SNAP23 is essential for platelet and mast cell development and required in connective tissue mast cells for anaphylaxis

Rodolfo A Cardenas,Ricardo Gonzalez,Elizabeth Sanchez,Marco A Ramos,Eduardo I Cardenas,Alejandro I Rodarte,Roberto J Alcazar-Felix,Alejandro Isaza,Alan R Burns,Ruth Heidelberger,Roberto Adachi

doi:10.1016/j.jbc.2021.100268

Rodolfo A Cardenas, Ricardo Gonzalez + Show 9 more

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https://doi.org/10.1016/j.jbc.2021.100268

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Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.

Highlights

Mast cells (MCs) modulate local and systemic inflammation through the release of inflammatory mediators stored in their large secretory granules [1]
The expression of Cre recombinase is driven in megakaryocytes and platelets by the promoter of platelet factor 4 (Pf4) [27], and synaptosomal-associated protein of 23 KDa (SNAP23) is deleted in the platelets of their progeny (SNAP23pΔ/Δ mouse)
Cre is expressed in MCs under the control of the promoter of chymase 1 (Cma1), known as mouse MC protease 5, deleting SNAP23 in a subset of MCs of the pups (SNAP23mΔ/Δ mouse)

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Mast cells (MCs) modulate local and systemic inflammation through the release of inflammatory mediators (e.g., histamine) stored in their large secretory granules [1]. We did not detect expression of SNAP25 despite loading 10x more total protein from MC and platelet lysates than from control tissues (Fig. 1A). When we studied the platelets from our mutant mice under EM, we observed that SNAP23-deficient platelets appeared to be larger and to have fewer alpha granules (Fig. 2, A and B).

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