Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis

Abstract

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.

Keywords: Munc18; SNARE proteins; exocytosis; hemostasis; platelet; secretion; thrombosis.

Figure 1.

Expression and deletion of Munc18 proteins. A, RT-qPCR of all Munc18 proteins relative to β-actin in C57BL/6J platelets. n = 3–4; bar, mean; error bar, S.E. B, representative immunoblots of platelet and control tissue lysates probed with anti-mouse Munc18-1, Munc18-2, or Munc18-3 antibody. Bands for the three Munc18 isoforms ran at ∼67 kDa. β-Actin (∼42 kDa) was used as loading control. PCMCs, peritoneal cell-derived mast cells; black triangles, 75-kDa molecular mass marker; white triangles, 50-kDa molecular mass marker; gray triangles, 37-kDa molecular mass marker.

Figure 2.

Deletion of Munc18-2 impairs dense, alpha, and lysosomal granule release in thrombin-stimulated platelets. Samples from Munc18-1 (A–C), Munc18-2 (D–F), and Munc18-3 (G–I) mutant mice were stimulated with thrombin. A, D, and G, ATP release (dense granules) measured by luminometry in whole blood. n = 6–9. B and C, E and F, and H and I, mean fluorescence intensity over baseline (ΔMFI) of P-selectin (alpha granules) (B, E, and H) and LAMP-1 (lysosomal granules) (C, F, and I) translocated to the surface of washed platelets measured by flow cytometry. n = 8–10. Color legends in A, D, and G apply to A–C, D–F, and G–I, respectively. White line, mean; box, 25th–75th percentile; whiskers, 5th–95th percentile. #, p < 0.05; †, p ≤ 0.01; *, p ≤ 0.001; comparisons are to WT controls (+/+) unless otherwise specified.

Figure 3.

Defective exocytosis in Munc18-2–deficient platelets is independent of the agonist used and cannot be rescued by exogenous ADP. Samples from Munc18-2 mutant mice were stimulated with collagen (10 μg/ml unless otherwise specified) or thrombin (1 unit/ml) in the absence or presence of ADP (10 μm). A, ATP release (dense granules) measured by luminometry in whole blood. n = 6. B and C, PF4 release (alpha granules) measured by ELISA. n = 5–6. D and E, mean fluorescence intensity over baseline (ΔMFI) of P-selectin (alpha granules) (D) and LAMP-1 (lysosomal granules) (E) translocated to the surface of washed platelets measured by flow cytometry. n = 6. The color legend in A applies to all panels. White line, mean; box, 25th–75th percentile; whiskers, 5th–95th percentile. †, p ≤ 0.01; *, p ≤ 0.001; comparisons are to WT controls (+/+) unless otherwise specified.

Figure 4.

Deletion of Munc18-2 impairs granule release but not granule biogenesis. Washed platelets from Munc18-2 mutant mice were activated with thrombin (0.1 unit/ml). A, representative EM cell profiles before (top row) and after (bottom row) stimulation. Black triangle, example of a dense granule; white triangle, example of an alpha granule; scale bars, 1 μm. B–D, values obtained by design-based stereology from ∼200 platelet profiles from three animals of each genotype. B and C, volume density (Vv) of alpha granules (B) and dense granules (C) in resting and activated platelets. D, surface density (Sv) for both types of granules in resting platelets. Color legend in B applies to B–D. White line, mean; box, 25th–75th percentile; whiskers, 5th–95th percentile. †, p ≤ 0.01; *, p ≤ 0.001; comparisons are to WT controls (+/+) unless otherwise specified.

Figure 5.

Deletion of Munc18-2 impedes platelet aggregation and interferes with thrombus formation in vitro. A and B, samples from Munc18-2 mutant mice were stimulated with collagen. Representative tracings of platelet aggregation (A) and maximum aggregation measured by light transmittance (B) on platelet-rich plasma. n = 6. C–F, whole blood was labeled fluorescently and perfused over collagen-coated plates at low (C and D) or high (E and F) shear stress. Thrombus buildup (C and E) was monitored by the change in fluorescence intensity over baseline (ΔMFI) and compared at 200 s (D and F). n = 5–8; AU, arbitrary units. The color legend in A applies to all panels. Circle or white line, mean; error bar, S.E.; box, 25th-75th percentile; whiskers, 5th–95th percentile. #, p < 0.05; †, p ≤ 0.01; *, p ≤ 0.001; comparisons are to WT controls (+/+) unless otherwise specified.

Figure 6.

Deletion of Munc18-2 in platelets disrupts hemostasis and thrombosis. A and B, tail bleeding times that depends mostly on arterial (A) and venous (B) bleeding were recorded in Munc18-2 mutant mice. n = 8–10. C, time to carotid occlusion after applying FeCl₃ abluminally for 3 min. n = 6–8. Circle, individual mouse; horizontal line, mean. #, p < 0.05; †, p ≤ 0.01; *, p ≤ 0.001; comparisons are to WT controls (+/+) unless otherwise specified.