Introduction: Thromboxane A2 [TxA2] is generated from arachidonic acid by cyclooxigenase-1 (COX-1) (prostaglandin H synthase-1) and thromboxane synthase. Aspirin, which irreversibly inhibits COX-1, is a widely used antiplatelet therapy with proven clinical efficacy. Inherited platelet disorders (IPD) are rare diseases caused by alterations of relevant genes in platelet formation and/or function. Despite the relevance of the TxA2 pathway in platelet physiology, few patients with mutations in PTGS1, the gene encoding COX-1, have been identified (<5 cases worldwide).
Objective: 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 IPD".
Methods: The index case is a 13-year-old adopted girl of Asian origin, referred because of moderate chronic bleeding (BAT-ISTH=6) and an aspirin-like platelet dysfunction. No coagulation defect or other relevant clinical symptoms were present. Platelet phenotyping included: blood count, PFA-100; platelet aggregation [LTA], glycoproteins (GP), activation and secretion of granules by flow cytometry (FC), TxA2 synthesis by enzyme-immunoassay, synthesis of eicosanoids by tandem gas chromatography with mass spectrometry (LC-MS), western-blot (WB) of platelet lysates, and immunofluorescence (IF) assays. The patient's DNA was analyzed with a HTS-gene panel (Bastida et al, Haematologica 2018). A HEK 293T cell transfection model was established to further assess the pathogenicity of the candidate variant found in the patient.
Results: The index case has normal platelet size and count (206x109/L; 11.4 fL). PFA-100 times were normal for COL-ADP and prolonged for COL-EPI (>300s). The FC analysis showed normal expression of GPs (Ib/IX, IIb/IIIa, Ia, GPVI) and reduced fibrinogen*488 binding (20-30%) in response to ADP, TRAP and low dose CRP (2ug/mL). P-selectin and CD63 secretion with agonists was comparable to those of controls. LTA was normal with ristocetin (1.25mg/mL) and TRAP (25uM), reduced by 40-50% with ADP (10uM) and collagen (3ug/mL) and absent with epinephrine (10uM), low dose collagen (1ug/mL) and arachidonic acid (1.6mM). LTA with U46619 (5uM), a direct agonist of the TxA2 receptor, was normal, suggesting a defect in TxA2 synthesis. Indeed, TxA2 levels in LTA supernatants in the patient were very low (5ng/mL; <10% vs. two controls). A significant reduction (50-90%) in TxA2 production was confirmed in the patient whole blood stimulated with collagen or TRAP, as measured by LC-MS. HTS analysis revealed that the patient is a heterozygous carrier of the variant c.428A>G, [p.Asn143Ser] in PTGS1. This variant, not previously described, affects a conserved residue in the catalytic domain of COX-1, which is one of the three N-glycosylation sites in the enzyme. The variant was not associated with reduced COX-1 expression as evaluated by WB in platelet lysates, and by IF in spread washed platelets and leukocytes. HEK 293T cells transfected with wild-type COX-1 construct (validated by RT-PCR and WB), displayed substantial TxA2 synthesis (500ng/mL; 2.5x105 transfected cells) in response to arachidonic acid. In contrast, similar transfection of p.143Ser COX-1 mutant almost abrogated this TxA2 production (≈50-75ng/mL in 2.5x105 transfected cells).
Conclusion: We have identified a novel autosomal dominant COX-1 variant, p.Asn143Ser, associated with functional haploinsufficiency of the enzyme and platelet aggregation defects. To our knowledge, this case represents the third description of variants in PTGS1 (Nance, JTH 2016; Sivapalaratnam, Blood 2018), which cause platelet dysfunction and bleeding.
Almarza:Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.