TMEM16F enhances procoagulant activity and thrombosis through platelet microparticle generation

Date
2016
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Publisher
University of Delaware
Abstract
Increased procoagulant plasma microparticles are detected in prothrombotic and hypercoagulable states and may contribute significantly to thrombosis, the single greatest cause of death worldwide. Platelet microparticles are strongly procoagulant because of the exposed membrane phosphatidylserine (PS) that interacts with the coagulation cascade enabling rapid, localized and efficient thrombin generation subsequently resulting in fibrin formation and further platelet activation. Recently, TMEM16F, a novel putative 8-transmembrane spanning calcium-activated calciumpermeable channel, emerged as an essential component required for calciumdependent PS exposure on platelet membranes, procoagulant activity and thrombosis, yet the role of TMEM16F in microparticle formation and subsequent TMEM16Fdependent microparticle generation on thrombosis has been unclear. ☐ To determine whether TMEM16F is required for optimal microparticle formation and PS exposure, platelets from mice lacking (TMEM16F KO) or expressing TMEM16F (WT) were treated with physiological agonists or calcium ionophore and microparticle numbers and PS exposure on platelets were assessed by flow cytometry. TMEM16F KO platelets displayed an impaired ability to generate optimal PS+ and total microparticles and also PS exposure in response to either thrombin and collagen or A23187. Moreover, both CD41+ (platelet- and megakaryocyte- derived) and total microparticles in healthy circulation were lower in TMEM16F KO mice compared to WT mice, underscoring the physiological requirement for TMEM16F in microparticle formation in vivo. ☐ It was demonstrated that TMEM16F-dependent platelet microparticles are important for procoagulant activity and thrombosis, since both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from TMEM16F KO mice had diminished thrombin generation compared to those isolated from WT mice. Subsequent addition of platelet-derived microparticles to either TMEM16F-deficient PRP or PPP completely restored thrombin generation to the level of that observed in WT PRP. Furthermore, TMEM16F KO mice displayed defective thrombosis in vivo using a FeCl3-injury to the carotid artery model and the thrombotic defect was overcome with injection of isolated platelet microparticles, underscoring the requirement of TMEM16F-dependent platelet microparticles for thrombosis. Notably, number of platelets, leukocytes and erythrocytes in addition to platelet aggregation responses remained intact in TMEM16F KO mice, though mean platelet volume was slightly higher compared to WT mice, suggesting that loss of TMEM16F does not interrupt the steady-state levels of platelets and other circulating cells or platelet aggregation pathways, but platelets may be larger on average because of reduced microparticle formation. ☐ Taken together, these data indicate that TMEM16F is required for platelet microparticle formation and that TMEM16F-dependent platelet microparticles subsequently play a role in procoagulant activity that can contribute to thrombosis.
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