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dependent platelets.40 Therefore, partial defects in ADP release would be expected to translate into thrombi that weaken and break as shear stress increases, and this is exactly what we observed in a perfusion chamber in hypomorphic Munc13-4F/F platelets (Figure 5). Moreover, we obtained a strong hemostatic defect in Munc13-4F/F mice when we transected the tail artery, but no prolonged bleeding time when we sectioned only the tail dorsal vein (Figure 6). Taken together, these results show that by con- trolling platelet dense granule release, Munc13-4-depen- dent exocytosis is a limiting factor for platelet aggregation, thrombus stabilization and hemostasis.
Allergic airway inflammation
Asthma is a chronic airway disease characterized by excessive airway constriction to non-specific stimuli (also known as AHR), airway eosinophilic inflammation, and epithelial mucous metaplasia.41 Although hyperproduc- tion of mucins and secretion of mucus by airway epithelial cells have been shown to be a component of AHR,42 we found that platelet exocytosis mediated by Munc13-4 did not influence airway mucous metaplasia. Instead, the decrease in AHR in animals with platelets deficient in Munc13-4 correlated with a reduction in the number of eosinophils recruited to the airways (Figure 6).
Platelets store in their alpha and dense granules many mediators and membrane-bound proteins linked to aller- gic airway inflammation. Platelet P-selectin mediates the recruitment of eosinophils and lymphocytes into the air- ways of mice subjected to a model of asthma,43 and recent evidence suggests a similar function in humans.44,45 Additionally, exocytosis of alpha granule contents may influence inflammation through many chemokines; for example, PF4 can induce AHR in rats.46
Platelets actively take up serotonin from plasma and concentrate it in their dense granules, becoming the main source of serotonin outside the central nervous system.47 Serotonin modulates adhesion, migration, and cytokine and chemokine production in inflammatory cells and lung epithelial cells.48 Serotonin also induces bronchoconstric- tion, and mice unable to synthesize serotonin in peripher- al tissues had a marked decrease in platelet serotonin and AHR.26 ADP can induce changes on the endothelium that result in leukocyte transendothelial migration via the P2Y1 and P2Y2 receptors,49,50 and global deletion of the ADP receptor P2Y12 in mice has been shown to partially protect from asthma.27 Even though treatment with the P2Y12 inhibitor clopidogrel had contradictory outcomes in mouse models of asthma,27-29 it is currently being tested in asthmatic patients (clinicaltrials.gov identifier: 01955512). On the other hand, the PRINA trial failed to show a significant decrease in mannitol-induced AHR in patients with asth- ma treated with prasugrel.51 The mechanism of action of anti-P2Y12 thienopyridines in allergic inflammation needs to be clarified, because it may be acting on eosinophils and
not necessarily on platelets.28 Other molecules present in dense granules such as ATP,52 glutamate53 and polyphos- phates54 can influence different steps of the allergic inflam- matory response. Therefore, the lack of Munc13-4 in platelets could significantly affect the development of asthma by directly impeding dense granule exocytosis and indirectly hindering alpha granule release.
Up until now all the evidence supporting a role for platelets in asthma has come from in vitro studies,55 in vivo experimentation on platelet-depleted animals24,25,56 or glob- al KO mice,23,26,27,43 and from pharmacological studies that targeted receptors expressed in multiple cell types.28-30 Here we show in mice with normal numbers of circulating platelets that selective deletion of Munc13-4 in platelets results in reduced AHR and lung eosinophilia, most likely because of impaired dense granule release.
It has been proposed that platelet activation could lead to either a hemostatic or an inflammatory response inde- pendently of each other.18 A recent publication suggests that low concentrations of collagen are capable of induc- ing platelet secretion of soluble factors, without causing aggregation or any of the other characteristic features of platelet activation.35 Studies on mouse models of allergic and bacterial airway inflammation suggest that platelet P2Y12 participates mostly in hemostasis, P2Y14 in inflam- mation, and P2Y1 in both.29,30 The ligand of P2Y1 (ADP) is stored in platelet dense granules, but it is unclear whether the ligand for P2Y14 (UDP-glucose) comes from platelets57 or from other cells.58 Hence, the defect in dense granule exocytosis we noted in platelets lacking Munc13-4 could result in failure to activate P2Y1, P2Y12 and perhaps P2Y14. The simultaneous failure to activate all these receptors could explain why our mutant animals have both abnor- mal hemostasis and decreased allergic airway inflamma- tion.
Acknowledgments
The authors would like to thank Margaret M. Gondo (University of Houston) for her assistance with the electron microscopy, Kimberly Langlois and Ngoc-Anh Bui-Thanh (Baylor College of Medicine) for their assistance with the in vivo thrombosis assay, and Terry Hoppe (Texas A&M Institute of Biosciences and Technology) for his assistance manufacturing the tail-bleeding device.
Funding
This project was supported by the National Institutes of Health AI093533A, CA016672 and HL116524, and the Department of Veterans Affairs Merit Review Award I01 BX002551. EIC received support from Instituto Tecnológico y de Estudios Superiores de Monterrey and Consejo Nacional de Ciencia y Tecnología (CONACyT, PhD grant scholarship # 352566). The contents of this manuscript do not represent the views of the Department of Veterans Affairs or the United States Government.
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