Alterations in platelet secretion differentially affect thrombosis and hemostasis by Smita Joshi, Meenakshi Banerjee, Jinchao Zhang, Akhil Kesaraju, Irina D. Pokrovskaya, Brian Storrie, and Sidney W. Whiteheart BloodAdv Volume 2(17):2187-2198 September 11, 2018 © 2018 by The American Society of Hematology

Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology

Generation of V2Δ 3Δ and V2Δ 3Δ 8−/− animals. Generation of V2Δ3Δand V2Δ3Δ8−/−animals. (A) A collage of representative western blots comparing protein levels among V2Δ3Δ, V2Δ3Δ8+/−, and V2Δ3Δ8−/− platelets. Washed platelets (5 × 107 platelets per lane) were prepared from WT, V2Δ3Δ, V2Δ3Δ8+/−, and V2Δ3Δ8−/− mice, and the indicated proteins were probed for. The blots for VAMP7, VAMP8, and Rab GDI were from the same membrane. (B) The VAMP isoforms in V2Δ3Δ, V2Δ3Δ8+/−, and V2Δ3Δ8−/− platelets were quantified using Rab GDI as a loading control and ImageQuant TL software (v 7.0). The data were plotted as the ratio of knockout/WT. The vertical dashed line indicates a ratio of 1. Data are representative of platelets collected from individual mice from each strain and ≥3 independent experiments (see supplemental Figure 1 for additional blots for VAMP7 and VAMP8). A Student t test was used to analyze the data. ***P < .001. Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology

Loss of VAMP2, VAMP3, and VAMP8 affects the kinetics and the extent of platelet secretion. [3H]-5-[HT] (serotonin)–labeled platelets from WT, V8−/−, V2Δ3Δ, and V2Δ3Δ8−/− mice were prepared as described in supplemental Methods. Loss of VAMP2, VAMP3, and VAMP8 affects the kinetics and the extent of platelet secretion. [3H]-5-[HT] (serotonin)–labeled platelets from WT, V8−/−, V2Δ3Δ, and V2Δ3Δ8−/− mice were prepared as described in supplemental Methods. The release of [3H]-5-[HT] from dense granules (A,D), PF4 from α granules (B,E), and β-hexosaminidase from lysosomes (C,F) was measured, and percentage secretion was calculated as described in supplemental Methods. (A-C) For the thrombin dose-response experiment, platelets were stimulated for 2 minutes with the indicated concentrations of thrombin. (D-F) For the time-course experiments, platelets were stimulated with 0.05 U/mL thrombin for the indicated times Data are mean ± standard error of the mean of triplicate measurements and are representative of ≥3 independent experiments. Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology

Depletion of VAMP2, VAMP3, and VAMP8 in platelets affects ATP/ADP release but not aggregation. Depletion of VAMP2, VAMP3, and VAMP8 in platelets affects ATP/ADP release but not aggregation. Aggregation (A-C) and ATP/ADP release (D-F) were monitored simultaneously in a lumi-aggregometer. Washed platelets (4 × 108/mL) from WT (black traces), V2Δ3Δ (dark gray traces), and V2Δ3Δ8−/− (light gray traces) platelets were stimulated with 0.05 U/mL (A), 0.1 U/mL (B), or 0.5 U/mL (C) thrombin (Thr), and ATP release and aggregation were measured for 5 minutes. Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology

V2Δ 3Δ 8−/− platelets have lower P-selectin and LAMP-1 exposure and reduced integrin αIIbβ3 surface levels but normal activation. V2Δ3Δ8−/−platelets have lower P-selectin and LAMP-1 exposure and reduced integrin αIIbβ3 surface levels but normal activation. Washed platelets (5 × 107) from WT, V2Δ3Δ, and V2Δ3Δ8−/− mice were held resting or stimulated with thrombin (0.1 U/mL) for 2 minutes and then incubated with fluorescein isothiocyanate–conjugated anti–P-selectin (A), PE-conjugated LAMP-1 (B), PE-conjugated JonA (C), or fluorescein isothiocyanate–conjugated CD41/61 (D) antibodies for 20 minutes at room temperature. Fluorescence intensities were measured by flow cytometry. Shown are representative data and geometric mean fluorescence intensity (GMFI) (mean ± standard error of the mean) of ≥2 independent experiments. A Student t test was used to analyze the data. *P < .05, **P < .01, and ***P < .001. Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology

The loss of VAMP8 delays fusion pore formation and dilation. The loss of VAMP8 delays fusion pore formation and dilation. Platelets from the indicated strains were isolated and fixed in resting or thrombin-stimulated states (0.1 U/mL thrombin for 90 or 300 seconds). EM tomographs were analyzed for fusion pore structures, and the diameters were measured in z-series. Representative EM images of WT platelets at 90 seconds (A) and at 300 seconds (B), V8−/− platelets at 300 seconds (C), and V2Δ3Δ8−/− platelets at 90 seconds (D). The arrows indicate fusion pores. (E) The distribution of fusion pore size in platelets at early (90 seconds) and late (300 seconds) times poststimulation for the indicated strains is graphed as a scatter plot. Mean pore sizes are as follows: (WT: 90 seconds, 82.4 nm; WT: 300 seconds, 166 nm; V8−/−: 90 seconds, 72 nm; V8−/−: 300 seconds, 160.8 nm; V2Δ3Δ8−/−: 90 seconds, 21 nm, V2Δ3Δ8−/−: 300 seconds, pores could not be detected). Pores detected per platelet profile are indicated. ND, not detected. Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology

Hemostasis and thrombus formation are impaired in V2Δ 3Δ 8−/− animals. Hemostasis and thrombus formation are impaired in V2Δ3Δ8−/−animals. (A) Tail bleeding times were measured after tail transection of WT (n = 28), V3−/− (n = 38), V2Δ3Δ (n = 20), V8+/− (n = 26), V8−/− (n = 31), V2Δ3Δ8+/− (n = 41), and V2Δ3Δ8−/− (n = 34) mice. (B) Thrombus formation in the carotid artery was induced by topical application of 5% FeCl3, and blood flow was monitored in WT (n = 6), V8+/− (n = 6), V2Δ3Δ (n = 7), V2Δ3Δ8+/− (n = 9), V8−/− (n = 7), and V2Δ3Δ8−/− (n = 7) mice. For panels A and B, data are mean ± standard error of the mean. The P values (log-rank test) are indicated and ***P < .001. The red line indicates the mean, and the black line indicates the median. The box represents 25th-75th percentiles. Whiskers/error bars above and below the box indicate the 90th and 10th percentiles, respectively. Outliers are as shown in tail bleeding figure. (C) APTT and PT were measured in platelet-poor plasma prepared from WT (n = 7), V8−/− (n = 5), V2Δ3Δ (n = 8), V2Δ3Δ8+/− (n = 3), and V2Δ3Δ8−/− (n = 5) mice. (D) PS exposure on the surface of thrombin-stimulated platelets from WT, V8+/−, V8−/−, V2Δ3Δ, V2Δ3Δ8+/−, and V2Δ3Δ8−/− mice was measured by fluorescein isothiocyanate–lactadherin binding and flow cytometry. The data are representative of 2 independent assays. *P < .05, Student t test. (E) A representative western blot showing the levels of TMEM16F in the indicated strains. Rab GDI was used as a loading control. Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology

Correlating VAMP-mediated exocytosis to platelet function. Correlating VAMP-mediated exocytosis to platelet function. VAMP levels, secretion, and function are graphically correlated using our data (closed circles) and that from Koseoglu et al15 (open circles). (A) Calculated VAMP levels remaining in each of the strains tested, relative to WT, were based on the data in Figure 1B and from Graham et al.12 (B) The VAMP levels in each strain are represented by green shading. The red line indicates α-granule secretion, and the blue line indicates dense granule secretion. Secretion levels are from Figure 2 (0.05 U/mL thrombin for 2 minutes). Secretion from dense and α granules of V7−/− platelets (open symbols) were estimated based on ATP release and P-selectin exposure, respectively.15 All values are normalized to WT. Dense granule release (C) and α-granule release (D) are as in panel B and are plotted as green shading. The red line indicates tail bleeding times, and the blue line indicates the occlusive thrombosis times in the FeCl3-injury model. Data are normalized to WT. These data were taken from other figures in this article in which variations were indicated; therefore, error indicators were omitted from these graphs to improve figure clarity. Smita Joshi et al. Blood Adv 2018;2:2187-2198 © 2018 by The American Society of Hematology