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mation, and organ infiltration by CD8+ T cells and macrophages.15 Platelets from FHL3 patients present defective degranulation,16 and bleeding diathesis has been reported in some patients with FHL3.17
There is mounting evidence that platelets participate in inflammation, including allergic airway disease.18 Patients with asthma have lower platelet counts19 and increased levels of markers of platelet activation20 after allergen exposure. Platelets have been found extravascularly in the airways,21 and platelet products have been measured in bronchoalveolar lavage (BAL) fluid of asthmatic patients.22 This suggests that platelets migrate to the lungs during asthma, which has been experimentally confirmed in mice.23 In animal models of asthma, platelet depletion has been shown to decrease the number of leukocytes infil- trating the airways24 and bronchoconstriction induced by allergen.25 Serotonin and/or ADP released from platelet dense granules may be responsible for these findings. In a mouse model of asthma, deletion of the enzyme that syn- thesizes serotonin in peripheral tissues caused a significant reduction in asthmatic symptoms.26 Multiple studies in mouse models of asthma have targeted the ADP receptor P2Y12 with mostly favorable outcomes,27,28 but there is evi- dence that the pro-inflammatory effects of platelets may be mediated by P2Y1 (another ADP receptor) and P2Y14 (a UDP-glucose receptor), and not P2Y12.29,30
Given that platelets participate in highly distinct physi- ological responses (hemostasis and airway inflammation), we investigated whether exocytosis of dense granules, so crucial for hemostasis, is also important for the develop- ment of asthma. We manipulated the expression of Munc13-2 and -4 in platelets in vivo. While absence of Munc13-2 had no significant effect, absence or reduced levels of Munc13-4 altered platelet dense granule secretion directly and alpha granule exocytosis indirectly, impairing platelet aggregation and thrombus formation. By using platelet-specific knockout (KO) mice, we proved that Munc13-4 from platelets, and not from other tissues, is required for venous and arterial hemostasis, and for arteri- al thrombosis in vivo. Finally, we observed that Munc13-4- dependent platelet exocytosis is essential for the full development of allergic airway inflammation.
Methods
A detailed description of the blood collection and platelet isola- tion, expression studies, secretion and activation assays, electron microscopy and stereology, aggregometry and flow-chamber assays, in vivo thrombosis model, and statistical analysis is provid- ed in the Online Supplementary Methods.
Mice
Munc13-2 KO and Munc13-4 global and conditional KO mice have been described previously.31,32 In short, we flanked exon 3 of the mouse Munc13-4 gene (Unc13d) with two loxP sites (“floxed” or F allele). Due to this genetic manipulation, mice homozygous for the F allele (Munc13-4F/F) had a reduced expression of Munc13-4 globally (i.e. Munc13-4F/F mice are hypomorphs). Exon 3 contains the start codon of Unc13d, its sequence is present in all described splice variants of mouse Munc13-4, and its removal by Cre-mediated recombination eliminates Munc13-4 expression.32 We crossed Munc13-4F/F mice with B6.C-Tg(CMV-cre)1Cgn/J mice (The Jackson Laboratory #006054), which express Cre recombi- nase ubiquitously, to delete Unc13d in the germ line33 and generate
our Munc13-4 global KO line (Munc13-4−/−). This line was propa- gated by heterozygote (Munc13-4+/−) crossings to generate Munc13-4−/−, Munc13-4+/− and control Munc13-4+/+ littermates. We also generated megakaryocyte/platelet-specific KO mice (Munc13-4Δ/Δ) by crossing Munc13-4F/F mice with C57BL/6– Tg(Pf4–icre)Q3Rsko/J mice (Munc13-4+/+ Cre+; The Jackson Laboratory #008535), which selectively express Cre recombinase in megakaryocytes. Munc13-4+/+ Cre+ mice were also used as addi- tional controls.
All lines were on a C57BL/6J background, as confirmed by speed-congenics scanning for 105 SNPs. Mice of both sexes were used in all experiments. Mice were kept in a pathogen-free facility and handled in accordance with the Institutional Animal Care and Use Committees of the University of Texas MD Anderson Cancer Center and Baylor College of Medicine.
Bleeding time tests
We used mice (20±2 weeks old) of the same weight (30±3 g) anesthetized with Avertin (tribromoethanol in tert-amyl alcohol) 0.4 mg/g intraperitoneally (i.p.). In the transection model, tails were cut 5 mm from the tip with a razor blade, and bleeding depended mainly on the tail artery. For the incision model, we cre- ated a device to make a reproducible transversal dorsal tail incision 0.8 mm in depth at a point where the tail has a diameter of 3.8 mm (Online Supplementary Figure S1), sectioning only the dorsal tail venous plexus. In both models, the tails were immediately immersed in 37°C saline and the time to cessation of bleeding was recorded. All animals were euthanized after bleeding stopped or at 20 minutes (min).
Asthma model
Mice (9±1 weeks old) were sensitized i.p. on days 0 and 7 with 10 mg ovalbumin (OVA; grade V) adsorbed to 1 mg of aluminum potassium sulfate dodecahydrate (both from Sigma-Aldrich) in 100 mL of saline. They were challenged once a day on days 19-21 in a nebulization chamber with 1% OVA in PBS for 30 min using an Aerotech II jet nebulizer (Biodex) at 10 L/min. Then, they were studied on day 22. A detailed description of the airway mechanics assessment, airway mucin quantification, histology and BALs is provided in the Online Supplementary Methods.
Results
Expression and targeting of Munc13 proteins in platelets
By qPCR, we found that C57BL/6J platelets express Munc13-1, -2 and -4 (Figure 1A). We had created Munc13-4 global and conditional KO lines,32 and we obtained Munc13-2 KO mice (Dr. Christian Rosenmund, Charité Universitaetsmedizin).31 We did not study Munc13-1 because its global deletion is perinatally lethal34 and a conditional KO line was not available. We confirmed lack of Munc13-2 and normal expression lev- els of Munc13-1 and -4 in Munc13-2−/− platelets (Figure 1B). Immunoblots of tissues and platelets from all Munc13-4 mutants confirmed the global reduction and absence of Munc13-4 expression in Munc13-4+/− and Munc13-4−/−, respectively, and the specific deletion of Munc13-4 in platelets in Munc13-4Δ/Δ mice (Figure 1C). The decreased expression in Munc13-4F/F mice to approx- imately 20% has been reported,32 and we used it to inter- rogate dose-response relationships between Munc13-4 expression (0, 20, 50, 100%) and the outcomes of our experiments.
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