Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis

Abstract

Edited by Peter CresswellPlatelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis. 3 The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; FHL-5, familial hemophagocytic lymphohistiocytosis type 5; KO, knockout; LAMP, lysosomal-associated membrane protein; MFI, mean fluorescence intensity; Munc, mammalian homolog of C. elegans uncoordinated gene; PF4, platelet factor 4; PRP, platelet-rich plasma; Stx, syntaxin; Stxbp, Stx-binding protein; VAMP, vesicle-associated membrane protein; qPCR, quantitative PCR.cro ARTICLE 4784

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

“…In our previous studies on platelet and MC exocytosis, we documented that removal of proteins crucial for this process did not affect cell ultrastructure, development or activation, a fundamental step before studying any functional defect ( 5 , 6 , 7 , 8 , 9 , 10 , 28 ).…”

“…Due to the thrombocytopenia, altered expression of plasma membrane receptors, significant changes in platelet structure, and faulty activation present when SNAP23 was removed, it made no sense to proceed to in vivo studies of thrombosis and hemostasis as we have done before ( 9 , 10 ), since they would not lead to any clear conclusion.…”

“…We have reported that deletion of Munc13-4 and Munc18-2 affects specifically platelet degranulation, based on the fact that the deletions did not perturb platelet development and activation ( 9 , 10 ). Although it has been suggested that the removal of SNAP23 renders the exocytic machinery of platelets nonfunctional ( 21 ), the severe alterations in platelet morphology and activation we observed in SNAP23-deficient platelets prevent us from drawing any clean conclusion from exocytic assays.…”

“…Several homologs of these proteins participate in regulated exocytosis in other cell types, and we have studied the roles of Stx-3 and -4, Syt-2, Munc13-1 and -2, and Munc18-1, -2, and -3 in MCs and platelets. The MC provides a system to study single-vesicle and compound exocytosis at high resolution, and platelets allow the study of the differential regulation of three different exocytic compartments simultaneously ( 5 , 6 , 7 , 8 , 9 , 10 ).…”

Cytoskeletal Transport, Reorganization, and Fusion Regulation in Mast Cell-Stimulus Secretion Coupling

“…This concurs also with results indicating that STX3 is a major t-SNARE in MC exocytosis. Munc18-2 KD also affects secretion in other hematopoietic cells, being responsible for the secretory defect in FLH5 patients carrying different mutations ( Cote et al, 2009 ; Cardenas et al, 2019 ). By contrast, no roles for Munc18-1 and Munc18-3 could be delineated in MC, although Munc18-3 seemed to play a role in neutrophil exocytosis ( Martin-Verdeaux et al, 2003 ; Brochetta et al, 2008 ; Gutierrez et al, 2018 ).…”