Maintaining flippase activity in procoagulant platelets is a novel approach to reducing thrombin generation.
Publication Date
2022-04Journal Title
J Thromb Haemost
ISSN
1538-7933
Publisher
Elsevier BV
Language
en
Type
Other
This Version
AO
VoR
Metadata
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Millington-Burgess, S. L., & Harper, M. T. (2022). Maintaining flippase activity in procoagulant platelets is a novel approach to reducing thrombin generation.. [Other]. https://doi.org/10.1111/jth.15641
Abstract
BACKGROUND: During thrombosis, procoagulant platelets expose phosphatidylserine (PS), which enhances local thrombin generation. Reducing platelet PS exposure could be a novel anti-thrombotic approach. PS is confined to the inner leaflet of the plasma membrane in unstimulated platelets by ATP-dependent "flippase" activity. Ca2+ ionophores trigger all platelets to expose a high level of PS by activating a scramblase protein and inactivating the flippase. Although R5421 was previously shown to reduce Ca2+ ionophore-induced PS exposure, its mechanism of action is unknown. OBJECTIVES: To determine the mechanism by which R5421 reduces platelet PS exposure. METHODS: Washed human platelets were stimulated with the Ca2+ ionophore, A23187, to induce procoagulant platelet formation while bypassing proximal receptor signalling. Platelets PS exposure was detected using annexin V or lactadherin in flow cytometry. NBD (7-nitro-2-1,3-benzoxadiazol-4-yl)-PS was used to assess scramblase and flippase activity. Thrombin generation was monitored using a fluorogenic substrate. RESULTS AND CONCLUSIONS: R5421 reduced the extent of A23187-stimulated platelet PS exposure, as demonstrated with annexin V or lactadherin binding. R5421 also maintained flippase activity in procoagulant platelets. Although R5421 appeared to inhibit scramblase activity in procoagulant platelets, it did not once the flippase had been inhibited, demonstrating that scramblase activity is not directly inhibited. Furthermore, R5421 inhibited the contribution of A23187-stimulated platelets to thrombin generation. Together these data demonstrate that R5421 reduces the extent of PS exposure in procoagulant platelets by maintaining flippase activity. Maintaining flippase activity in procoagulant platelets is a novel and effective approach to reducing thrombin generation.
Keywords
BRIEF REPORT, BRIEF REPORTS, blood platelets, pharmacology, phosphatidylserine, phospholipid transfer proteins, thrombosis
Sponsorship
British Heart Foundation (PG/20/12/34982)
Identifiers
jth15641
External DOI: https://doi.org/10.1111/jth.15641
This record's DOI: https://doi.org/10.17863/CAM.80851
Rights
Licence:
http://creativecommons.org/licenses/by/4.0/
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