Additional information regarding plasmids, TMEM16F KO mouse generation, and detailed procedures for flow cytometry, electrophysiology, and in vivo experiments are described in the Supplemental Information
. All animal procedures were approved by the UCSF Institutional Animal Care and Use Committee and were performed according to the guidelines provided.
Generation of TMEM16F Knockout Mice
Using recombineering technology, loxP site was inserted in the 3′ intronic region of exon 2 and a frt-neo-frt-loxP cassette was inserted in the 5′ intronic region of exon 2. Linearized targeting vector was electroporated into embryonic stem cells. G418 and Gancyclovir were used in the ES cell media to select for ES cell clones with proper homologous recombination, which were confirmed by Southern blot analysis. Correctly targeted ES cells were then injected into blastocysts, and germline transmission was confirmed by PCR. The resulting mouse was bred with β-Actin-Flp mouse to delete neomycin cassette. Male β-Actin-Cre deleter mice were then bred with female TMEM16F flox/+ neomycin-deleted mice to generate founding TMEM16F heterozygous mice. TMEM16F heterozygous mice (het) were crossed with each other to generate TMEM16F KO mice. PCR genotyping was performed on tail DNA extracted from such offsprings. All experimental mice were backcrossed between two and five generations to C57Bl6.
Immunoblotting of Washed Mouse Platelet Lysates
Blood was collected from the vena cava of anesthetized mice in ACD (2.5 g Trisodium citrate, 1.37 g citric acid monohydrate, 2.0 g dextrose in 100 ml of ddH2O), mixed with 20 mM PIPES in saline (pH 6.5), and centrifuged at 100 × g in room temperature. The platelet-containing upper phase was mixed with low-pH washing buffer (140 mM NaCl, 10 mM NaHCO3, 2.5 mM KCl, 0.5 mM Na2HPO4, 1 mM MgCl2, 22 mM Trisodium Citrate, 0.1% dextrose, and 0.35% BSA [pH 6.5]) with 3 U/ml apyrase (Sigma) and then centrifuged at 500 × g at room temperature. Pelleted platelets were resuspended in Ca2+-free Tyrode’s HEPES (CFTH) buffer (134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1.0 mM MgCl2, 10 mM HEPES, 0.9% wt/vol dextrose, and 0.35% wt/vol BSA [pH 7.4]) then lysed using RIPA buffer. Western blot analysis was performed using a rabbit polyclonal antibody raised against a peptide corresponding to the N terminus of mouse TMEM16F protein. The β-actin antibody was used as loading control.
Flow Cytometric Analysis of PS Exposure
To assess the A23187 Ca2+ ionophore-induced PS exposure on the platelet surface, 1 × 106 platelets were resuspended in 24 μl total volume of CFTH buffer with 2 mM Ca2+ with or without A23187 (Sigma-Aldrich). This was immediately followed by addition of 1 μl FITC-conjugated Annexin-V (BD Biosciences) and incubation in 37°C water bath for 30 min. The incubation was quenched using 500 μl of chilled CFTH buffer, and platelets were analyzed using FACSCalibur (Becton Dickinson). Red blood cells were collected from the lower phase following whole-blood centrifugation and were similarly used for flow cytometric analysis.
For B cell analysis, mouse spleens from WT and KO mice were gently mashed, filtered, and washed once using PBS. The cells (1 × 106) were resuspended in 1X Annexin-V binding buffer (BD Biosciences) and were incubated with A23187 in 37°C water bath for 15 min. Cells were subsequently stained using Annexin-V FITC and CD19 APC on ice for 15 min followed by 500 μl of annexin-V binding buffer containing 7-AAD (1 μg/ml final).
Thrombin Generation Measured by a Thrombinoscope
Thrombin generation was measured using a fluorogenic thrombin substrate on a multiwell automated fluorescent plate reader (ThrombinoSCOPE, Maastricht, The Netherlands).
A small tip (1–2 mm) of the tail was transected and immersed in 37°C saline water. The time for the bleeding to stop was measured up to 5 min. For mice bleeding more than 5 min, pressure was applied near the wound to stop the bleeding. The mice were genotyped after the experiment. Statistical significance was determined with the Student’s t test.
FeCl3-Induced Carotid Thrombosis Model
Eight- to fourteen-week-old mice were anesthetized. The carotid was dissected free, a flow probe was placed around the left common carotid artery proximal to the bifurcation, and the artery was exposed to filter papers soaked in FeCl3 for 3 min. Arterial flow was measured continuously until clotting occurred (defined as no flow for ≥ 2 min). If no stoppage occured, measurements were ceased at 20 min. Percentage of arteries without occlusion (i.e., with continued flow) at the end of the assay was determined. Kaplan-Meier-type data were compared by log rank test.
Molecular Biology and Channel Expression
cRNA (5–100 ng) was injected into defolliculated oocytes. HEK293 cells were transfected with corresponding plasmids using FuGENE6 (Roche) and were cultured for 1–3 days before recording.
Axolotl or Xenopus oocytes were stained with 50 μg/ml Alexa-633-conjugated Concanavalin A for 15 min at room temperature. TMEM16F-eGFP proteins in HEK293 cells were detected using Alexa-488-conjugated anti-rabbit antibody. All images were acquired using a Leica SP5 laser confocal microscope.
Electrophysiology and Data Analysis
Macroscopic currents were recorded from inside-out patches. Data were acquired using an Axopatch 200-B patch-clamp amplifier and pClamp9 software (Molecular Devices). All experiments were performed at room temperature (22°C –24°C).
To determine the reversal potentials while the NaCl concentration of the internal solution (without buffer) was varied, the osmolality of each solution was adjusted to about 330 mOsm/kg by addition of sucrose, except for the 280 mM and 420 mM NaCl solutions, which had higher osmolalities and contained no sucrose. To minimize leak currents, patches with seal resistance lower than 3 GΩ were discarded. Similar reversal potential values were obtained by two different protocols (mTMEM16F in with the ramp protocol and with the voltage-step protocol).
For noise analysis, the currents were recorded at +80 mV using a gap-free protocol. The data were filtered at 1 KHz with an eight-pole low-pass Bessel filter were and sampled at 5 KHz.
For megakaryocyte recording, we used a protocol adopted from that of Mahaut-Smith (Mahaut-Smith, 2004
Clampfit10 (Molecular Devices) and Origin (OriginLab) were used for data analysis.