Protease-activated receptor 4 variant p.Tyr157Cys reduces platelet functional responses and alters receptor trafficking

OBJECTIVE—: Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect on platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. APPROACH AND RESULTS—: We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to affect most on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. CONCLUSIONS—: Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists

A new pedigree with thrombomodulin-associated coagulopathy in which delayed fibrinolysis is partially attenuated by co-inherited TAFI deficiency

ACKNOWLEDGEMENTS We thank NIHR BioResource volunteers for their participation, and gratefully acknowledge NIHR BioResource centres, 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 authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. S.K.W. was supported during this work by the Medical Research Council (MR/K023489/1) and is now funded through an NIHR-funded Academic Clinical Lectureship. K.D. is supported as a HSST trainee by NHS Health Education England. N.J.M. and C.S.W. are supported by the British Heart Foundation (PG/15/82/31721). J.C.M. is a fellow of the Research Foundation Flanders (FWO Vlaanderen; 1137717N). A.D.M. is supported by the NIHR Biomedical Research Centre at the University Hospitals Bristol National Health Service Foundation Trust and the University of Bristol. We thank Prof Paul Declerck and Prof Ann Gils, University Leuven, Belgium for the kind gift of the MA-T12D11 antibody. We acknowledge technical assistance from Dorien Leenaerts, University of Antwerp, Belgium and Michela Donnarumma, University of Aberdeen, UK.Peer reviewedPublisher PD

Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy

Thrombomodulin-associated coagulopathy (TM-AC) is a newly recognised dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor-induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared to controls, but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with rFVIIa or aPCC did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognised fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of haemostasis