Platelets play a critical role in hemostasis but, more recently, it was demonstrated that they also modulate the inflammatory response by establishing direct and indirect interactions with immune cells. Although platelets are anucleated cells, they contain functional transcription factors (TFs) that modulate their activation via non-transcriptional functions. In particular, it has been shown that platelets contain NF-κB, a TF that controls important functions of immune cells during the inflammatory process, and that NF-κB regulates platelet activation. We investigated the possibility that other TFs different from NF-κB might play regulatory roles during platelet activation. We found that platelets contain another TF typically expressed by immune cells: nuclear factor of activated T cells (NFAT). In particular, we found that upon PAR4 ligation, the calcineurin/NFATc2 pathway is activated and regulates platelet functions. This pathway is activated both in murine and human platelets, and its inhibition results in enhancement of platelet aggregation, integrin activation, granules release and spreading on fibrinogen. By using murine in vivo models, we demonstrated that NFATc2 activation in platelets modulates hemostasis and thrombosis. Moreover, platelet NFATc2 activation regulates the interaction between platelets and neutrophils and impacts the severity of bacterial sepsis development. Our in vitro experiments support the capacity of NFATc2 to act downstream of PAR4 via a mechanism that involves both the ADP receptor and the outside-in integrin α2bβ3 pathway. Finally, we found that inhibition of the calcineurin/NFATc2 pathway partially rescues platelet activation defects in two rare human platelet pathologies, the Hermansky-Pudlak syndrome and the Glanzmann thrombasthenia. Our data suggest that the calcineurin/NFATc2 pathway can be targeted to develop innovative therapeutic approaches to treat platelet defects with poor prognosis. Our work reveals for the first time that the activation of the calcineurin/NFATc2 pathway in platelets has a non-transcriptional role and it is a key negative regulator of platelet responses. Further studies are needed to characterize the mechanism of action through which NFATc2 exerts its non-transcriptional function in modulating platelets activation and to understand if NFATc2 could have a non-transcriptional role also in nucleated cells.

Le piastrine ricoprono un ruolo fondamentale nei processi di emostasi, ma, recentemente, è stato dimostrato che hanno anche un ruolo nel modulare la risposta infiammatoria, interagendo con le cellule del sistema immunitario in modo diretto e indiretto. Nonostante le piastrine siano delle cellule anucleate, esse contengono fattori trascrizionali (TF) che modulano la loro attivatione attraverso attività non-trascrizionali. In particolare, è stato dimostrato che le piastrine contengono NF-κB, un TF che controlla importanti funzioni delle cellule immunitarie durante i processi infiammatori, e che NF-κB regola l’attivazione delle piastrine. In questo lavoro, abbiamo investigato la possibilità che altri TF oltre a NF-κB possano avere un ruolo regolatorio durante l’attivazione piastrinica. Abbiamo osservato che le piastrine contengono un altro importante TF tipicamente espresso dalle cellule immunitarie: il fattore nucleare delle cellule T attivate (NFAT). In particolare, abbiamo osservato che, a seguito dell’attivazione del recettore PAR4, la via calcineurina/NFATc2 viene attivata e regola varie funzioni piastriniche. Questa via viene attivata sia nelle piastrine murine sia umane e la sua inibizione determina un aumento dell’aggregazione piastrinica, dell’attivazione dell’integrina, del rilascio di granule e dell’adesione su fibrinogeno. Attraverso modelli murini in vivo, abbiamo dimostrato che l’attivazione di NFATc2 nelle piastrine modula l’emostasi e la trombosi. Inoltre, l’attivazione di NFATc2 nelle piastrine regola l’interazione tra piastrine e neutrofili, influenzando la severità dello sviluppo di sepsi batterica. I nostril esperimenti in vitro supportano la capacità di NFATc2 di agire a valle del recettore PAR4 attraverso un meccanismo che coinvolge il recettore dell’ADP e l’outside-in signaling dell’integrina α2bβ3. Infine, abbiamo osservato che l’inibizione della via calcineurina/NFATc2 è in grado di compensare in parte i difetti di attivazione piastrinica in due rare patologie, la sindrome di Hermansky-Pudlack e la trombastenia di Glanzmann. I nostri dati suggeriscono che la via calcineurina/NFATc2 può essere bersaglio per lo sviluppo di approcci terapeutici innovativi per il trattamento di difetti piastrinici con prognosi infausta. Il nostro lavoro mostra per la prima volta che l’attivatione della via calcineurina/NFATc2 nelle piastrine ha un ruolo non-trascrizionale e è un regolatore negativo delle risposte piastriniche. Ulteriori studi sono necessari per caratterizzare il meccanismo di azione attraverso il quale NFATc2 agisce nel modulare l’attivazione piastrinica e per capire se NFATc2 possa avere un ruolo non-trascrizionale anche in cellule nucleate.

(2019). A non-transcriptional role of NFAT in regulating platelets functions. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).

A non-transcriptional role of NFAT in regulating platelets functions

POLI, VALENTINA
2019

Abstract

Platelets play a critical role in hemostasis but, more recently, it was demonstrated that they also modulate the inflammatory response by establishing direct and indirect interactions with immune cells. Although platelets are anucleated cells, they contain functional transcription factors (TFs) that modulate their activation via non-transcriptional functions. In particular, it has been shown that platelets contain NF-κB, a TF that controls important functions of immune cells during the inflammatory process, and that NF-κB regulates platelet activation. We investigated the possibility that other TFs different from NF-κB might play regulatory roles during platelet activation. We found that platelets contain another TF typically expressed by immune cells: nuclear factor of activated T cells (NFAT). In particular, we found that upon PAR4 ligation, the calcineurin/NFATc2 pathway is activated and regulates platelet functions. This pathway is activated both in murine and human platelets, and its inhibition results in enhancement of platelet aggregation, integrin activation, granules release and spreading on fibrinogen. By using murine in vivo models, we demonstrated that NFATc2 activation in platelets modulates hemostasis and thrombosis. Moreover, platelet NFATc2 activation regulates the interaction between platelets and neutrophils and impacts the severity of bacterial sepsis development. Our in vitro experiments support the capacity of NFATc2 to act downstream of PAR4 via a mechanism that involves both the ADP receptor and the outside-in integrin α2bβ3 pathway. Finally, we found that inhibition of the calcineurin/NFATc2 pathway partially rescues platelet activation defects in two rare human platelet pathologies, the Hermansky-Pudlak syndrome and the Glanzmann thrombasthenia. Our data suggest that the calcineurin/NFATc2 pathway can be targeted to develop innovative therapeutic approaches to treat platelet defects with poor prognosis. Our work reveals for the first time that the activation of the calcineurin/NFATc2 pathway in platelets has a non-transcriptional role and it is a key negative regulator of platelet responses. Further studies are needed to characterize the mechanism of action through which NFATc2 exerts its non-transcriptional function in modulating platelets activation and to understand if NFATc2 could have a non-transcriptional role also in nucleated cells.
GRANUCCI, FRANCESCA
ZANONI, IVAN
NFAT; piastrine; non trascrizionale; infiammazione; sistema immunitario
NFAT; platelets; non transcriptional; inflammation; sistema immunitario
MED/04 - PATOLOGIA GENERALE
English
12-feb-2019
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
31
2017/2018
open
(2019). A non-transcriptional role of NFAT in regulating platelets functions. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/241337
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