Katharina Grundler1, Raffaela Rotter2, Sloane Tilley2, Joachim Pircher3, Thomas Czermak3, Mustaf Yakac3, Erik Gaitzsch2, Steffen Massberg4, Florian Krötz5, Hae-Young Sohn6, Ulrich Pohl7, Hanna Mannell2, Bjoern F Kraemer8. 1. Walter Brendel-Zentrum, Ludwig-Maximilians Universitaet Muenchen, Schillerstr. 44, 80336 Muenchen, Gemany; Medizinische Klinik und Poliklinik I, Klinikum der Universitaet Muenchen, Ziemssenstr. 1, 80336 Muenchen, Germany. 2. Walter Brendel-Zentrum, Ludwig-Maximilians Universitaet Muenchen, Schillerstr. 44, 80336 Muenchen, Gemany. 3. Medizinische Klinik und Poliklinik I, Klinikum der Universitaet Muenchen, Ziemssenstr. 1, 80336 Muenchen, Germany. 4. Medizinische Klinik und Poliklinik I, Klinikum der Universitaet Muenchen, Ziemssenstr. 1, 80336 Muenchen, Germany; DZHK (German Center for Cardiovascular Research) partner site Munich Heart Alliance, Munich, Germany. 5. Klinikum Starnberg, Osswaldstr.1, 82319 Starnberg, Germany. 6. MediCenter Germering, Hartstr. 52, 82110 Germering, Germany. 7. Walter Brendel-Zentrum, Ludwig-Maximilians Universitaet Muenchen, Schillerstr. 44, 80336 Muenchen, Gemany; DZHK (German Center for Cardiovascular Research) partner site Munich Heart Alliance, Munich, Germany. 8. Medizinische Klinik und Poliklinik I, Klinikum der Universitaet Muenchen, Ziemssenstr. 1, 80336 Muenchen, Germany. Electronic address: bjoern.kraemer@med.uni-muenchen.de.
Abstract
INTRODUCTION: Platelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. MATERIAL AND METHODS: Using fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. RESULTS: We show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. CONCLUSIONS: This work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.
INTRODUCTION: Platelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. MATERIAL AND METHODS: Using fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. RESULTS: We show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. CONCLUSIONS: This work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.
Authors: Marion Mussbacher; Manuel Salzmann; Christine Brostjan; Bastian Hoesel; Christian Schoergenhofer; Hannes Datler; Philipp Hohensinner; José Basílio; Peter Petzelbauer; Alice Assinger; Johannes A Schmid Journal: Front Immunol Date: 2019-02-04 Impact factor: 7.561
Authors: Bjoern F Kraemer; Marc Geimer; Mirita Franz-Wachtel; Tobias Lamkemeyer; Hanna Mannell; Stephan Lindemann Journal: Int J Mol Sci Date: 2020-10-31 Impact factor: 5.923