Defects in Filamin A (FLNa) in Women with X-Linked Filaminopathies: Abnormalities in Platelet Production and Platelet Function

Abstract 1152

Filamin (FLNa) is a major component of the platelet cytoskeleton. Platelet FLNa has a critical role in attaching the Von Willebrand Factor (VWF) receptor GPIb-IX-V and integrins to the actin cytoskeleton. Recently, in an animal model, FlnA has been shown to play an important role in platelet function involving ITAM-containing receptor signalling and also in platelet production. Filaminopathies linked to mutations in the X-linked FLNA gene are associated with a range of rare diseases such as periventricular nodular heterotopia (PVNH), otopalatidigital and cardiac valvular dystrophy. We have studied three women with FLNA mutations. For two of them (P1and P2) mutations were associated with PVNH, for the third patient (P3) only macrothrombocytopenia was detected. For patient P1 a heterozygous frameshift point mutation (c.4573_4insA, p.Tyr1525X) was present in exon 27 of FLNA. For patient 2, comparative genomic hybridization (array-CGH) revealed a heterozygous intragenic deletion involving FLNA exons 31–32 to 48. Patient P3 has a novel heterozygous point mutation c.5407G>A, p.Glu1803Lys affecting a glutamic acid residue that is highly conserved between species. Electron microscopy showed for all the patients a population of large platelets with a round shape and abnormal distribution of alpha-granules. In vitro culture of megakaryocytes (MKs) showed an abnormal and faster CD34+ platelet production compared to controls. Many MKs showed signs of abnormal fragmentation (confocal microscopy and EM) indicating an important role of FlnA in the early formation of proplatelets and in the localisation of granules. For patients P1 and P2 a minor population of platelets and also of MKs was found to be devoid of FLNa; for patient P3 with the missense mutation the localization of FLNa was heterogeneous with zones of individual MKs lacking FLNa. Western Blotting revealed a degraded form of FLNa in platelets that was particularly evident for P3 but residual intact FLNa always remained present in this X-linked syndrome.

Platelet function testing in all patients showed an abnormal response to collagen and VWF. Thrombus formation under flow (300s⁻¹) on collagen, was significantly decreased for the P1 and P3 but was partially restored for P1 at 1500s⁻¹. The signalling pathway of GPVI, as shown by the phosphorylation of the tyrosine kinase Syk, was reduced. On a VWF matrix at 1500s⁻¹, thrombus formation was identical between control and P1 and P2 but increased for P3. However, at higher shear rates (5000s⁻¹), thrombus formation was drastically diminished for P1 while normal for P2 and remained increased for P3.

In comparison with published animal studies where Flna is specifically deleted in the MK lineage (Jurak Begonja A, et al, Blood 2011 June 7, online) our results for women with different FLNA mutations suggest milder defects of the megakaryocytic-platelet lineage than in the mouse model and depending on the location of the mutation. For all patients we found giant platelets, and an abnormal distribution of FLNa. More variability was found for platelet function. These results resemble those obtained for the mice that linked the GPVI signalling pathway to FLNa. Nevertheless, with all the different mutations an altered platelet production led to the presence of giant platelets with an abnormal granule distribution. These results showing that platelet production was affected for all cases imply that platelet production is very dependant on FLNa concentration and integrity.