Filamin A Deficiency in Platelets Reveals Functional Impairment in ITAM-Based Signaling.

Abstract

Abstract 769Filamin A (FlnA), encoded by the FLNA gene on the X-chromosome, crosslinks F-actin and controls the topology of the Von Willebrand factor (VWF) receptor and the integrin αIIbβ3 on platelets by connecting them to the underlying cytoskeleton. In mice, FlnA deficiency is embryonic lethal to hemizygous males. Heterozygous (HT) females are born, but experience premature mortality. FlnA confers survival value to platelets since HT female mice have a mild thrombocytopenia and 95% of their circulating platelets are FlnA positive. Mice specifically lacking FlnA expression in platelets were generated by breeding FlnAloxP/loxP females with GATA1-Cre males. FlnAloxP/y GATA1-Cre males have a severe macrothrombocytopenia and increased tail bleeding time. Their platelets lack a normal cytoskeleton and have decreased GPIbα expression, which lacks cytoskeletal linkage and has an altered surface distribution by electron microscopy. Binding of VWF to FlnA-null platelets was decreased in response to botrocetin. Under arterial shear conditions (shear rate 1,500/s), fluorescent FlnA-null platelets covered ∼70% less of the collagen-coated area via collagen-bound VWF than WT platelets. Unexpectedly, immunoreceptor tyrosine-based activation motif (ITAM)-mediated signals were severely compromised in the absence of FlnA. FlnA-null platelets had decreased α-granule secretion and integrin αIIbβ3 activation in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2 (CLEC-2). Protein tyrosine phosphorylation, including that of the protein tyrosine kinase Syk and phospholipase C-γ2, was severely impaired in FlnA-null platelets stimulated through GPVI and CLEC-2. Interaction between FlnA and Syk was detected using immunoprecipitation and purified proteins. The data reveal a novel interaction between FlnA and Syk regulating ITAM-containing receptor signaling and platelet function. Disclosures:No relevant conflicts of interest to declare.