D3 Phosphoinositides and Outside-in integrin Signaling by Glycoprotein IIb-IIIa Mediate Platelet Actin Assembly and Filopodial Extension Induced by Phorbol 12-Myristate 13-Acetate

Abstract

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by 5 M thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 M) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.Extracellular perturbations cause cells to change cell shape and translocate by remodeling diverse intracellular actin-based structures. The relative functional simplicity of these changes in the anucleate human blood platelet makes it a useful subject for working out the pathways leading to this remodeling. Of particular utility is the fact that actin remodeling events in the platelet predominantly take place sequentially in time rather than the actin rearrangements that occur simultaneously in space during cell locomotion.The resting platelet is a rigid disc stabilized by an actin filament gel that links to a submembrane skeleton and to transmembrane proteins (1, 2). Over 98% of the actin filaments in resting platelets have their fast growing ("barbed" as defined by myosin head fragment binding) ends stabilized by specific barbed-end capping proteins. In response to thrombin, a calcium transient in the platelet activates gelsolin, which then severs actin filaments in the periphery of the actin filament gel and caps the newly formed barbed ends of the severed filaments (3). Subsequently, phosphoinositides accumulate and inactivate gelsolin as well as another abundant barbed end binding factor, capping protein. Phosphoinositide-mediated uncapping of at least 25% of the filament barbed ends eventuates in massive actin assembly as micromolar quantities of monomeric subunits, previously prevented from spontaneous nucleation by sequestering proteins, add onto the uncapped filament barbed ends (4 -6).The actin assembly following thrombin-induced platelet activation mediates the appearance of two types of surface protrusions, ruffling lamellae that spread circumferentially and threadlike filopodial protrusions. The fi...

Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton

“…Because the activation of small GTP-binding proteins is required for actin polymerization [16,17,30,31,38], we investigated the effects of the FC analogues FTA and AGGC on actin filament formation in platelets. Our results show that both FTA and AGGC clearly inhibited actin polymerization stimulated by TG.…”

“…The F-actin contents of resting and activated platelets were determined in accordance with the modifications [33] of a procedure described previously [38]. In brief, washed platelets (2i10) cells\ml) were activated in Hepes-buffered saline.…”

Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase

“…Direct activation of PKC by phorbol esters induces PI 3-kinasedependent signaling pathways not only in neutrophils. Actin filament formation in platelets [56], cell transformation [57], DNA synthesis [58], noradrenalin release [59], as well as glucose transport in adipocytes [60], are all processes activated by PMA in a PI 3-kinase-dependent manner. The positioning of PI 3-kinase both upstream and downstream of PKC is not necessarily a contradiction.…”

A Role for the Actin Cytoskeleton in the Initiation and Maintenance of Store-mediated Calcium Entry in Human Platelets

“…Thrombin-evoked Ca 2ϩ influx was measured as the integral of the rise in [Ca 2ϩ ] i above basal for 1 min after the addition of thrombin in the presence of external Ca 2ϩ , corrected by subtraction of the integral over the same period for stimulation in the absence of external Ca 2ϩ (with 1 mM EGTA). Measurement of F-actin Content-The F-actin content of resting and activated platelets was determined according to the modifications (18) of a previously published procedure (19). Briefly, washed platelets (2 ϫ 10 8 cells/ml) were activated in HEPES-buffered saline.…”