Recombinant scinderin, an F-actin severing protein, increases calcium- induced release of serotonin from permeabilized platelets, an effect blocked by two scinderin-derived actin-binding peptides and phosphatidylinositol 4,5-bisphosphate

Abstract

In response to vessel injury or exposure to different substances, platelets undergo activation which consists of shape changes, formation of cellular pseudopodia, aggregation, and secretion. These dramatic changes are accompanied by cycles of actin depolymerization and polymerization. Previous work has shown the presence in platelets of gelsolin and scinderin, two Ca(2+)-dependent F-actin severing proteins. Recent published evidence suggests that scinderin is a component of the exocytotic machinery in chromaffin cells. The present work describes the preparation of recombinant scinderin and peptides Sc-ABP1 and Sc-ABP2 with sequences corresponding to two actin-binding sites of scinderin. Recombinant scinderin and peptides Sc-ABP1 and Sc-ABP2 were tested for their effects on Ca(2+)-induced serotonin release from digitonin permeabilized platelets. The results indicated that recombinant scinderin potentiates Ca(2+)-evoked serotonin release, an effect blocked in the presence of Sc-ABP1, Sc-ABP2, exogenous gamma-actin, or the addition of phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of a mismatched peptide (MMP) the potentiating effect of recombinant scinderin was not affected. Moreover, Sc-ABP1, Sc-ABP2, and gamma-actin inhibited Ca(2+)-induced release of serotonin in the absence of recombinant scinderin, suggesting an inhibition of platelet endogenous scinderin. MMP was ineffective under these conditions. The results suggest that F-actin disassembly, perhaps at a specific site, is required for platelet secretion and that scinderin might be an important component of the exocytotic machinery in platelets.

Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis

“…Proteins were obtained by osmotic shockate to a purity of at least 80%, as described previously . The r-Sc was further purified by actin-Dnase-I-sepharose 4B affinity chromatography as described by Marcu et al 1996). Fusion proteins were dialyzed against de-ionized water and lyophilized as described previously Zhang et al 1996).…”

The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

“…However, unlike Gelsolin, Ads's F-actin severing activity is inhibited by a wider range of membrane lipids [20] – [22] , and its activation requires lower intracellular calcium concentrations [23] . Ads has been heavily implicated in regulating exocytosis through a rapid depolymerization of cortical actin in a number of biological systems, including chromaffin cells [24] , airway goblet cells [25] , murine pancreatic β-cell [26] and platelets [27] . In addition, Ads has been identified as a member of a multi-protein complex required for the trafficking of the water channel aquaporin-2 in rat renal collecting ducts [28] and has been shown to play a role in the differentiation of platelets [29] and chondrocytes [30] .…”

Intracellular function in rehydrated lyophilized platelets

“…Our finding that RL platelets both degranulate and increase surface membrane thrombogenicity in an activation‐dependent manner provides evidence for intracellular signalling and positive feedback (unpublished observations). Degranulation in platelets is the end‐point of intracellular signalling events that involve protein kinase C activation (Elzagallaai et al , 2000), actin reorganization (Marcu et al , 1996) and the assembly of VAMP‐SNAP‐syntaxin membrane fusion complexes (Reed et al , 1999; Chen, 2000). Increases in surface membrane thrombogenicity occur when an activation‐dependent intracellular calcium signal stimulates ‘scrambalase’ activities that flip phosphatidylserine from the inner to the outer surface of the platelet (Bevers et al , 1996).…”