title: SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice.
abstract: Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.
authors.affiliation: Department of Experimental Medicine, University Hospital Würzburg and Rudolf Virchow Center for Experimental Biomedicine, Würzburg, Germany;
authors.forename: Deya
authors.initials: D
authors.lastname: Cherpokova
authors.affiliation: Department of Experimental Medicine, University Hospital Würzburg and Rudolf Virchow Center for Experimental Biomedicine, Würzburg, Germany;
authors.forename: Markus
authors.identifier: http://orcid.org/0000-0002-2381-116X
authors.initials: M
authors.lastname: Bender
authors.affiliation: Department of Experimental Medicine, University Hospital Würzburg and Rudolf Virchow Center for Experimental Biomedicine, Würzburg, Germany;
authors.forename: Martina
authors.initials: M
authors.lastname: Morowski
authors.affiliation: Department of Neurology, University Hospital Würzburg, Würzburg, Germany;
authors.forename: Peter
authors.initials: P
authors.lastname: Kraft
authors.affiliation: Department of Neurology, University Hospital Würzburg, Würzburg, Germany;
authors.forename: Michael K
authors.initials: MK
authors.lastname: Schuhmann
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, and.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Akbar
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, and.
authors.forename: Cheryl S
authors.initials: CS
authors.lastname: Sultan
authors.affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; and.
authors.forename: Craig E
authors.initials: CE
authors.lastname: Hughes
authors.affiliation: Department of Neurology, University Hospital Würzburg, Würzburg, Germany;
authors.forename: Christoph
authors.initials: C
authors.lastname: Kleinschnitz
authors.affiliation: Department of Neurology, University Hospital Würzburg, Würzburg, Germany;
authors.forename: Guido
authors.initials: G
authors.lastname: Stoll
authors.affiliation: Departments of Pediatrics and Immunology, University of Colorado, School of Medicine, Aurora, CO.
authors.forename: Leonard L
authors.initials: LL
authors.lastname: Dragone
authors.affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; and.
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, and.
authors.forename: Michael G
authors.identifier: http://orcid.org/0000-0002-1189-0091
authors.initials: MG
authors.lastname: Tomlinson
authors.affiliation: Department of Experimental Medicine, University Hospital Würzburg and Rudolf Virchow Center for Experimental Biomedicine, Würzburg, Germany;
authors.forename: Bernhard
authors.initials: B
authors.lastname: Nieswandt
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001792:Blood Platelets / Q000166:cytology; D020520:Brain Infarction / Q000378:metabolism*; D002339:Carotid Arteries / Q000473:pathology; D002462:Cell Membrane / Q000378:metabolism; D003435:Crotalid Venoms / Q000737:chemistry; D007249:Inflammation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D037181:Lectins, C-Type / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D020768:Middle Cerebral Artery / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D015398:Signal Transduction; D012910:Snake Venoms / Q000737:chemistry; D000072377:Syk Kinase; D013927:Thrombosis / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003435:Crotalid Venoms; D047908:Intracellular Signaling Peptides and Proteins; C491734:Lat protein, mouse; D037181:Lectins, C-Type; D008565:Membrane Proteins; D010750:Phosphoproteins; D010980:Platelet Membrane Glycoproteins; C095148:Sla protein, mouse; C486695:Sla2 protein, mouse; D012910:Snake Venoms; C098341:platelet membrane glycoprotein VI; C028890:convulxin; D050721:Proto-Oncogene Proteins c-cbl; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D000072377:Syk Kinase; C000606897:Syk protein, mouse; C493849:Cbl protein, mouse