The complementary roles of VAMP-2, -3, and -7 in platelet secretion and function

Abstract

Platelet secretion requires Soluble N-ethylmaleimide Sensitive Attachment Protein Receptors (SNAREs). Vesicle SNAREs/Vesicle-Associated Membrane Proteins (v-SNAREs/VAMPs) on granules and t-SNAREs in plasma membranes mediate granule release. Platelet VAMP heterogeneity has complicated the assessment of how/if each is used and affects hemostasis. To address the importance of VAMP-7 (V7), we analyzed mice with global deletions of V3 and V7 together or platelet-specific deletions of V2, V3, and global deletion of V7. We measured the kinetics of cargo release, and its effects on three injury models to define the context-specific roles of these VAMPs. Loss of V7 minimally affected dense and α granule release but did affect lysosomal release. V3^-/-7^-/- and V2^Δ3^Δ7^-/- platelets showed partial defects in α and lysosomal release; dense granule secretion was unaffected. In vivo assays showed that loss of V2, V3, and V7 caused no bleeding or occlusive thrombosis. These data indicate a role for V7 in lysosome release that is partially compensated by V3. V7 and V3, together, contribute to α granule release, however none of these deletions affected hemostasis/thrombosis. Our results confirm the dominance of V8. When it is present, deletion of V2, V3, or V7 alone or in combination minimally affects platelet secretion and hemostasis.

Keywords: TI-VAMP; V-SNAREs; cellubrevin; endobrevin; platelet exocytosis; platelet granules.

Figure 1.. SNARE levels in platelets from V3^-/−7^−/− and V2^Δ3^Δ7^−/− animals.

(A) A collage of representative western blots (n>3) comparing protein levels among V3^-/−7^−/− and V2^Δ3^Δ7^−/− platelets. Washed platelets (5 × 10⁷ platelets per lane) were prepared from wild-type (WT), V3^-/−7^−/− and V2^Δ3^Δ7^−/− mice and the indicated proteins were probed for by immuno-blotting. The actin loading control in A was run on the same gel as SNAP23 and that in B was run on the same gel as fibrinogen. (C) Ratio of fibrinogen/actin in each of the strains studied-WT, V3^-/−7^−/− and V2^Δ3^Δ7^−/− . Each data point represents an individual experiment. Data are from n>3. Statistical significance was calculated using a two-tailed, paired t-test. ns not significant, * p≤0.05.

Figure 2.. Loss of V3, and V7 together affects the kinetics and extent of α granule and lysosomal secretion.

[³H]-5-[HT] (serotonin)–labeled platelets from wild type (WT) and V3^-/−7^−/− mice were prepared as described in 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. (A-C) For the thrombin dose-response experiment, platelets were stimulated for 2 min 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 is representative of ≥3 independent experiments. Statistical significance was calculated using a two-way ANOVA. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 3.. Loss of V2, V3 and V7 together affects the kinetics and extent of α granule and lysosomal secretion.

[³H]-5-[HT] (serotonin)–labeled platelets from wild type (WT) and V2^Δ3^Δ7^−/− mice were prepared as in 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. (A-C) For the thrombin dose-response experiment, platelets were stimulated for 2 min with the indicated concentrations of thrombin. (D-F) For the time-course experiments, platelets were stimulated with 0.05 U/mL thrombin. Data are mean ± standard error of the mean of triplicate measurements and represent ≥3 independent experiments. Statistical significance was calculated using a two-way ANOVA. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001

Figure 4:. V3^-/−7^−/− and V2^Δ3^Δ7^−/− platelets have lower P-selectin and LAMP-1 exposure but normal integrin α_IIbβ₃ activation.

Washed platelets from wild type (WT), V3^-/−7^−/−, and V2^Δ3^Δ7^−/− mice were held resting or stimulated with thrombin (0.1 U/mL) for 2 min and then incubated with FITC–conjugated anti–P-selectin (A), PE-conjugated LAMP1 (B), FITC–conjugated CD41/61 (C, D), or PE-conjugated JonA (E, F) antibodies for 20 min at room temperature. Fluorescence intensities were measured by flow cytometry, and the ratios of stimulated vs. resting GMFI were calculated and plotted. The data represents ≥ 3 independent experiments. Statistical significance was calculated using a two-way ANOVA. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001

Figure 5:. Hemostasis and thrombosis are not impaired in V3^-/−7^−/− and V2^Δ3^Δ7^−/− animals.

Thrombosis and hemostasis were measured in wild type (WT), V3^-/−7^−/−, and V2^Δ3^Δ7^−/− mice using three different injury models: tail bleeding times after tail transection, WT (n=37), V3^-/−7^−/− (n=8), and V2^Δ3^Δ7^−/− (n=36); (A) occlusive thrombosis in the carotid artery injury, induced by topical application of 6% FeCl₃, WT (n=10), V3^-/−7^−/− (n=8), and V2^Δ3^Δ7^−/− (n=9); (B) and bleeding cessation after puncture of the jugular vein, WT (n=10), V7^−/− (n=6), V3^-/−7^−/− (n=10), and V2^Δ3^Δ7^−/− (n=16). (C) Sex-based distribution of the data is shown in A’, B’, and C’ respectively. The data were analyzed using a log-rank test and pairwise multiple comparisons by Holm-Sidak. P values are indicated; ns not significant; * p ≤ 0.05.