TY - THES A1 - Reil, Lucy Honor T1 - The role of WASH complex subunit Strumpellin in platelet function T1 - Die Rolle der WASH-Komplexuntereinheit Strumpellin in der Thrombozytenfunktion N2 - Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets. Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin. In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear. N2 - Strumpellin ist Teil des hoch konservierten, pentameren WASH-Komplexes, der den Arp2/3-Komplex auf Endosomen aktiviert und somit die Bildung eines verzweigten Aktinnetzwerkes ermöglicht. Der WASH-Komplex beteiligt sich an der Bildung und Sta-bilisierung von endosomalen Retrieval-Subdomänen und Transportvesikel. In letztere werden Proteine verpackt und anschließend zu ihrem Bestimmungsort innerhalb der Zelle, z.B. der Zellmembran, transportiert. Die Rolle von Strumpellin in der Thrombozytenfunktion und im endosomalen Transport wurde bislang noch nicht untersucht. Hierfür wurde eine konditionale Knockout-Mauslinie generiert, die weder in Megakaryozyten noch in Thrombozyten Strumpellin aufwies. Der konditionale Knockout von Strumpellin hatte nur einen milden Thrombozytenphänotyp zur Folge. Der Verlust von Strumpellin resultierte in einem verminderten Gesamt-proteingehalt von αIIbβ3-Integrin in Thrombozyten, einschließlich einer ca. 20-prozentigen Reduktion der Oberflächenexpression von αIIbβ3 und einer verringerten αIIbβ3-Aktivierung nach Thrombozytenaktivierung. Die reduzierte Oberflächenexpression von αIIbβ3 konnte auch in Megakaryozyten nachgewiesen werden. Die Expression anderer Oberflächenglykoproteine war nicht betroffen. Thrombozytenzahl, -größe und -morphologie blieben unverändert. Die reduzierte αIIbβ3-Expression in Thrombozyten führte zu einer verminderten Fibrinogenbindungskapazität nach Thrombozytenaktivierung. Die Fibrinogenaufnahme unter ruhenden Bedingungen, trotz initialer Verzögerung, und der Gesamtproteingehalt von Fibrinogen waren hingegen vergleichbar mit Kontrollproben. Interessanterweise verursachte die reduzierte αIIbβ3-Expression keine in vitro Spreading- und Aggregationsdefekte der Thrombozyten. Ein verminderter WASH1-Proteingehalt konnte ebenfalls nachgewiesen werden. Abschließend lässt sich sagen, dass der Verlust von Strumpellin die Thrombozytenfunktion, zumindest in vitro, nicht beeinträchtigt. Die Daten zeigen jedoch, dass Strumpellin eine selektive Rolle in der Regulierung der αIIbβ3-Oberflächenexpression spielt. Als WASH-Komplexuntereinheit könnte Strumpellin möglicherweise das Recycling von αIIbβ3 zurück zur Thrombozytenoberfläche regulieren. Zudem könnten verbleibende WASH-Komplexuntereinheiten trotz fehlendem Strumpellin weiterhin einen funktions- fähigen Komplex bilden. Dies könnte unter anderem die nur 20-prozentige Reduktion der αIIbβ3 Oberflächenexpression erklären. Der genaue Mechanismus ist jedoch noch nicht bekannt. KW - Strumpellin KW - WASH complex KW - endosomal trafficking KW - alpha-IIb beta-3 KW - platelet Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242077 ER - TY - JOUR A1 - Osmanoglu, Özge A1 - Gupta, Shishir K. A1 - Almasi, Anna A1 - Yagci, Seray A1 - Srivastava, Mugdha A1 - Araujo, Gabriel H. M. A1 - Nagy, Zoltan A1 - Balkenhol, Johannes A1 - Dandekar, Thomas T1 - Signaling network analysis reveals fostamatinib as a potential drug to control platelet hyperactivation during SARS-CoV-2 infection JF - Frontiers in Immunology N2 - Introduction Pro-thrombotic events are one of the prevalent causes of intensive care unit (ICU) admissions among COVID-19 patients, although the signaling events in the stimulated platelets are still unclear. Methods We conducted a comparative analysis of platelet transcriptome data from healthy donors, ICU, and non-ICU COVID-19 patients to elucidate these mechanisms. To surpass previous analyses, we constructed models of involved networks and control cascades by integrating a global human signaling network with transcriptome data. We investigated the control of platelet hyperactivation and the specific proteins involved. Results Our study revealed that control of the platelet network in ICU patients is significantly higher than in non-ICU patients. Non-ICU patients require control over fewer proteins for managing platelet hyperactivity compared to ICU patients. Identification of indispensable proteins highlighted key subnetworks, that are targetable for system control in COVID-19-related platelet hyperactivity. We scrutinized FDA-approved drugs targeting indispensable proteins and identified fostamatinib as a potent candidate for preventing thrombosis in COVID-19 patients. Discussion Our findings shed light on how SARS-CoV-2 efficiently affects host platelets by targeting indispensable and critical proteins involved in the control of platelet activity. We evaluated several drugs for specific control of platelet hyperactivity in ICU patients suffering from platelet hyperactivation. The focus of our approach is repurposing existing drugs for optimal control over the signaling network responsible for platelet hyperactivity in COVID-19 patients. Our study offers specific pharmacological recommendations, with drug prioritization tailored to the distinct network states observed in each patient condition. Interactive networks and detailed results can be accessed at https://fostamatinib.bioinfo-wuerz.eu/. KW - signaling network KW - controllability KW - platelet KW - SARS-CoV-2 KW - fostamatinib KW - drug repurposing KW - COVID-19 Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-354158 VL - 14 ER -