Abstract 8: TMEM16f Regulates PhosphatidylSerine (PS) Exposure and PS+ Microparticle Generation From Mouse Platelets

Abstract

Transmembrane (TMEM)16f is a protein from the family of TMEM16 Ca++-dependent Chloride channels that is essential for optimal Ca2+-dependent phospholipid scrambling activity in platelets. Recently, it has been shown that TMEM16f-dependent exposure of phosphatidylserine (PS) on platelet surfaces is required for platelet-dependent pro-coagulant activity and hemostasis. However, relatively little is known about the effect of TMEM16f on platelet signaling, functional activity, and microparticle formation. TMEM16f-/- mice were generated in the laboratory of Andrea Vortkamp and bred in our laboratory as heterozygous crosses. When assessed by flow cytometry, platelets from TMEM16f-/- mice exposed 27.8% of the PS exposed by WT platelets in response to thrombin and convulxin, 23.1% of the PS in response to 1 uM A23187 , and 38% of the PS exposed by WT platelets in response to 10 uM A23187. Similar trends were obtained using high resolution immunofluorescent microscopy (TMEM16f-/- exposed 49.2% of the PS detected on WT platelets in response to thrombin + convulxin; 25.6% PS in response to 1 uM A23187 ). Microparticle generation from TMEM16f-/- platelets compared to WT platelets was also evaluated by both high resolution immunofluorescent microscopy and flow cytometry. The proportion of PS+αIIb+ microparticles generated per platelet was lower in platelets from TMEM16f-/- mice compared to WT when evaluated by both of these methods. Given that TMEM16f-/- platelets elicit similar calcium responses upon thrombin and convulxin exposure to those of WT platelets, these data may suggest that PS scrambling activity is required not only for PS exposure on the outer leaflet of platelet membranes, but also for the shedding of PS+ microparticles. Surprisingly, TMEM16f-/- platelets were less sensitive to AYPGKF-induced fibrinogen binding than WT platelets (n= 4, p<.05). Finally, TMEM16f-/- mice form less stable occlusive thrombi following 10% ferric chloride injury of the carotid artery (0/4 stable thrombi of TMEM16f-/-) than WT mice (7/9 stable thrombi). The loss of procoagulant microparticles may contribute to the lower sensitivity of TMEM16f-/- mice to ferric chloride-induced thrombosis.