The role of TMEM16F in calcium flux, PS exposure and microparticle formation in platelets.

摘要

In platelets, TMEM16F, an eight-transmembrane protein, is a calcium activated-cation channel highly permeable to calcium. The TMEM16F gene has been shown to be mutated in Scott's syndrome, a rare bleeding disorder characterized by a defect in the scrambling activity of platelets and microparticle generation. In this thesis, I investigated the effect of TMEM16F on calcium signaling, microparticle formation and phosphatidylserine exposure in platelets under physiological conditions. First, I investigated whether TMEM16F was required for optimum calcium signaling in platelets. TMEM16F+/- mouse platelets treated with a combination of thrombin and collagen displayed a significant decrease in calcium entry measured as percent above basal calcium entry compared to the wild-type platelets (p=0.02). Further analysis of calcium entry revealed that there was a delay in the SOCE for the TMEM16F+/- platelets compared to the WT platelets (p=0.05). To determine whether TMEM16F is required for maximal PS exposure, binding of Annexin V to activated platelets from WT vs. TMEM16F knockout mice (TMEM16F KO) was compared. To measure PS exposure, Annexin V binding to platelet surfaces was quantified by flow cytometry: when treated with thrombin and convulxin, there was a significant decrease in maximal Annexin V binding achieved by 10muM A23187 of TMEM16F knock-out (KO) platelets compared to the WT (p=0.0005). Microparticle generation from TMEM16F KO platelets compared to WT platelets was also analyzed by flow cytometry. On average, after thrombin and convulxin treatment, there was a significant decrease of the percentage of microparticles over total particles (microparticles and platelets) generated by the TMEM16F KO mice compared to the WT mice (p=0.05). These data demonstrate that TMEM16F regulates calcium influx, PS exposure and microparticle formation in platelets. Interestingly, when the channel function of TMEM16F was blocked with CaCCinhA01, there was a significant decrease in the calcium influx compared to the untreated platelets (p=0.01) while no significant change was seen in the PS exposure, suggesting that TMEM16F might have two independent functions in platelets: one as a channel and the other as a regulator of the scrambling activity of platelets. Furthermore, preliminary results of calcium entry analyzed in HEK293T cells and CHOK cells stimulated with ionophore showed an increase in calcium entry when cells transfected with TMEM16F were compared to untransfected cells. However, analysis of the scrambling activity revealed that heterologous expression of TMEM16F was not sufficient to induce PS exposure after treatment with A23187. These results suggest that there are additional mechanisms to promote TMEM16F scramblase activity in platelets.