Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function

Figures

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  Figure 1.. Pedigree phenotype. (A) Pedigree of the affected family, in which black, white and grey symbols indicate presence of the bleeding phenotype, absence of the bleeding phenotype and unknown bleeding phenotype, respectively. The genotype, where known, is shown under each symbol, where G is the mutant allele and C is the reference allele. Double lines indicate consanguinity and strike-through lines are used to indicate deceased individuals. (B) Human Phenotype Ontology (HPO) annotation of the three affected family members. (C) A ribbon diagram of the crystal structure of aspirin-acetylated cyclo-oxygenase 1 (COX-1) showing the location of the variant for the proband which results in a missense substitution of tryptophan to serine at amino acid 322.

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  Figure 2.. COX-1 protein in control, proband and relatives. (A) Western blots and quantification of cyclo-oxygenase 1 (COX-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression in platelet lysates, isolated from the controls, the proband (IV-1), homozygous (III-1 and III-2) and unaffected (III-3, III-4, III-5 and IV- 2) family members. Representative immunohistochemical analysis of COX-1 expression in (B) control, (C) proband and (D) homozygous relative (i) platelets and (ii) leukocytes. Washed platelets were identified by tubulin (green) staining and COX-1 (magenta) was present in control but not in the proband or affected relative. In washed leukocytes, nuclear staining was confirmed by DAPI (blue), LAMP-3 (green) and COX-1 (magenta) was expressed in all samples.

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  Figure 3.. Analysis of unstimulated whole blood acquired using an ImageStreamX Mark II incorporating a 60x objective lens. Scale bars represent 7 μm and identified (A) platelet-monocyte and (B) platelet-neutrophil aggregates. Percentage of (C) platelet-monocyte (CD14+) and (D) platelet-neutrophil (CD66b+) aggregates are quantified. Platelets identified by anti-CD61, leukocytes by anti-CD45, monocytes by anti-CD14 and neutrophils by anti-CD66b.

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  Figure 4.. Effect of PTGS1 variant on platelet aggregation, secretion, and adhesion responses. (A) Aggregation responses to arachidonic acid (AA; 1 mmol/L), adenosine diphosphate (ADP; 10 mmol/L), collagen (0.1-3 mg/mL), epinephrine (10 mmol/L), ristocetin (1.5 mg/mL), U46619 (3 mmol/L) and TRAP-6 amide (25 mmol/L) and (B) ATP secretion to AA (1 mmol/L), ADP (10 mmol/L), collagen (3 mg/mL) and TRAP-6 amide (25 mmol/L). n=20 (healthy controls; range with median); n=1 (proband); n=2 (homozygous relatives); n=4 (unaffected relatives). (C) P-selectin expression as measured by flow cytometry in whole blood stimulated by ADP (40 mmol/L), U46619 (0.5 μmol/L) or ADP plus U46619

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  Figure 5.. Platelet spreading on fibrinogen-coated surfaces. (A) a control, (B) the proband and (C) a homozygous relative with (D) quantification of adhered platelet, filopodia, lamellipodia, fully spread platelet frequency and total platelets per field of view.

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  Figure 6.. Contribution of PTGS1 to eicosanoid synthesis in whole blood and urine. Platelet-derived eicosanoid levels in whole blood from healthy volunteers or from the proband stimulated with collagen (30 mg/mL) (A) or TRAP-6 amide (30 mmol/L) (B). Levels are expressed as increase over levels in vehicle-treated blood. Urinary (C) prostaglandin (PG) D2 and (D) PGE2 (C) PGI2 and (D) thromboxane A2 (TXA2) metabolite levels in proband, homozygous and unaffected relatives. n=4 (healthy volunteers); n=1 (proband), n=2 (homozygous relatives), n=3 (unaffected relatives). Normal control ranges are indicated.