L’étude de l’identification des défauts moléculaires mis jeu dans les pathologies héréditaires plaquettaires est d’un apport considérable pour améliorer la compréhension des mécanismes physiologiques. Durant ma thèse, j’ai étudié les plaquettes d’individus appartenant à deux familles distinctes souffrant de dysfonctions plaquettaires à l’origine d’hémorragies sévères. Par séquençage entier des exons, nous avons identifié pour la première famille une mutation du gène RASGRP2 à l’origine de la substitution Gβ48W empêchant l’activation de CalDAG-GEFI. Les plaquettes des individus porteurs de la mutation à l’état homozygote ont une capacité réduite à activer Rap1 et l’intégrine αIIbß3 en réponse à de faibles doses d'agonistes. La présence d'un allèle non muté (hétérozygotie) est suffisante pour prévenir lessaignements mais ne permet pas de rétablir totalement une fonction plaquettaire normale. La deuxième famille est porteuse d’une mutation du gène FERMT3 (pN54RfsX142) conduisant à une absence complète de kindline-3. Les plaquettes homozygotes pour cette mutation sont incapables d’activer l’intégrine αIIbß3. Elles forment des filopodes et desnodules d’actine mais ne peuvent étendre des lamellipodes même en présence de Mn2+. La kindline-3 s’est révélée essentielle à la régulation de l’activité de Cdc4β et au réarrangement au cytosquelette d'actine lors de la signalisation «outside-in» de l’intégrineαIIbß3. Seule la kindline-3 a jusqu’ici été impliquée dans l'activation des intégrinesplaquettaires. Nous mettons en évidence la présence de kindline-2 dans les plaquettes et les mégacaryocytes humains. Des localisations différentes ont été mises en évidence pour ces deux kindlines. Dans le mégacaryocyte la kindline-2 se situe dans les zones d’adhérence focales et s’associe préférentiellement avec les intégrines ß3. Dans les plaquettes, seule la kindline-3 est présente dans nodules d’actine. Ces résultats sont en faveur de rôles non redondants des kindlines-2 et -γ et d’une implication potentielle de la kindline-2 dans la mégacaryopoïèse. / Inherited platelet disorders are rare diseases that give rise to severe bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Identifying the molecular mechanisms involved brings important insight into platelet pathophysiology. During my PhD, I studied platelets isolated from members of two families suffering severe bleedings among those one had no established diagnosis. In the first family, using whole exome sequencing, we identified a RASGRP2 mutation causing a G248W substitution leaving CalDAG-GEFI inactive. Platelets from individualscarrying the mutation exhibit a reduced ability to activate Rap1 and to perform proper Inherited platelet disorders are rare diseases that give rise to severe bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Identifying the molecular mechanisms involved brings important insight into platelet pathophysiology. During my PhD, I studied platelets isolated from members of two families suffering severe bleedings among those one had no established diagnosis. In the first family, using whole exome sequencing, we identified a RASGRP2 mutation causing a G248W substitution leaving CalDAG-GEFI inactive. Platelets from individuals carrying the mutation exhibit a reduced ability to activate Rap1 and to perform proper αIIbß3 integrin inside-out signaling in response to low doses agonists. The presence of a single normal allele is sufficient to prevent bleeding but does not allow normal platelet function. integrin inside-out signaling in response to low doses agonists. The presence of a single normal allele is sufficient to prevent bleeding but does not allow normal platelet function. Members of the second family carry a FERMT3 mutation leading to a completekindlin-3 deficiency (pN54RfsX142). Platelets from the homozygous patient are unable to perform proper integrin αIIbß3 activation. We now observe that kindlin-3 deficient platelets form filipodia and actin nodules but are unable to extend lamellipodia even in presence of Mn2+. We demonstrate that kindlin-3 is essential for Cdc42 activity regulation and actincytoskeleton remodeling during αIIbß3 integrin outside-in signaling To date, only the kindlin-3 has been involved in integrin activation. We show that kindlin-2 is present in human platelets and megakaryocytes. Both kindlins exhibit distinctlocalizations. In megakaryocytes, kindlin-2 specifically localizes within focal adhesion and associates preferentially with ß3 integrins. In platelets, unlike kindline-2, kindline-3 is located in actin nodule. All together these data argue in favor of specific roles played by each kindlins and a possible implication of kindlin-2 in megakaryocytopoiesis.

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Background: Selective serotonin reuptake inhibitor (SSRI) antidepressants prevent serotonin (5-HT) uptake by the serotonin transporter (SERT). Since blood platelets express SERT, SSRIs may modify platelet function and the risk of cardiovascular disease. However, the beneficial or adverse effects of SSRIs on arterial thrombosis are poorly characterised and detailed in vitro experimental data is limited. The SSRI citalopram is a racemate, the (S)-isomer being the more potent SERT inhibitor. Although citalopram has been shown to inhibit platelets in vitro, it is unclear whether this is mediated via SERT blockade. Aim: To determine if citalopram inhibits platelet function via SERT blockade, or through a novel mechanism of action. Findings: 5-HT uptake into platelets was blocked by both citalopram isomers at concentrations that had no apparent effect on platelet function. Despite the (S)-citalopram isomer being the more potent SERT inhibitor, (R)-citalopram was equally potent at inhibiting other platelet functions. These findings strongly suggest that inhibition of platelet function by citalopram in vitro is not mediated by blocking SERT. Subsequent experiments identified two putative mechanisms for citalopram-mediated platelet inhibition: 1) citalopram did not inhibit calcium store release induced by the platelet agonist U46619, despite blocking subsequent Rap1 activation. A credible target for this inhibitory mechanism is the calcium and diacylglycerol guanine nucleotide exchange factor-1 (CalDAG-GEFI): 2) citalopram suppressed early protein phosphorylation within the GPVI pathway, resulting in the inhibition of subsequent platelet responses. Further experiments show that other commonly used antidepressants also inhibit platelets. As with citalopram, inhibition was only observed at concentrations above those required to block SERT, suggesting that alternative inhibitory mechanism(s) are responsible. Conclusions: Data presented in this thesis support two novel putative mechanisms of citalopram-induced platelet inhibition. These findings demonstrate that citalopram and other antidepressants inhibit platelets independently of their ability to block SERT-dependent 5-HT transport. The identification of thesemechanisms provides a pharmacological approach to develop novel antiplatelet agents based on current antidepressants.