REGULAR ARTICLE IkB kinase 2 is not essential for platelet activation Manuel Salzmann, 1 Sonja Bleichert, 1,2 Bernhard Moser, 1 Marion Mussbacher, 1 Mildred Haase, 1 Bastian Hoesel, 1 Waltraud C. Schrottmaier, 1 Julia B. Kral-Pointner, 1 Makoto Itakura, 3 Katy Schmidt, 4 Alice Assinger, 1 and Johannes A. Schmid 1 1 Institute of Vascular Biology and Thrombosis Research and 2 Department of Surgery, General Hospital, Medical University of Vienna, Vienna, Austria; 3 Department of Biochemistry, Kitasato University School of Medicine, Kanagawa, Japan; and 4 Division of Cell and Developmental Biology, Medical University of Vienna, Vienna, Austria Key Points • NF-kB and its activator, IkB kinase 2 (IKK2, IKKb), are central in- flammatory molecules in various cell types, including anucleate platelets. • Neither platelet-specific deletion of IKK2 by ex- cision of exon 3 nor pharmacological inhibi- tion of IKK2 affects platelet function. Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IkB kinase 2 (IKK2), the central enzyme of the inflammatory NF-kB pathway, is involved in platelet activation, because megakaryocyte/ platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function. Introduction Platelets are key players in hemostasis, and granule secretion is essential for their function. Although platelets lack a nucleus, it has been postulated that the pathway that leads to activation of the inflammatory transcription factor NF-kB is important for their activation and degranulation. 1 In general, NF-kB is kept inactive by binding to inhibitory molecules (IkBs). A plethora of stimuli leads to phosphorylation of I kBs by IkB kinases (IKKs), triggering their proteasomal degradation and the release of NF-kB. Most of these activating pathways converge at IKK2, which is the main IkB- phosphorylating enzyme in the course of NF-kB activation. 2,3 In platelets, adenosine 59-diphosphate (ADP), thrombin, epinephrine, and collagen have been reported to cause activation of the IKK2/IkB/ NF-kB axis. 3 However, although some investigators claim an activating role for this pathway, 1,4 others suggest that it has inhibitory effects, 5 leaving its role in platelet activation incompletely understood. We aimed to resolve these conflicting findings for the nongenomic link between the NF-kB pathway and platelet signaling by using a mouse model with a platelet-specific deletion of IKK2, 6 combined with in-depth analysis of hemostatic and immunomodulatory platelet functions in vitro and in vivo. Methods Detailed information is provided in supplemental Methods. Submitted 1 October 2019; accepted 15 January 2020; published online 19 February 2020. DOI 10.1182/bloodadvances.2019001044. Data sharing requests should be sent to Johannes A. Schmid (johannes.schmid@ meduniwien.ac.at) or Alice Assinger (alice.assinger@meduniwien.ac.at). The full-text version of this article contains a data supplement. © 2020 by The American Society of Hematology 638 25 FEBRUARY 2020 x VOLUME 4, NUMBER 4 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/4/638/1716798/advancesadv2019001044.pdf by guest on 07 March 2023