Bernard-Soulier syndrome (BSS) is a severe bleeding disorder, classically described as an autosomal recessive disease caused by biallelic mutations affecting the GPIb-IX glycoprotein complex, a major platelet receptor. It combines moderate to severe thrombocytopenia, giant platelets and platelet dysfunction, with an estimated incidence of approximately one case per million. While the formation of abnormally large platelets has long been linked to defective proplatelet production by megakaryocytes, the precise mechanisms remained poorly understood, particularly in the hitherto little-studied heterozygous (monoallelic) forms.
To investigate these forms, the authors established induced pluripotent stem cells (iPSCs) from a patient carrying a novel heterozygous GP1BA p.N103D mutation, located in the LRR4 domain of the GPIbα subunit and responsible for moderate macrothrombocytopenia. Unlike murine or iPSC models of biallelic BSS, which exhibit an absence or marked reduction of the GPIb-IX complex at the cell surface, this mutation alters neither megakaryocyte differentiation, nor complex expression, nor the GPIbα/filamin A interaction. It does, however, reduce affinity for von Willebrand factor (VWF) and induces, independently of VWF, enhanced signaling through integrin αIIbβ3, leading to a profound defect in proplatelet formation following adhesion to fibrinogen.
The analysis revealed pre-activation of integrin αIIbβ3 and increased stress fiber formation, linked to overactivation of the RhoA pathway. Proteomic arrays highlighted increased phosphorylation of the SRC kinase at tyrosine Y419, downstream of GPIbα. SRC may activate the RhoA pathway via GEF-H1, either directly or through STAT3. Consistent with this hypothesis, dasatinib, a SRC inhibitor, restores normal stress fiber formation. The authors recall that RhoA must be partially inactivated at the end of megakaryocyte maturation to ensure normal thrombopoiesis, a balance that is here dysregulated by the mutant.
Using a three-dimensional silk-based bone marrow model that reproduces platelet release under flow, the team showed that the ROCK1/2 inhibitor Y27632 increases platelet number and restores platelet size in megakaryocytes carrying the N103D mutation, as well as in two other heterozygous patients (p.L160P and p.N150S mutations). In contrast, Y27632 had no additional effect on platelet production from megakaryocytes of two patients with biallelic BSS, indicating a distinct molecular mechanism. The authors conclude that monoallelic BSS results, at least in part, from a defect in the late stages of megakaryopoiesis, through dysregulated, VWF-independent GPIbα signaling to αIIbβ3, whereas biallelic forms more closely resemble the phenotype observed in mice deficient in Gp1ba and Gp1bb.