Increased RhoA pathway activation downstream of αIIbβ3/SRC contributes to heterozygous Bernard Soulier syndrome

Bernard Soulier syndrome (BSS) is a severe bleeding disorder with moderate to severe thrombocytopenia, giant platelets, and platelet dysfunction, caused by biallelic mutations in GP1BA, GP1BB, or GP9 genes. We generated induced pluripotent stem cells (iPSC) from a BSS patient with a novel heterozygous GP1BA p.N103D mutation, resulting in moderate macrothrombocytopenia. The mutation does not affect megakaryocyte (MK) differentiation or GPIb-GPIX complex expression but reduces affinity to von Willebrand factor (VWF). It induces increased signaling independent of VWF and αIIbβ3-mediated outside-in signaling, causing a profound defect in proplatelet formation after adhesion on fibrinogen. Pre-activation of αIIbβ3 integrin and heightened stress fiber formation linked to RhoA pathway overactivation were observed, likely due to increased phosphorylation of SRC at Y419 downstream of GPIbα. Dasatinib, a SRC inhibitor, restored stress fiber formation. Using a 3D bone marrow model to mimic platelet release under flow, we demonstrated that the ROCK1/2 inhibitor Y27632 increased platelet number and restored platelet size in GPIbαN103D MK, as well as in MK from two other patients with heterozygous GP1BA mutations (p.L160P and p.N150S). However, Y27632 had no additional effect on platelet generation from MK of two patients with biallelic BSS, suggesting a distinct molecular mechanism in biallelic cases.

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Journal
Haematologica
Published
2025-03-06
DOI
https://doi.org/10.3324/haematol.2024.286424
Primary Topic
Platelet Disorders and Treatments
Type
article
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article

Increased RhoA pathway activation downstream of αIIbβ3/SRC contributes to heterozygous Bernard Soulier syndrome

Isabelle Plo, Rémi Favier, Hana Raslová, Francesco Baschieri et al.
Haematologica
Platelet Disorders and Treatments
article

Increased RhoA pathway activation downstream of αIIbβ3/SRC contributes to heterozygous Bernard Soulier syndrome

Isabelle Plo, Rémi Favier, Hana Raslová, Francesco Baschieri, Nicolas Papadopoulos, Christian A. Di Buduo, Stefan N. Constantinescu, Valérie Proulle, Paola Ballerini, Cecilia P. Marín Oyarzún, Alessandra Balduini, Nathalie Balayn, Larissa Lordier, Ida Biunno, Alexandre Kauskot, Cécile Lavenu‐Bombled, Tudor Manoliu, William Vainchenker, Francesca Careddu, Philippe Rameau, G. Nguyen
article en

Abstract

Bernard Soulier syndrome (BSS) is a severe bleeding disorder with moderate to severe thrombocytopenia, giant platelets, and platelet dysfunction, caused by biallelic mutations in GP1BA, GP1BB, or GP9 genes. We generated induced pluripotent stem cells (iPSC) from a BSS patient with a novel heterozygous GP1BA p.N103D mutation, resulting in moderate macrothrombocytopenia. The mutation does not affect megakaryocyte (MK) differentiation or GPIb-GPIX complex expression but reduces affinity to von Willebrand factor (VWF). It induces increased signaling independent of VWF and αIIbβ3-mediated outside-in signaling, causing a profound defect in proplatelet formation after adhesion on fibrinogen. Pre-activation of αIIbβ3 integrin and heightened stress fiber formation linked to RhoA pathway overactivation were observed, likely due to increased phosphorylation of SRC at Y419 downstream of GPIbα. Dasatinib, a SRC inhibitor, restored stress fiber formation. Using a 3D bone marrow model to mimic platelet release under flow, we demonstrated that the ROCK1/2 inhibitor Y27632 increased platelet number and restored platelet size in GPIbαN103D MK, as well as in MK from two other patients with heterozygous GP1BA mutations (p.L160P and p.N150S). However, Y27632 had no additional effect on platelet generation from MK of two patients with biallelic BSS, suggesting a distinct molecular mechanism in biallelic cases.

HaematologicaVol. 110(7)
Tufts University (US), Innsbruck Medical University (AT), Inserm (FR), Universität Innsbruck (AT), Ludwig Cancer Research (BE), Université Paris Cité (FR), University of Pavia (IT), Université Paris-Saclay (FR), Institut Gustave Roussy (FR), Sorbonne Université (FR), University of Oxford (GB), Hôpital Armand-Trousseau (FR), Centre de Recherche des Cordeliers (FR), Hôpital Cochin (FR), La Ligue Contre le Cancer (FR), Assistance Publique – Hôpitaux de Paris (FR), de Duve Institute (BE), Integrated Systems Solutions (United States) (US), Walloon Excellence in Lifesciences and Biotechnology (BE), Bicêtre Hospital (FR), UCLouvain (BE)
Openalex Percentile: Top 94%
Platelet Disorders and Treatments
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