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M.V. Chan et al.
other groups have found that aspirin does not have a sig- nificant effect on spreading.33
The proband had a more severe bleeding phenotype than the other family members homozygous for the PTGS1 variant which might be attributable to an addition- al diagnosis of CF and antibiotic use34 but is more likely due to an additional dysfunctional pathway.35,36 Indeed, ristocetin-induced platelet aggregation was impaired even though VWB factor antigen (VWF:Ag) and function (VWF:RCo) levels were in the normal range (83.2 IU/dL and 71.7 IU/dL respectively). No coagulation defect was identified which could contribute to bleeding: prothrom- bin time (9.6 seconds) and activated partial thromboplas- tin time (28 seconds) were in the normal range and the proband’s factor VIII level was 0.98 IU/mL, above the minimum required for normal haemostasis. Furthermore, no variants were found in GP1b or P-selectin in the proband. Interestingly, the proband and homozygous rel- atives had significant changes in platelet spreading on col- lagen where 70% fewer platelets adhered than samples taken from controls, a response which is dependent upon platelet integrin αIIbb3 (which also binds VWF and fibrino- gen). Also, of the platelets that did adhere, fewer reached the stage of being fully spread.
As expected, COX-1-deficient platelets in whole blood failed to produce any COX-derived prostanoids, namely PGE2, PGD2, 11-HETE, 15-HETE and the stable metabolite of TXA2 (TXB2) following exposure to platelet ago- nists.32,37–39 Notably, the individuals supplying these platelets had thromboxane metabolite (TX-M) levels within the normal range indicating that urinary TX-M is not a valid or reliable measure of platelet function; con- trary to its frequent use for this purpose. This finding sup- ports our recent report that in humans basal TX-M is not derived from platelets but from other sources such as the kidneys,16 and provides further rebuttal to challenges of this interpretation.40 As urinary TX-M levels are reduced in humans consuming low dose aspirin8, the findings also demonstrate that low dose aspirin is not specific for platelets and inhibits COX at other sites. Previous studies have measured the urinary eicosanoid profile in CF patients and reported higher levels of TX-M than in healthy comparators. This is in agreement with our find- ings in the proband who had higher levels than other homozygous family members. This implies that the ele- vated production of TXA2 in CF leading to increased TX- M cannot be explained by increased platelet activation.41 Indeed, COX-2 inhibitors reduced urinary TX-M levels in CF patients consistent with a source other than platelet COX-1.42
Previous cases have been reported variants in PTGS1 which have been associated with autosomal dominant inheritance of enhanced bleeding, some impairment of platelet aggregation and changes in protein levels. There have been no reports of absence of COX-1 protein and/or ablation of associated eicosanoid production as reported here.39,43,52,53,44–51 Nance et al.50 identified a pedigree with a non-synonymous variant in the signal peptide of PTGS1 (rs3842787; c.50C>T, p.Pro17Leu) that segregated with an aspirin-like platelet function defect. The proband also car- ried a variant in the F8 causing hemophilia A (rs28935203; c.5096A>T; p.Y1699F). The affected family members with both variants had more severe bleeding than expected from mild hemophilia A alone. In this study, extensive platelet function testing was performed demonstrating
impaired platelet aggregation induced by AA, epinephrine and low dose ADP and reduced platelet TXB2 release.50 Two compound heterozygous cases have been reported. The first in a patient with post-procedural bleeds and an aspirin-like defect who carried two high frequency vari- ants (R8L and P17L) which had previously been reported not to have an effect on function.31,52 Analysis of the sec- ond case identified a rare variant (c.337C>T, p.Arg113Cys; gnomAD frequency 6.134x10-5) in compound heterozy- gosity with a common variant (c.1003G>A, p.Val481Ile; gnomAD frequency 0.007) which was classified as proba- bly pathogenic and accompanied reduced plasma TXB2 levels.51 Finally, Bastida et al. reported two cases with vari- ants in PTGS1 (c.35_40delTCCTGC, p.Leu13_Leu14del and c.428A>G, p.Asn143Ser) by sequencing 82 patients with an inherited platelet disorder on their high-through- put sequencing platform to investigate the unknown molecular pathology. They did not, however, perform in- depth platelet phenotyping53. Consequently, none of these previous reports describe complete loss of platelet PTGS1 function.
In conclusion, we describe the first case of a well charac- terized family with autosomal recessive inheritance pro- ducing an aspirin-like platelet function defect due to a rare variant in PTGS1. This case models the specific loss of platelet COX-1 activity and provides a benchmark of COX- 1’s role in platelet function and eicosanoid metabolism.
Disclosures
No conflicts of interest to disclose.
Contributions
MVC, MAH, SS, MC, MEA, MLE, DCZ, GLM, JS, DG, MH, VBO, LD, MGM, CL, KW, MS, KD, DPH and KF designed and performed experiments; MVC, MAH, SS, MC, MEA, MLE, GLM, VBO and LD performed data analysis; MVC, MAH, SS, MAL and TDW wrote the manuscript; VBO, WHO, ET, KF, MAL and TDW supervised the study, and all authors reviewed the manuscript.
Acknowledgments
The authors would like to thank research nurse Amy Frary at the University of Cambridge for her contributions to the clinical phenotyping of National Institute for Health Research (NIHR) BioResource participants. The authors would also like to thank NIHR BioResource volunteers for their participation, and grate- fully acknowledge NIHR BioResource centers, NHS Trusts and staff for their contribution. We thank the National Institute for Health Research and NHS Blood and Transplant. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.
Funding
The NIHR BioResource was mainly funded by grants from the NIHR in England (NIHR, grant number RG65966). Additional funding was provided by the British Heart Foundation (BHF), Medical Research Council (MRC), National Health Service (NHS) England, the Wellcome Trust and many other fund providers (also see Funding acknowledgment for individual researchers below). This work was supported by the British Heart Foundation (PG/15/47/31591 to TDW for MVC, PG/17/40/33028 to TDW for MC, and RG/12/11/29815 to VBO), Barts & the London School of Medicine and Dentistry, Queen Mary University of London (to TDW for MAH and
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