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E.I. Cardenas et al.
A
Figure 4. Deletion of Munc13-4 impairs dense granule release. Washed platelets from Munc13-4 mutant mice were activated with thrombin 0.1 U/mL. (A) Representative electron microscopy (EM) cell profiles. Red arrows: dense granules. Scale bar: 1 mm. Mean volume density (Vv) of alpha granules (AG) (B) and dense granules (DG) (C) obtained by
BCD stereology. (D) Mean fluorescence intensity over baseline (ΔMFI) of Jon/A antibody binding to activated integrin αIIbβ3 on stimulated washed platelets. N=5. Color leg- end in (B) applies to all panels. Bar: mean; error bar: Standard Error of Mean. #P<0.05; †P<0.01; *P<0.001; comparisons are with Munc13-4+/+ unless otherwise
specified.
Table 1. Blood cell counts from Munc13-4 mutant mice. Munc13-4+/+
Munc13-4−/−
9.1 ± 0.5 2.8 ± 0.6 611 ± 42
Munc13-4F/F
8.5 ± 0.6 3.1 ± 0.4 668 ± 47
Munc13-4Δ/Δ 8.0 ± 0.3
2.6 ± 0.3 645 ± 50
Red blood cells (x1012/L) White blood cells (x109/L)
Platelets (x109/L)
Mean ± Standard Error of Mean. N=7-12.
8.3 ± 0.3 2.2 ± 0.3 613 ± 28
and D). However, there was a significant decrease in eosinophilic inflammation, which was also independent of Cre expression. Qualitatively, Munc13-4Δ/Δ and Munc13-4−/− mice had reduced histological evidence of tis- sue eosinophilia (Figure 7E), and quantitatively, a decreased number of eosinophils in BAL fluid (Figure 7F).
Discussion
Platelet exocytosis
Exocytosis occurs constitutively in all eukaryotic cells, but some cell types possess a parallel regulated process to release pre-made mediators upon stimulation in a spa- tiotemporally-controlled manner. All known forms of reg- ulated exocytosis require a Munc13 protein.31,34 Platelets and mast cells rely on regulated exocytosis as their main effector mechanism, and both express Munc13-2 and -4. Like in mast cells, we found that Munc13-4 plays an important role in granule release, but in contrast to mast cells we documented no role for Munc13-2 in platelet exo- cytosis (Figures 2 and 3). An alternative explanation is that a minor role for Munc13-2 could not be resolved by our methods, which cannot yield the high resolution attained by single-cell membrane capacitance recordings in mast cells.32
Previous studies, as well as our current findings in secre- tion assays and morphometry (Figures 2-4), indicate that Munc13-4 regulates exocytosis of platelet dense granules directly.8,9 No ATP release could be detected after Munc13-4 was deleted, and platelets with decreased expression of Munc13-4 released proportionally less ATP than their WT counterparts regardless of the agonist used. By studying the relationship between Munc13-4 expres- sion levels (0, 20, 50, 100%) and dense granule release (Figures 2 and 3), we have shown that Munc13-4 is an agonist-independent, rate-limiting factor in dense granule exocytosis. Interestingly, it has been proposed that, besides its known role in priming, Munc13-4 participates in dense granule tethering and conveys Ca2+ sensitivity to platelet exocytosis.7 We found no involvement of Munc13-4 in alpha or lysosomal granule exocytosis when we used collagen as agonist. With thrombin, we detected a partial exocytic defect in both types of granules, but only alpha granule release could be rescued by exogenous ADP. This confirms that deletion of Munc13-4 indirectly impairs alpha granule exocytosis,10 but that the alpha gran- ule exocytic machinery is intact.
There is evidence that the release of the three types of granules from platelets is differentially regulated,2 and it is possible that each requires different exocytic components.
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