Filamin-A susceptibility to calpain-mediated cleavage as a marker of dynamic conformational changes in intact platelets

Abstract

Filamin-A (FlnA), an actin-binding protein that organizes the actin cytoskeleton and mechanically links transmembrane glycoproteins to the cytoskeleton, associates with platelet receptors integrin IIb{beta}3, glycoprotein-Ib (GPIb), and integrin 2{beta}1. Fibrinogen, von Willebrand Factor (vWF) and collagen, binding to these receptors mechanically connect the extracellular matrix to the cytoskeleton. Here we identified that under standardized conditions, platelet activation and ligand binding to IIb{beta}3, GPIb, or 2{beta}1, generates reproducible patterns of FlnA cleavage after platelet lysis. We exploited this novel assay to study the impact of ligand binding and receptor activation on the platelet cytoskeleton. We identified that: i) the FlnA modification that renders it cleavable by calpain after platelet lysis, requires both ligand binding and either internal force (e.g; clot retraction) or external forces (e.g; stirring and aggregating), ii) FlnA modification depends on actin polymerization downstream of integrin IIb{beta}3 and integrin 2{beta}1, but not downstream of GPIb signaling, iii) FlnA modification is reversible in time when platelets are activated with convulxin (Cvx), collagen, von Willebrand factor (vWF) or thrombin receptor activating peptide (T6), and this reversibility correlates with platelet dissagregation, iv) in contrast to the reversible nature of platelet aggregation and FlnA modification by activation with Cvx, collagen, ristocetin or T6, when platelets are activated by thrombin the platelet do not disaggregate and FlnA remains cleavable. Our data demonstrate that IIb{beta}3, 2{beta}1 and GPIb can each exert tension on the cytoskeleton by virtue of binding ligand under conditions of shear. We further identified a unique role for IIb{beta}3-fibrin interactions in creating sustained cytoskeletal tension, with implications for thrombus stability and clot retraction.