TMEM16E regulates endothelial cell procoagulant activity and thrombosis

Figures

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  Figure 1. TMEM16E and TMEM16F regulate EC procoagulant activity. (A) Heatmap illustrating the relative positive or negative regulation of factor VIIa–catalyzed activation of factor X following silencing of the indicated genes in HUVECs. Indicated genes were silenced with a pool of 4 distinct siRNAs and tested in triplicate. Each box represents an independent experimental plate, with scale bar depicting percentage of factor Xa generated compared with cells transfected with untargeted control siRNA. Lighter color indicates lower percentage factor Xa generation compared with control. (B–G) HUVECs were transfected with individual siRNAs for 72 hours and assayed for their ability to support factor VIIa–catalyzed activation of factor X (B–D) or thrombin generation in plasma-treated ECs (E–G). Cells were stimulated with TNF-α (10 ng/mL) for 3.5 hours (B, C, E, and F), TNF-α for 3.5 hours plus Ca2+ ionophore A23187 (6 μM) for 20 minutes (D), or A23187 alone for 20 minutes (G). Representative experiments are depicted as mean absorbance for factor Xa generation (B) or the first derivative of arbitrary fluorescent units for thrombin generation (E) as a function of time. 16E, 16F, and TF denote siRNA…

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  Figure 2. TMEM16E and TMEM16F are required for PS externalization on ECs. HUVECs were transfected with indicated siRNAs for 72 hours, stimulated with TNF-α (10 ng/mL) for 16 hours (A) or Ca2+ ionophore A23187 (6 μM) for 20 minutes (B), and stained with annexin V (green) to detect PS externalization and Zombie Red (red) to detect cell death. Total annexin V fluorescence was normalized to number of nuclei (blue) and dead cells. (C) PS externalization following treatment with ionophore A23187 was detected using annexin V by flow cytometry. Histograms were generated after gating on live (DAPI negative) cells only. (D) HUVECs were transfected with indicated siRNAs for 72 hours, stimulated with TNF-α (10 ng/mL) for 3.5 hours, and stained for TF (green). Mean fluorescent intensity (MFI) was normalized to background for each image. Representative images are shown. (E and F) Intracellular Ca2+ flux was measured with Calbryte 520 AM in siRNA-transfected HUVECs following stimulation with thrombin (1 U/mL). Silencing of the store-operated Ca2+ regulator STIM1 served as a positive control. Time course of Calbryte 520 fluorescence after thrombin stimulation normalized to background fluorescence (E) and…

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  Figure 3. PS externalization visualized via intravital microscopy occurs on the vessel wall and is unaffected by platelet inhibition. Thrombus formation was monitored for 180 seconds in WT mice following laser injury of the cremasteric arteriole in the presence or absence of the platelet aggregation inhibitor eptifibatide (10 μg/g of body weight). (A) Representative images at indicated time points of the PS probe annexin V (red, Alexa Fluor 647), platelets (blue, anti-CD42b antibody, DyLight 405), and fibrin (green, anti-fibrin antibody, DyLight 488). Note annexin V positivity on the vessel wall and in the absence of platelet aggregation. Kinetics and magnitude of median integrated RFUs for platelet accumulation (B) and PS externalization (D) are shown following laser injury. AUC for fluorescence intensity was determined for platelets (C) and annexin V (E). Lines represent the median AUC for individual thrombi (vehicle n = 34, eptifibatide n = 35) analyzed by Mann-Whitney U test. ****P < 0.0001. A vessel-wall pattern for PS externalization is also observed using alternative PS probes pSIVA (F, red pseudocolor) and lactadherin-FITC (G, red pseudocolor), shown 180 seconds following laser…

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  Figure 4. TMEM16E supports vessel-wall fibrin formation. Thrombus formation following laser injury of the cremasteric arteriole was monitored for 180 seconds in TMEM16E–/– (Ano5–/–) or TMEM16E+/+ (Ano5+/+) littermate controls in the presence of vehicle (A–F and M) or eptifibatide (10 μg/g of body weight) (G–L and N). Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to DyLight 405 and 488, respectively. PS externalization was monitored with annexin V conjugated to Alexa Fluor 647. Kinetics and magnitudes of median integrated RFUs for platelet (A and G), annexin V (C and I), and fibrin (E and K) accumulation are shown following laser injury. AUC for fluorescence intensity was determined for platelets (B and H), annexin V (D and J), and fibrin (F and L) for each thrombus. Lines represent the median AUC for individual thrombi (vehicle 16E+/+ n = 41, 16E–/– n = 46; eptifibatide 16E+/+ n = 42 16E–/– n = 38) analyzed by Mann-Whitney U test, *P < 0.05; ***P < 0.001. Injury sizes associated with the thrombi analyzed above under vehicle (M) and eptifibatide (N), analyzed by Student’s t test. Time to cessation of bleeding (O) and total hemoglobin loss…

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  Figure 5. TMEM16 antagonists reduce EC procoagulant activity by inhibiting PS externalization. (A) HUVECs were treated with Ca2+ ionophore A23187 for 20 minutes, and PS externalization was measured by flow cytometry. Histograms were generated after gating on live (DAPI negative) cells only. (B) HUVECs were treated with TNF-α (10 ng/mL) for 3.5 hours and/or A23187 for 20 minutes at indicated concentrations and analyzed for factor VIIa–catalyzed activation of factor X. Anti-TF antibody (10 μg/mL) and lactadherin (100 nM) were used to block TF and PS, respectively. All cells not receiving anti-TF antibody were treated with IgG isotype control. Asterisks denoting statistical significance show comparison with cells treated with TNF-α, no ionophore, unless otherwise specified, with brackets to indicate pairwise comparison. (C) HUVECs were treated with A23187 (6 μM) for 20 minutes in the presence of TMEM16 inhibitors CaCCinh-A01 (A01) and BBR (both 10 μM) and analyzed for PS externalization as in A. (D) TMEM16 antagonists were assayed for their ability to inhibit factor VIIa–catalyzed activation of factor X on HUVECs stimulated with TNF-α (10 ng/mL, 3.5 hours) followed by A23187 (6 μM, 20…

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  Figure 6. BBR inhibits thrombosis without increasing bleeding following tail transection. Thrombus formation following laser injury of the cremasteric arteriole was monitored for 180 seconds in mice treated with BBR (5 μg/g of body weight) or vehicle (A–F and M) and BBR or vehicle in the presence of eptifibatide (10 μg/g of body weight) (G–L and N). Platelet and fibrin accumulation were monitored by anti-CD42b and anti-fibrin antibody conjugated to DyLight 405 and 488, respectively. PS externalization was monitored with annexin V conjugated to Alexa Fluor 647. Kinetics and magnitude of median integrated RFUs for platelet (A and G), annexin V (C and I), and fibrin (E and K) accumulation are shown following laser injury. AUC for fluorescence intensity was determined for platelets (B and H), annexin V (D and J), and fibrin (F and L) for each thrombus. Lines represent median AUC for individual thrombi (vehicle n = 41, BBR n = 47; eptifibatide + vehicle n = 34 eptifibatide + BBR n = 35) analyzed by Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (M and N) Injury sizes associated with the thrombi analyzed above, analyzed by Student’s t test. Time to cessation of…