VERÓNICA PALMA BARQUEROS1, Marilena Crescente2, Eugenia de la Morena Barrio1, NATALIYA BOHDAN 1, Melissa Chan2, Elena Almarza3, Nuria Revilla Calvo1, José Padilla Ruiz1, ANTONIA MIÑANO NAVARRO1, Matthew Edin4, Darryl Zeldin4, Cristina Mesa-Nuñez5, Carlos Damian3, Ana Marín Quílez6, Rocio Benito6, IRENE MARTINEZ MARTINEZ1, Nuria Bermejo7, Ignacio Casas7, Agustín Rodriguez Alen8, Jose Ramón González-Porras9, Jesús Maria Hernández-Rivas9, Vicente Vicente García1, Javier Corral de la Calle1, Timothy Warner2, Jose Maria Bastida9, José Rivera Pozo1


(2) Blizard Institute &London School of Medicine and Dentistry, Queen Mary University of London London, UK., Reino Unido
(3) Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD/UAM), España (Comunidad de Madrid), España (Comunidad de Madrid)
(4) National Institutes of Health, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina., Estados Unidos
(5) Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD/UAM), España (Comunidad de Madrid)
(6) IBSAL, IBMCC, CIC, Universidad de Salamanca-CSIC, Salamanca, España (Castilla y León)
(7) Servicio de Hematología, Hospital San Pedro de Alcántara, Complejo Universitario de Cáceres, España (Extremadura)
(8) Servicio de Hematología y Hemoterapia, Hospital Virgen de la Salud, Complejo Hospitalario de Toledo, España (Castilla la Mancha)
(9) Departamento de Hematología, Hospital Universitario de Salamanca, Salamanca-IBSAL, España (Castilla y León)



Thromboxane A2 [TxA2] is generated from arachidonic acid by cyclooxigenase-1 (COX-1, the aspirin target) and thromboxane synthase. Despite the clinical-biological relevance of COX-1, few patients with mutations in PTGS1, the gene encoding COX-1, have been identified (<5 cases worldwide). Our aim is the characterization of a patient with aspirin-like platelet defect and moderate bleeding, enrolled in the Spanish multicentric project "Functional and molecular characterization of patients with Inherited Platelet Disorder".


The index case is a 13-year-old adopted girl of Asian origin, referred because of moderate chronic bleeding. The proband has normal platelet size and count. No coagulation defect or other relevant clinical symptoms were present. Platelet phenotyping included: PFA-100; glycoprotein expression and platelet activation by flow cytometry; platelet aggregation [LTA]; TxA2 synthesis by enzyme-immunoassay, synthesis of eicosanoids by tandem gas chromatography with mass spectrometry (LC-MS) and western-blot (WB). The patient's DNA was analyzed with a HTS-gene panel (Bastida et al, Haematologica 2018). To further assess the pathogenicity of the candidate variant, we analyzed wild-type (WT) and mutant (generated by site-directed mutagenesis) recombinant proteins expressed in 293T-HEK cells. Moreover, a HEK 293 cells stably transfected with Ser143 were assessed. N-glycosylation analysis included N-glycosidase F treatment.


PFA-100 times were prolonged for COL-EPI (>300s). Patient’s platelets showed null aggregation with AA but normal U46619-induced aggregation, suggesting a defect in TxA2 synthesis. Indeed, TxA2 levels in LTA supernatants in the patient were <10% vs. controls, and the prostanoid profile was severely altered in collagen/TRAP-stimulated whole blood. DNA analysis identified a heterozygous variant c.428A>G (p.Asn143Ser) in PTGS1, affecting a conserved residue involved in N-glycosylation at the COX-1 catalytic domain. HEK 293T cells transfected with wild-type COX-1 construct, displayed substantial AA-induced TXA2 synthesis. In contrast, cells transfected with Ser143 COX-1 displayed negligible AA-induced TXA2 synthesis. In cells transfected with equal amounts of wild-type and Ser143 COX-1 constructs, the TXA2 synthesis was reduced by 70%, suggesting a dominant-negative effect for the p.Asn143Ser mutation (Fig A). Remarkably, disrupting this N-glycosylation site with a different variant, p.Ser145Ala, caused a similar effect (Fig A). Moreover, introduction of wild-type COX-1 in HEK 293 cells stably transfected with Ser143 recovered TXA2 production following a non-linear concentration-dependent pattern, suggesting increased competition for the substrate as the mechanism accounting for the dominant-negative effect of the mutation (Fig B). This dominant-negative effect was confirmed in a dose-response curve of AA at low doses [upon 1µM AA:WT: 35; Ser143: 1; co-transfected:5pg/mL) suggesting a greater sequestration of AA by the mutant (Fig C). A COX-1 isoform with lower molecular weight than wild-type COX-1 was expressed in patient’s platelets and in cells transfected with Ser143 or Ala145 variants. This mutant COX-1 was undetectable upon treatment of transfected cells with N-glycosidase F, suggesting that the reduced size is due to N-glycan loss (Fig D).


Overall, this work extends the genetic spectrum of COX-1 defects and reveals COX-1 hypo-glycosylation as a new dominant-negative mechanism causing platelet function disorder.

Image 1


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