The WASH-complex subunit Strumpellin regulates integrin αIIbß3 trafficking in murine platelets.

The platelet specific integrin αIIbß3 mediates platelet adhesion, aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbß3 is expressed on the membrane surface and in intracellular compartments. Upon activation, the number of surface-expressed αIIbß3 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 αIIbß3 surface expression. While exposure of the internal αIIbß3 pool after platelet activation was unaffected, the uptake of the αIIbß3 ligand fibrinogen was delayed. The number of platelet α-granules was slightly but significantly increased in Strumpellin-deficient platelets. Quantitative proteome analysis of isolated αIIbß3-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 αIIbß3 trafficking in murine platelets.

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 αIIbß3 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.