Cell, Vol. 82, 643-653, August 25, 1995, Copyright 0 1995 by Cell Press Thrombin Receptor Ligation and Activated Rat Uncap Actin Filament Barbed Ends through Phosphoinositide Synthesis in Permeabilized Human Platelets John H. Hartwig,* Gary M. Bokoch,t Christopher L. Carpenter,* Paul A. Janmey, l Lance A. Taylor,* Alex Toker,S and Thomas P. Stossel’ *Experimental Medicine Brigham and Women’s Hospital 221 Longwood Avenue Boston, Massachusetts 02115 tDepartment of Immunology and Cell Biology The Scripps Research Institute La Jolla, California 92037 *Department of Medicine Division of Hematology Laboratory of Signal Transduction Beth Israel Hospital Boston, Massachusetts 02115 Summary Cells respond to diverse external stimuli by polymeriz- ing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood plate- lets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting per- meabilized platelets. The thrombin receptor-activat- ing peptide (TRAP), GTP, and GTPyS, but not GDPPS, also uncap F-actin in permeabilized platelets. GDPf3S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mu- tant Rat, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin in- hibit F-actin uncapping by TRAP, Rat, and GTPyS. TRAP and Rat induce rapid Pl(4,5)P2 synthesis in per- meabilized platelets. Thefindingsestablish asignaling pathway for actin assembly involving Rat in which the final message is phosphoinositide-mediated F-actin uncapping. Introduction Human platelets are tiny oval discs, 7 fl in volume, that circulate in the blood at a concentration of 2.5 x lOa/ ml. They are subject to stimulation by numerous agents generated by tissue injury, following which they undergo a profound shape transformation. First, the disc expands into a sphere, and then it projects two types of surface protrusions, flat lamellae and finger-like filopodia. The la- mellae adhere to injured surfaces to plug vascular leaks, and the filopodia bind fibrin strands and other platelets to form a three-dimensional blood clot. These reactions are responsible for physiological hemostasis and also for thromboses associated with pathological states such as myocardial infarctions, peripheral vascular insufficiency, and strokes. Remodeling of the actin filament scaffolding that holds the resting platelet in the discoid configuration is responsi- ble for the cellular shape change, and the biochemistry and morphology of the actin remodeling have been the subject of much investigation (Fox, 1993; Fox et al., 1984; Hartwig, 1992; Nachmiaset al., 1993). The polymeric actin cytoskeleton of the resting platelet consists of 2000 actin filaments cross-linked by the actin gelation factor ABP-280 (platelet filamin) and utilizes 40% of the 0.5 mM actin con- tained within the cell. The remainder of the actin is largely in the form of individual monomeric subunits, bound as 1 :l complexes to a small polypeptide (thymosin 64) or to profilin. As presently conceptualized, thymosin 84 inhibits the spontaneous nucleation of monomeric actin that is required for polymerization (Safer et al., 1990). Based on biochemical data, however, the fast-growing ends of actin filaments (conventionally designated as barbed in refer- ence to arrowheads conferred by bound myosin head do- mains) have a higher affinity for actin monomers than thy- mosin 84, and profilin-actin complexes can add directly to the barbed end but not to the pointed end of filaments. Therefore, thymosin (34 can keep 60% of the total platelet actin unpolymerized only if the actin filament barbed ends in the cytoskeleton of the resting platelet are blocked (“capped”) (Nachmias et al., 1993; Pantaloni and Carlier, 1993). The barbed ends of actin filaments in detergent- permeabilized resting platelets do not accommodate addi- tion of actin subunits, consistent with these ends being capped, but barbed ends of actin filaments do become available to nucleate actin assembly following exposure of the cells to stimuli that elicit the platelet shape change (Hartwig, 1992; Nachmias et al., 1993). One interesting question concerns the signal transduc- tion mechanisms that regulate the actin remodeling in the activating platelet. Strong evidence indicates that calcium is required for the disc-to-sphere transformation that pre- cedes actin polymerization and for the protrusion of flat lamellae, because the nature of the shape change and the extent of exposure of cryptic barbed ends depend on the ability of the platelet to increase its intracellular calcium concentration. Following platelet stimulation, the intracel- lular calcium rises from the basal nanomolar to micromolar levels (Davies et al., 1989). The source of calcium in this reaction is both extracellular and intracellular. Release of calcium from intracellular stores depends on the hydroly- sisof phosphatidylinositol(4,5)bisphosphate (P1(4,5)P2) by phospholipase Cf3 to generate inositol trisphosphate, a reaction dependent upon a pertussis toxin-sensitive het- erotrimeric GTPase (Brass et al., 1986). In this setting, the usual disc-to-sphere transformation takes place, several hundred barbed actin filament ends uncap, and the plate- let extrudes lamellae and filopodia. An intermediate step in this reaction is a marked shortening of the actin fila- ments in the resting platelets, best explained by a calcium-Cdl 644 dependent actin filament-severing reaction mediated