Platelet lamellipodium formation is not required for thrombus formation and stability

During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow

Die Rolle der WASH-Komplexuntereinheit Strumpellin in der Thrombozytenfunktion

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.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

The WASH-complex subunit Strumpellin regulates integrin αIIbβ3 trafficking in murine platelets.

Acknowledgements: We thank Daniela Naumann as well as Petra Hauck for excellent technical assistance and Dr. Timo Vögtle for critical proofreading. We also thank Dr. Andreas Schlosser (mass spectrometry) from the Rudolf Virchow Center for providing technical infrastructure. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) project number 452622720 to M.B. LMM was funded by a CRUK core Grant A24452.Funder: Universitätsklinikum Würzburg (8913)The platelet specific integrin αIIbβ3 mediates platelet adhesion, aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbβ3 is expressed on the membrane surface and in intracellular compartments. Upon activation, the number of surface-expressed αIIbβ3 is increased by the translocation of internal granule pools to the plasma membrane. The WASH complex is the major endosomal actin polymerization-promoting complex and has been implicated in the generation of actin networks involved in endocytic trafficking of integrins in other cell types. The role of the WASH complex and its subunit Strumpellin in platelet function is still unknown. Here, we report that Strumpellin-deficient murine platelets display an approximately 20% reduction in integrin αIIbβ3 surface expression. While exposure of the internal αIIbβ3 pool after platelet activation was unaffected, the uptake of the αIIbβ3 ligand fibrinogen was delayed. The number of platelet α-granules was slightly but significantly increased in Strumpellin-deficient platelets. Quantitative proteome analysis of isolated αIIbβ3-positive vesicular structures revealed an enrichment of protein markers, which are associated with the endoplasmic reticulum, Golgi complex and early endosomes in Strumpellin-deficient platelets. These results point to a so far unidentified role of the WASH complex subunit Strumpellin in integrin αIIbβ3 trafficking in murine platelets