Cofilin-1 – Induced Actin Reorganization and Phosphatidylserine Exposure in Platelets
Authors: Dasgupta Swapan , Le Anhquyen , Haudek Sandra , Entman Mark , Thiagarajan Perumal 
Published: October 2019

Abstract
Background: Platelet activation leads to the transbilayer movement of phosphatidylserine (PS) from inner to the outer leaflet of membrane bilayer. Exteriorization of PS promotes platelet procoagulant activity by promoting the assembly of tenase and the prothrombinase complex on platelet membrane. In a previous study, we observed that Rho-associated coiled-coil kinase-1 (ROCK1) deficiency in murine platelets or ROCK inhibition by Y-27632 in human platelets resulted in increase in PS exposure and platelet procoagulant activity. ROCK1 deficient platelets had a marked decrease in phosphorylation of cofilin-1. Cofilin-1 decreases actin filament length by increasing the rate of dissociation of actin monomers and its activity is abolished by phosphorylation. These studies suggested a role for cofilin-1-induced actin reorganization in collagen-induced PS exposure. Cofilin-1 activity is also modulated by its interactions with phosphatidylinositol 4, 5-bisphosphate (PIP2) and a cofilin-1 binding protein, Wdr1, which enhances capacity of cofilin-1 to accelerate depolymerization by capping their barbed ends. Here, we studied the role of cofilin-1 phosphorylation and its interaction with PIP2 and Wdr1 in activation induced PS exposure in platelets.
Materials and Methods: We isolated platelet membrane, cytosol and cytoskeleton through differential centrifugation from resting and collagen-stimulated platelets and assessed the relative abundance of cofilin-1 and phosphocofilin-1 by mobility shift in phosphate-affinity polyacrylamide gel electrophoresis. This method allows simultaneous detection of relative proportions of phosphoproteins and its nonphospho counterparts. For cofilin-1 and Wdr-1 distribution, platelets were immobilized on a polylysine-coated cover slip or on a collagen-coated cover slip, fixed, permeabilized and immunostained with appropriate antibody. In addition, PS exposure (FITC lactadherin binding), F-actin (Alexa Fluor 488-phalloidin) and calcium concentration (Fura-2AM fluorescence) were also measured.
Results: Immunofluorescence images show that in resting platelets, cofilin-1 is present in distinct patches in the plasma membrane and following activation with collagen, cofilin-1 is redistributed in a discrete granular pattern throughout the cytoplasm. In parallel, we also studied the relative distribution and the phosphorylation status of cofilin-1 in various subcellular fractions of platelets. In resting platelets, cofilin-1 is present in the plasma membrane and in the cytosol but absent in the cytoskeleton. Following activation with collagen, cofilin-1 moves to the cytoskeleton with a concomitant decrease in the plasma membrane fraction. These results suggest that active cofilin-1 is incorporated into reorganizing actin cytoskeleton during platelet activation. In ROCK1-deficient mice, there is increased cofilin-1 in cytoskeletal fraction, which also correlates with increase in PS exposure. Blocking PIP2 hydrolysis by U73122 prevents cofilin-1 translocation and PS exposure. A cell permeable cofilin-1 peptide containing the actin binding site of cofilin-1 (amino acids Trp104 to Met115) at the carboxy terminus (that competitively inhibits cofilin-1 binding to F-actin), blocks cofilin-1 translocation and decreases PS exposure. Platelets from a Wdr1-deficient mice which express less than 2% of wild-type protein in platelets, showed impaired collagen-induced PS exposure despite equivalent increase in intra platelet calcium compared to wild-type platelets.
Conclusion: Our results show cofilin-1 trafficking from membranre to cytoskeleton and subsequent actin reorganization precedes PS exposure during platelet activation. In ROCK1-deficient platelets, there is increased cofilin-1 activity due to decreased serine-3 phosphorylation and it is associated with increased cofilin-1 translocation to actin filaments and enhanced PS exposure. In contrast, inhibiting cofilin-1 translocation, either by inhibiting PIP2 hydrolysis or by competitive inhibition by a cell permeable peptide, prevents cofilin-1 trafficking and decreases PS exposure. Furthermore, in Wdr1-deficient mouse platelets, which have diminished cofilin-1 activity, have impaired PS exposure during platelet activation.

Disclosures
No relevant conflicts of interest to declare.