S Hofmann1, T Vögtle, M Bender, S Rose-John, B Nieswandt. 1. Chair of Vascular Medicine, University of Würzburg, University Hospital and Rudolf Virchow Center for Experimental Biomedicine, Würzburg, Germany.
Abstract
BACKGROUND AND OBJECTIVE: Ectodomain shedding is a major mechanism to modulate platelet receptor signaling and to downregulate platelet reactivity. Proteins of the a disintegrin and metalloproteinase (ADAM) family are implicated in the shedding of various platelet receptors. The signaling lymphocyte activation molecule (SLAM) family receptor CD84 is highly expressed in platelets and immune cells, but its role in platelet physiology is not well explored. Because of its ability to form homodimers, CD84 has been suggested to mediate contact-dependent signaling and contribute to thrombus stability. However, nothing is known about the cellular regulation of CD84. METHODS: We studied the regulation of CD84 in murine platelets by biochemical approaches and use of three different genetically modified mouse lines. Regulation of CD84 in human platelets was studied using inhibitors and biochemical approaches. RESULTS: We show that CD84 is cleaved from the surface of human and murine platelets in response to different shedding inducing agents and platelet receptor agonists. CD84 downregulation occurs through ectodomain-shedding and intracellular cleavage. Studies in transgenic mice identified ADAM10 as the principal sheddase responsible for CD84 cleavage, whereas ADAM17 was dispensable. Western blot analyses revealed calpain-mediated intracellular cleavage of the CD84 C-terminus, occurring simultaneously with, but independently of, ectodomain shedding. Furthermore, analysis of plasma and serum samples from transgenic mice demonstrated that CD84 is constitutively shed from the platelet surface by ADAM10 in vivo. CONCLUSIONS: These results reveal a dual regulation mechanism for platelet CD84 by simultaneous extra- and intracellular cleavage that may modulate platelet-platelet and platelet-immune cell interactions.
BACKGROUND AND OBJECTIVE: Ectodomain shedding is a major mechanism to modulate platelet receptor signaling and to downregulate platelet reactivity. Proteins of the a disintegrin and metalloproteinase (ADAM) family are implicated in the shedding of various platelet receptors. The signaling lymphocyte activation molecule (SLAM) family receptor CD84 is highly expressed in platelets and immune cells, but its role in platelet physiology is not well explored. Because of its ability to form homodimers, CD84 has been suggested to mediate contact-dependent signaling and contribute to thrombus stability. However, nothing is known about the cellular regulation of CD84. METHODS: We studied the regulation of CD84 in murine platelets by biochemical approaches and use of three different genetically modified mouse lines. Regulation of CD84 in human platelets was studied using inhibitors and biochemical approaches. RESULTS: We show that CD84 is cleaved from the surface of human and murine platelets in response to different shedding inducing agents and platelet receptor agonists. CD84 downregulation occurs through ectodomain-shedding and intracellular cleavage. Studies in transgenic mice identified ADAM10 as the principal sheddase responsible for CD84 cleavage, whereas ADAM17 was dispensable. Western blot analyses revealed calpain-mediated intracellular cleavage of the CD84 C-terminus, occurring simultaneously with, but independently of, ectodomain shedding. Furthermore, analysis of plasma and serum samples from transgenic mice demonstrated that CD84 is constitutively shed from the platelet surface by ADAM10 in vivo. CONCLUSIONS: These results reveal a dual regulation mechanism for platelet CD84 by simultaneous extra- and intracellular cleavage that may modulate platelet-platelet and platelet-immune cell interactions.
Authors: Constance C F M J Baaten; Frauke Swieringa; Tomasz Misztal; Tom G Mastenbroek; Marion A H Feijge; Paul E Bock; Marjo M P C Donners; Peter W Collins; Renhao Li; Paola E J van der Meijden; Johan W M Heemskerk Journal: Blood Adv Date: 2018-09-25
Authors: Sebastian Hofmann; Attila Braun; Rastislav Pozgaj; Martina Morowski; Timo Vögtle; Bernhard Nieswandt Journal: PLoS One Date: 2014-12-31 Impact factor: 3.240
Authors: Michael K Schuhmann; Guido Stoll; Michael Bieber; Timo Vögtle; Sebastian Hofmann; Vanessa Klaus; Peter Kraft; Mert Seyhan; Alexander M Kollikowski; Lena Papp; Peter U Heuschmann; Mirko Pham; Bernhard Nieswandt; David Stegner Journal: Circ Res Date: 2020-07-30 Impact factor: 17.367