title: Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.
abstract: Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.
authors.affiliation: Oxford Molecular and Pathology Institute, South Parks Road, Oxford, OX1 3RE, United Kingdom.
authors.forename: Clive
authors.initials: C
authors.lastname: Metcalfe
authors.affiliation: Oxford Molecular and Pathology Institute, South Parks Road, Oxford, OX1 3RE, United Kingdom.
authors.forename: Anjana
authors.initials: A
authors.lastname: Ramasubramoni
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom.
authors.forename: Giordano
authors.initials: G
authors.lastname: Pula
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, United Kingdom.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Harper
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, United Kingdom.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Mundell
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, United Kingdom.
authors.forename: Carmen H
authors.initials: CH
authors.lastname: Coxon
mesh_terms: D052160:Benzothiazoles / Q000494:pharmacology; D001780:Blood Coagulation Tests / Q000379:methods; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D004220:Disulfides / Q000494:pharmacology; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010979:Platelet Function Tests / Q000379:methods; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D038981:Receptors, Collagen / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D013879:Thioredoxins / Q000494:pharmacology*; D013927:Thrombosis / Q000188:drug therapy* / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D004220:Disulfides; D006873:Hydroquinones; D007093:Imidazoles; C554438:PMX 464; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D038981:Receptors, Collagen; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D012310:Ristocetin; D013879:Thioredoxins; C412893:1-methylpropyl-2-imidazolyl disulfide