TY - JOUR A1 - Hofmann, Sebastian A1 - Braun, Attila A1 - Pozgaj, Rastislav A1 - Morowski, Martina A1 - Vögtle, Timo A1 - Nieswandt, Bernhard T1 - Mice lacking the SLAM family member CD84 display unaltered platelet function in hemostasis and thrombosis JF - PLoS One N2 - Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. Objective The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. Methods and Results We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. \(Cd84^{−/−}\) platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of \(Cd84^{−/−}\) mice. In vivo, \(Cd84^{−/−}\) mice exhibited unaltered hemostatic function and arterial thrombus formation. Conclusion These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions. KW - flow cytometry KW - CD coreceptors KW - integrins KW - blood KW - platelet aggregation KW - platelet activation KW - cytotoxic T cells KW - platelets Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126477 VL - 9 IS - 12 ER - TY - JOUR A1 - Nieswandt, Bernhard A1 - Morowski, Martina A1 - Brachs, Sebastian A1 - Mielenz, Dirk A1 - Dütting, Sebastian T1 - The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice N2 - Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. Objective Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. Methods and Results We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. Conclusion These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss. KW - adaptor protein Swiprosin-1/EFhd2 KW - platelets KW - platelet activation KW - platelet aggregation KW - cytoskeleton KW - thrombin KW - blood KW - actins KW - collagens Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113316 ER - TY - THES A1 - Morowski, Martina T1 - Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation T1 - Relevanz der Thrombozytenzahl und des ITAM-Signalwegs in Mausmodellen der Hämostase, Thrombose und Thromboinflammation N2 - Platelets are important players in haemostasis and their activation is essential to limit post-traumatic blood loss upon vessel injury. On the other hand, pathological platelet activation may lead to thrombosis resulting in myocardial infarction and stroke. Platelet activation and subsequent thrombus formation are, therefore, tightly regulated and require a well-defined interplay of platelet surface receptors, intracellular signalling molecules, cytoskeletal rearrangements and the activation of the coagulation cascade. In vivo thrombosis and haemostasis models mimic thrombus formation at sites of vascular lesions and are frequently used to assess thrombotic and haemostatic functions of platelets. In this dissertation, different in vivo models were used in mice to address the question at what level a reduced platelet count (PC) compromises stable thrombus formation. To study this, mice were rendered thrombocytopenic by low-dose anti-GPIbα antibody treatment and subjected to a tail bleeding time assay as well as to four different in vivo thrombosis models. Haemostasis and occlusive thrombus formation in small vessels were only mildly affected even at severe reductions of the PC. In contrast, occlusive thrombus formation in larger arteries required higher PCs demonstrating that considerable differences in the sensitivity for PC reductions exist between these models. In a second part of this study, mice were rendered thrombocytopenic by injection of high-dose anti-GPIbα antibody which led to the complete loss of all platelets from the circulation for several days. During recovery from thrombocytopenia, the newly generated platelet population was characterised and revealed a defect in immunoreceptor tyrosine-based activation motif (ITAM)-signalling. This defect translated into impaired arterial thrombus formation. To further investigate ITAM-signalling in vivo, genetically modified mice were analysed which display a positive or negative regulation of platelet ITAM-signalling in vitro. Whereas mice lacking the adapter Grb2 in platelets showed a delayed thrombus formation in vivo after acetylsalicylic acid treatment, Clp36ΔLIM bone marrow chimeric mice and SLAP/SLAP2-deficient mice displayed pro-thrombotic properties in vivo. Finally, mice lacking the adapter protein EFhd2 were analysed in vitro and in vivo. However, EFhd2-deficient platelets showed only a minor increase in the procoagulant activity compared to control. N2 - Thrombozyten sind wichtige Zellen für die Hämostase, die bei einer Verletzung der Gefäßwand aktiviert werden, um den Blutverlust zu begrenzen. Auf der anderen Seite kann die pathologische Aktvierung von Thrombozyten jedoch zu Thromboseereignissen und in Konsequenz zu Schlaganfall und Herzinfarkt führen. Die Aktivierung von Thrombozyten und die nachfolgende Thrombusbildung sind daher streng reguliert und setzen ein enges Zusammenspiel von Thrombozytenoberflächenrezeptoren, intrazellulären Signalmolekülen, Zytoskelettumstrukturierungen und die Aktivierung der Koagulationskaskade voraus. In vivo Thrombose- und Hämostase-Modelle ahmen die Thrombusbildung in Gefäßen nach und werden häufig benutzt um die hämostatische und thrombotische Funktion von Thrombozyten zu untersuchen. In dieser Dissertation wurden verschiedene in vivo Modelle in Mäusen genutzt, um zu klären, ab welcher Thrombozytenzahl eine stabile Thrombusbildung beeinträchtigt ist. Für diese Untersuchung wurden Mäuse mit geringen Dosen anti-GPIbα Antikörper behandelt, die zu einer Reduktion der Thrombozytenzahl führen. Anschließend wurden die Mäuse in einem Blutungszeitmodel und vier verschiedenen thrombotischen Modellen untersucht. Hämostase oder okklusive Thrombusbildung in kleinen Gefäßen waren auch bei einer sehr starken Reduktion der Thrombozytenzahl kaum beeinträchtigt. Im Gegensatz dazu war für eine normale Thrombusbildung in größeren Gefäßen eine höhere Thrombozytenzahl nötig. Verschiedene in vivo Modelle zeigen daher eine unterschiedliche Sensitivität gegenüber einer Reduktion der Thrombozytenzahl. Im zweiten Teil dieser Arbeit wurden Mäuse mit einer hohen Dosis anti-GPIbα Antikörper behandelt, die zu einem völligen, mehrtägigen Verlust aller Thrombozyten im Blutkreislauf führte. Während sich die Thrombozytenzahl erholte, wurden neu generierte Thrombozyten charakterisiert, die einen deutlichen Defekt im ITAM-Signalweg zeigten. Dieser Defekt führte zu einer verminderten arteriellen Thrombusbildung. Um Auswirkungen eines Defektes im ITAM-Signalweg detaillierter in vivo zu untersuchen, wurden genetisch veränderte Mäuse, die in vitro eine reduzierte oder verstärkte ITAM-Signaltransduktion in Thrombozyten zeigen, untersucht. Während Mäuse, die in Thrombozyten kein Grb2 exprimieren nach Acetylsalicylsäurebehandlung in vivo eine verlangsamte Thrombusbildung zeigten, war die Thrombusbildung in Clp36ΔLIM Knochenmarkchimären und SLAP/SLAP2-defizienten Mäusen beschleunigt. Darüber hinaus wurden EFhd2-defiziente Mäuse in vitro und in vivo analysiert. EFhd2-defiziente Thrombozyten zeigten jedoch nur eine geringe Steigerung in der prokoagulatorischen Aktivität im Vergleich zu Kontrolltieren. KW - Thrombozyt KW - Platelet count KW - ITAM-signalling KW - Murine models of thrombosis and haemostasis KW - Maus KW - Blutstillung KW - Thrombose Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-99193 ER -