A gain‐of‐function filamin A mutation in mouse platelets induces thrombus instability

Abstract

Background: Filaminopathies A are rare disorders affecting the brain, intestine, or skeleton, characterized by dominant X-linked filamin A (FLNA) gene mutations.Macrothrombocytopenia with functionally defective platelets is frequent. We have described a filaminopathy A male patient, exhibiting a C-terminal frame-shift FLNa mutation (Berrou et al., Arterioscler Thromb Vasc Biol. 2017;37:1087-1097. Contrasting with female patients, this male patient exhibited gain of platelet functions, including increased platelet aggregation, integrin αIIbβ3 activation, and secretion at low agonist concentration, raising the issue of thrombosis risk.Objectives: Our goal is to assess the thrombotic potential of the patient FLNa mutation in an in vivo model. Methods:We have established a mutant FlnA knock-in mouse model. Results:The mutant FlnA mouse platelets phenocopied patient platelets, showing normal platelet count, lower expression level of mutant FlnA, and gain of platelet functions: increased platelet aggregation, secretion, and αIIbβ3 activation, as well as increased spreading and clot retraction. Surprisingly, mutant FlnA mice exhibited a normal bleeding time, but with increased re-bleeding (77%) compared to wild type (WT) FlnA mice (27%), reflecting hemostatic plug instability. Again, in an in vivo thrombosis model, the occlusion time was not altered by the FlnA mutation, but arteriolar embolies were increased (7-fold more frequent in mutant FlnA mice versus WT mice), confirming thrombus instability. Conclusions:This study shows that the FlnA mutation found in the male patient induced gain of platelet functions in vitro, but thrombus instability in vivo. Implications for the role of FLNa in physiology of thrombus formation are discussed.

Over‐expression of Dyrk1A affects bleeding by modulating plasma fibronectin and fibrinogen level in mice

“…Quantification of αIIbβ3 activation in conditions of unstirred platelets in whole blood was assessed by flow cytometry using the JON/A monoclonal antibody specific for the activated form of αIIbβ3. Isolated platelets were obtained as previously described 24 . Platelet surface β‐galactose exposure was determined using fluorescein isothiocyanate (FITC)‐conjugated RCA or ECL (RCA #FL‐1081; ECL #FL‐1141; Vector Laboratories) as described 25 .…”

“…Isolated platelets were obtained as previously described. 24 Platelet surface β‐galactose exposure was determined using fluorescein isothiocyanate (FITC)‐conjugated RCA or ECL (RCA #FL‐1081; ECL #FL‐1141; Vector Laboratories) as described. 25 Washed platelets (3 × 10 8 /mL) were stimulated with a range of activators (convulxin, ADP, or thrombin).…”

Filamin A in platelets: Bridging the (signaling) gap between the plasma membrane and the actin cytoskeleton

“…The patient’s platelets exhibited increased aggregation, secretion, and αIIbβ3 integrin activity, as well as an increased association between talin and the β3 subunit ( Berrou et al, 2017 ). The same research group recently generated a knock-in mouse that recapitulates the FLNA mutation; platelets from this knock-in mouse essentially replicate the gain-of-function phenotype observed in the human subject ( Adam et al, 2022 ). These data clearly point to the functional importance of the FLNA-αIIbβ3 integrin association with regards to hemostasis and thrombosis.…”