología , Tromboxanos/sangre , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/antagonistas & inhibidores , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/sangre , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/deficiencia , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Animales , Plaquetas/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Ratones Noqueados , Fosforilación , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proto-Oncogenes Mas , Embolia Pulmonar/sangre , Embolia Pulmonar/genética , Embolia Pulmonar/prevención & control , Pirimidinas/farmacología , Transducción de Señal , Tiofenos/farmacología , Trombosis/sangre , Trombosis/genética , Trombosis/prevención & controlRESUMEN
BACKGROUND: Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood. OBJECTIVES: Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far. METHODS AND RESULTS: Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b. CONCLUSIONS: The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.
Asunto(s)
Plaquetas/enzimología , Traumatismos de las Arterias Carótidas/enzimología , Gránulos Citoplasmáticos/enzimología , Proteínas Inmediatas-Precoces/sangre , Activación Plaquetaria , Proteínas Serina-Treonina Quinasas/sangre , Vesículas Secretoras/enzimología , Trombosis/enzimología , Adenosina Trifosfato/sangre , Adenosina Trifosfato/metabolismo , Animales , Plaquetas/metabolismo , Plaquetas/ultraestructura , Traumatismos de las Arterias Carótidas/sangre , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Células Cultivadas , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Modelos Animales de Enfermedad , Femenino , Genotipo , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Masculino , Megacariocitos/enzimología , Megacariocitos/metabolismo , Ratones Noqueados , Fenotipo , Agregación Plaquetaria , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructura , Serotonina/sangre , Serotonina/metabolismo , Transducción de Señal , Tetraspanina 30/sangre , Tetraspanina 30/metabolismo , Trombosis/sangre , Trombosis/genética , Trombosis/patología , Factores de Tiempo , Transfección , Regulación hacia Arriba , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
BACKGROUND: Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets and induce filopodia and lamellipodia formation and actin polymerization to strongly increase the platelet surface upon activation. Moreover, they are important for platelet secretion, integrin activation and arterial thrombus formation. OBJECTIVES: Rho GTPases are regulated by GTPase-activating proteins (GAPs) that stimulate their GTPase activity to terminate Rho signaling. The regulation of Rho GTPase activity in platelets is not well defined. Recently, we identified oligophrenin1 (OPHN1), a RhoGAP in platelets that exhibits strong GTPase-stimulating activity towards RhoA, Cdc42 and Rac1. RESULTS: In the present study we show for the first time, that deficiency of OPHN1 led to abnormal Rho activation and increased platelet cytoskeletal reorganization, including cell adhesion and lamellipodia formation on fibrinogen. Furthermore, platelets from ophn1(-/-) mice showed enhanced susceptibility to platelet activation with alterations in actin distribution and early release of granules. Platelet activation was enhanced following GPVI and PAR4 stimulation. This translated into elevated platelet thrombus formation and promoted arterial thrombosis under low shear conditions with altered hemostasis, as detected by tail bleeding time. CONCLUSIONS: The results of the present study identified OPHN1 as an important regulator of platelet cytoskeletal reorganization and demonstrate that abnormal regulation of Rho proteins leads to increased platelet adhesion and thrombus formation under low shear conditions in vitro and in vivo, suggesting a prothrombotic phenotype of mice critical for acute thrombotic occlusions.
Asunto(s)
Coagulación Sanguínea , Plaquetas/enzimología , Proteínas del Citoesqueleto/deficiencia , Proteínas Activadoras de GTPasa/deficiencia , Proteínas Nucleares/deficiencia , Trombosis/enzimología , Proteínas de Unión al GTP rho/sangre , Animales , Proteínas del Citoesqueleto/genética , Citoesqueleto/enzimología , Modelos Animales de Enfermedad , Activación Enzimática , Femenino , Proteínas Activadoras de GTPasa/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/sangre , Proteínas Nucleares/genética , Activación Plaquetaria , Seudópodos/enzimología , Transducción de Señal , Trombosis/sangre , Trombosis/genética , Factores de Tiempo , Proteína de Unión al GTP cdc42/sangre , Proteína de Unión al GTP rac1/sangre , Proteína de Unión al GTP rhoARESUMEN
Platelet secretion and aggregation as well as thrombus formation of blood platelets critically depend on increase of cytosolic Ca2+ concentration ([Ca2+]i) mainly resulting from intracellular Ca2+ release followed by store operated Ca2+ entry (SOCE) through Ca2+ release activated channels (CRAC). SOCE is in part accomplished by the pore forming unit Orai and its regulator stromal interaction molecule (STIM). Orai1 and STIM1 transcription is stimulated by NF-κB (nuclear factor kappa B). Serum- and glucocorticoid-inducible kinase 1 (SGK1) up-regulates NF-κB-activity in megakaryocytes and thus Orai1-expression and SOCE in platelets. SGK1 is thus a powerful regulator of platelet Ca2+-signalling and thrombus formation and presumably participates in the regulation of platelet activation by a variety of hormones as well as clinical conditions (e.g. type 2 diabetes or metabolic syndrome) associated with platelet hyperaggregability and increased risk of thromboocclusive events. SOCE in platelets is further regulated by scaffolding protein Homer and chaperone protein cyclophilin A (CyPA). Additional potential regulators of Orai1/STIM1 and thus SOCE in platelets include AMP activated kinase (AMPK), protein kinase A (PKA), reactive oxygen species, lipid rafts, pH and mitochondrial Ca2+ buffering. Future studies are required defining the significance of those mechanisms for platelet Orai1 abundance and function, for SOCE into platelets and for platelet function in cardiovascular diseases.