Functional redundancy between RAP1 isoforms in murine platelet production and function

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we conditionally deleted Rap1a and/or Rap1b in the megakaryocytic lineage (mKO). Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired pro- platelet formation by megakaryocytes. In platelets, RAP isoforms had both redundant and isoform-ˇspecific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α- granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. While important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation

Apport de pathologies plaquettaires rares à la compréhension des rôles de CalDAG-GEFI et des kindlines dans l'activation de l'intégrine αIIbß3

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

A new heterozygous mutation in GP1BA gene responsible for macrothrombocytopenia

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Bernard-Soulier syndrome: first human case due to a homozygous deletion of GP9 gene

International audienceBernard–Soulier Syndrome (BSS) is a rare (1:1 million) hereditary bleeding disorder caused by defects in the platelet glycoprotein (GP)‐Ib/IX/V complex, a receptor for von Willebrand factor (VWF) and thrombin (Lanza, 2006; Berndt & Andrews, 2011). Patients typically present with epistaxis, petechial or gingival bleeding with onset already in infancy. They present with macrothrombocytopenia and their platelets do not agglutinate in response to ristocetin, while maintaining a normal aggregation in response to a variety of aggregating agents. GPIb/IX/V complex consists of two GPIbα and four GPIbβ subunits stabilized by disulphide bonds (Luo et al., 2007). This heterodimer is non‐covalently associated with two GPIX and one GPV subunits. The N‐terminal residues of GPIbα form seven leucine‐rich repeats (LRRs) and include the binding sites for VWF and thrombin. BSS is due to biallelic loss‐of‐function pathogenic variants (deletions, insertions and nonsense mutations) in GPIBA, GPIBB or GP9 genes encoding GPIb/IX/V complex (Savoia et al., 2014). However, so far, no mutation in GP5 causing BSS has been reported yet. Most of the mutations prevent the formation of the complex or trafficking it through the endoplasmic reticulum and Golgi apparatus and alter receptor expression (Salles et al., 2008; Savoia et al., 2011; Nurden et al., 2012)

First report of a CalDAG-GEFI gene (RASGRP2) mutation in humans that affects platelet function and causes severe bleeding

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Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to co-repressors. We also observed large expansion of CFU-MKs derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton Cdc42 and RhoA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.status: publishe

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.

International audienceVariants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34(+) cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34(+) cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels