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Munc13-4 in hemostasis and airway inflammation
Munc13-4 regulates platelet dense granule exocytosis
To test how different Munc13 isoforms contribute to platelet granule secretion, we assessed exocytosis of each type of platelet granule. We measured the translocation of P-selectin (alpha granules) and LAMP-1 (lysosomal gran- ules) to the plasma membrane, and the release of ATP (dense granules). To interrogate potential agonist-depen- dent differences, we activated platelets with collagen (Figure 2) and thrombin (Figure 3). Based on dose-response curves (Online Supplementary Figure S2), we chose a high and a low dose for each agonist. In agreement with previ- ous reports, the concentration of collagen necessary to induce translocation of membrane-associated proteins (P-selectin and LAMP-1) was higher than that required to induce release of soluble mediators (ATP).35 There were no differences among all the wild-type (WT) controls from our Munc13-2, Munc13-4 and double KO (DKO) colonies, therefore we pooled them in a single control group (WT in Figures 2 and 3).
We observed that dense granule exocytosis was com- pletely abolished in platelets lacking Munc13-4 regardless of the agonist used (Figures 2A and B, and 3A and B). With collagen, we documented a clear correlation between the level of expression of Munc13-4 (nil in Δ/Δ, −/− and DKO, approx. 20% in F/F, approx. 50% in +/−, and 100% in WT) and the secretion of ATP (Figure 2B). Although we observed a significant decrease in P-selectin translocation only at the lower dose of thrombin (Figures 2C and D, and 3C and D), we found a complete thrombin dependent defect in lysosomal granule exocytosis in platelets lacking Munc13-4 (Figures 2E and F, and 3E and F). Munc13-2−/− platelets showed no defects in any of these tests. To assess if the consequences of removing Munc13-2 would mani- fest only in the absence of Munc13-4, we tested platelets from DKO mice and did not observe any additional exo- cytic defects (Figures 2 and 3).
Figure 1. Munc13 expression and deletion. (A) RT-qPCR of all Munc13 proteins relative to β-actin in C57BL/6J platelets. N=3. (B) RT-qPCR from Munc13-2−/− platelets. All values are relative to β-actin and Munc13 iso- form levels in Munc13-2+/+ platelets (WT). N=3. Bar: mean; error bar: Standard Error of Mean. (C) Representative immunoblots of platelet and spleen lysates from all Munc13-4 mutant mice probed with anti-mouse Munc13-4 antibody. β-actin was used as loading control. Numbers below blots represent densitometry results relative to β-actin and Munc13-4+/+. ND: not detected.
Given the secondary effect of Munc13-4 on alpha gran- ule release,9,10 we decided to further interrogate alpha gran- ule exocytosis by measuring the release of platelet factor 4 (PF4). Interestingly, Munc13-4−/− platelets had a significant reduction in PF4 release when stimulated with thrombin (Figure 3G). To test if the defects observed in alpha and lysosomal granule exocytosis could be indirectly caused by an impaired release of ADP from dense granules, we added exogenous ADP to stimulated platelets and showed that it was capable of rescuing PF4 release (Figure 3G) and P-selectin translocation (Figure 3H), but not LAMP-1 translocation (Figure 3I). Once again, Munc13-2−/− platelets had no exocytic defect and DKO platelets failed to show any additional phenotype (Figure 3G); we therefore decid- ed to drop these two lines from subsequent experiments.
Platelet granule biogenesis and platelet activation are Munc13-4-independent
The exocytic defects observed in platelets lacking Munc13-4 could be due to defective platelet granule bio- genesis. Therefore, we analyzed resting platelets by elec- tron microscopy (EM) (Figure 4A). Stereological analysis of platelet profiles before stimulation revealed no differ- ence in the volume densities (Vv) of alpha or dense gran- ules among all genotypes (Figure 4B and C). EM also pro- vided an additional method to assess platelet exocytosis. After stimulation, Vv of alpha and dense granules in Munc13-4+/+ platelets decreased due to loss of granules through exocytosis. However, Munc13-4-deficient (Munc13-4−/− and Munc13-4Δ/Δ) platelets lost no dense gran- ules (Figure 4C) and only a fraction of alpha granules (Figure 4B). Munc13-4F/F platelets showed an intermediate phenotype. To rule out the possibility that lack of Munc13-4 might interfere with inside-out platelet activa- tion, we measured integrin αIIbβ3 activation, and found no differences (Figure 4D).
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