β1-tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying six β1-tubulin variants: p.Cys12Leufs12*, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size and a minority no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant, displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets, while had negligible effect on platelet activation, secretion or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.

<b><i>Background:</i></b> The mechanisms underlying neonatal platelets hyporesponsiveness are not fully understood. While previous studies have demonstrated developmental impairment of agonist-induced platelet activation, differences in inhibitory signaling pathways have been scarcely investigated. <b><i>Objective:</i></b> To compare neonatal and adult platelets with regard to inhibition of platelet reactivity by prostaglandin E1 (PGE₁). <b><i>Methods:</i></b> Platelet-rich plasma from umbilical cord (CB) or adult blood was incubated with PGE₁ (0–1 μM). We assessed aggregation in response to adenosine diphosphate (ADP), collagen, and thrombin receptor activating peptide as well as cyclic adenosine 3′5′-monophosphate (cAMP) levels (ELISA). G_αs, G_αi2, and total- and phospho-protein kinase A (PKA) were evaluated in adult and CB ultrapure and washed platelets, respectively, by immunoblotting. <b><i>Results:</i></b> Neonatal (vs. adult) platelets display hypersensitivity to inhibition by PGE₁ of platelet aggregation induced by ADP and collagen (PGE₁ IC₅₀: 14 and 117 nM for ADP and collagen, respectively, vs. 149 and 491 nM in adults). They also show increased basal and PGE₁-induced cAMP levels. Mechanistically, PGE₁ acts by binding to the prostanoid receptor IP (prostacyclin receptor), which couples to the G_αs protein-adenylate cyclase axis and increases intracellular levels of cAMP. cAMP activates PKA, which phosphorylates different target inhibitor proteins. Neonatal platelets showed higher basal and PGE₁-induced cAMP levels, higher G_αs protein expression, and a trend to increased PKA-dependent protein phosphorylation compared to adult platelets. <b><i>Conclusion:</i></b> Neonatal platelets have a functionally increased PGE₁-cAMP-PKA axis. This finding supports a downregulation of inhibitory when going from neonate to adult contributing to neonatal platelet hyporesponsiveness.

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