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M.V. Chan et al.
Introduction
Genotyping
The proband and her parents underwent whole genome sequencing (WGS). Variants were called and annotated as described previously.13 In all other family members variants in PTGS1 were called by Sanger sequencing.14 Furthermore, the vari- ant was expressed in cells and COX-activity was measured using a Clark type oxygen electrode.15
Sample collection
Midstream flow urine was collected and stored for subsequent eicosanomic analysis.16 Blood was collected by venepuncture into trisodium citrate (BD Diagnostics, UK). Platelet-rich plasma (PRP) was obtained by centrifugation at 175xg for 15 min. Platelet-poor plasma (PPP) was obtained by centrifugation of PRP at 12,000xg for 2 min. COX-1 protein presence was determined by western blotting in platelets and confocal microscopy in platelets and leukocytes. In addition, the number of platelet-monocyte and platelet-neutrophil aggregates were quantified to assess whether this variant modulates interactions of platelets with other blood cells using an ImageStream®X imaging flow cytometer (Merck Millipore, UK).
Platelet function studies
Platelet reactivity by light transmission aggregometry (LTA) and Optimul methods was completed within 2 hours of blood collec- tion.17,18 In parallel, ATP release and P-selectin levels were deter- mined to establish markers of platelets release and activation respectively and platelet spreading on collagen-coated surfaces were performed.19
Data analysis
Statistical summaries are presented as mean±standard deviation (SD). One-way ANOVA was performed using GraphPad Prism version 8.1.1 for Mac OS X (GraphPad Software, CA, USA) where appropriate. Statistically significant differences in means are pre- sented as *P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001. Percentiles of control values were generated, and pedigree mem- ber data was compared to this where only one value was obtained.
Results
Whole genome sequencing and phenotyping of the pedigree
The proband, a female of Iranian descent, aged 37 at enrolment (Figure 1, IV-1) was referred to the hemophilia outpatient clinic because of perioperative bleeding follow- ing a sinus operation. She had a history of cystic fibrosis (CF), C6 complement deficiency resulting in chronic infec- tions, b-thalassemia trait and normoprolactinemic galactor- rhoea. She had more extensive hemoptysis than expected from her CF and also suffered from frequent nosebleeds. At presentation she had a normal platelet count of 234x109/L.
Upon taking the family history we appreciated that she was part of a large consanguineous family. Moreover, her mother (III-2) and maternal aunt (III-1) also had a clinical bleeding phenotype including easy bruising and menor- rhagia. The two uncles (III-4 and III-5) and a cousin (IV-2) did not have any clinical bleeding (Figure 1A and B). Depending on the severity of bleeding, the proband received desmopressin, tranexamic acid and, very occa- sionally, platelets.
Sequencing demonstrated that the proband (IV-1), her mother (III-2) and aunt (III-1) were homozygous for a vari-
Platelets are central to the processes of hemostasis and thrombosis, the latter of which can lead to cardiovascular events such as myocardial infarction or stroke. At sites of vascular injury, platelets are activated upon interaction with collagen, von Willebrand factor (VWF) and fibrino- gen and undergo shape change. In order to form a platelet plug, platelets first adhere, then pseudopodia extend from the surface. Following this, lamellipodia spread between these protrusions, resulting in a fully spread platelet with- in 30 minutes (min).1,2 Platelets release the contents of their dense and α-granules, reinforcing activation and leading to the recruitment of further platelets to form a hemostatic plug in a positive feedback loop.3 A second feedback loop comprises liberation of arachidonic acid (AA) from mem- brane phospholipids by phospholipase A2 (PLA2) to form thromboxane A2 (TXA2).
AA is the substrate for three groups of eicosanoid-pro- ducing enzymes: lipoxygenase (LOX) which leads to hydroxyeicosatraenoic acids (HETE) and leukotrienes, cytochrome P450 (CYP450) which leads to epoxye- icosatrienoic acids (EET) and cyclo-oxygenase (COX) which leads to prostanoids. COX exists in two isoforms, the constitutively expressed COX-1 (more precisely known as prostaglandin endoperoxide synthase 1 [PTGS1]) and the (generally) inducible COX-2 [PTGS2], which both con- vert AA into prostaglandin (PG) G2 via an oxygenation reaction and then PGH2 via a peroxidase reaction.4–6 In platelets, PGH2 is then converted by thromboxane syn- thase to the pro-aggregatory TXA2.7 TXA2 is a key part of the positive feedback loop mentioned above. Irreversible blockade of platelet COX-1 by aspirin abolishes the pro- duction of TXA2 by platelets, explaining its efficacy in anti- thrombotic prophylaxis.8 Because of aspirin’s short half-life within the body and its irreversible effects upon COX, a low dose (75-100 mg per day) demonstrates a more selec- tive effect upon platelets than upon the rest of the body, where nucleated cells can regenerate COX-1 protein.9–11
Here, we describe the first case of autosomal recessive inheritance of a rare variant in PTGS1 which reproduces the selective anti-platelet effect of aspirin and provides insight into the normal balance of prostanoid production.
Methods
Additional methods can be found in the Online Supplementary Appendix.
Ethics and consent
The proband and relatives were enrolled in the National Institute for Health Research (NIHR) BioResource under the Bleeding, Platelet and Thrombotic Disease domain after providing informed written consent12,13. The NIHR BioResource projects were approved by Research Ethics Committees in the UK and appropriate national ethics authorities in non-UK enrolment cen- ters. Extensive phenotyping included coding of clinical and labora- tory phenotypes with Human Phenotype Ontology (HPO) terms and collection of numerical and family history data was per- formed as described previously12. Healthy volunteer studies were approved by the NHS St. Thomas’ Hospital Research Ethics Committee (07/Q702/24). Healthy volunteers and family mem- bers abstained from non-steroidal anti-inflammatory drug (NSAID) use for 2 weeks before sample collection.
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haematologica | 2021; 106(5)