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

John H Hartwig,Sophia Kung,Tibor Kovacsovics,Paul A Janmey,Lewis C Cantley,Thomas P Stossel,Alex Toker

doi:10.1074/jbc.271.51.32986

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 microM 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 microM) 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.

Highlights

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets
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
In keeping with the association between platelet filopodia and GPIIb-IIIa, we show that PMA-activated filopodial actin assembly requires signaling through this integrin

Summary

EXPERIMENTAL PROCEDURES

Materials—Human thrombin, PMA, prostaglandin E1, wortmannin, bovine serum albumin, fibrinogen, phallacidin, FITC-phalloidin, iodomethacin, PtdIns-4,5-P2 from bovine brain, and other general chemical reagents were purchased from Sigma. Phospholipid labeling experiments were performed in stirred cells in the presence of 500 ␮g/ml of fibrinogen In these [32P]orthophosphoric acid-labeling experiments, platelets were purified from plasma by two centrifugations at 800 ϫ g for 10 min in the presence of 1 ␮M prostaglandin E1. Measurement of F-actin Content and Barbed End Nuclei—The Factin content of resting and activated cells was determined by quantitation of FITC-phalloidin binding to formaldehyde fixed platelets after permeabilization with Triton X-100 [14, 15]. Platelet suspensions (2 ϫ 108/ml) were either PMA and TRAP activated or first preincubated with 4 ␮M LIBS3 Fab for 5 min exposed to 30 ␮g/ml of fibrinogen at 37 °C. All phospholipid solutions were made in 10 mM Tris-HCl, pH 7.4, from lyophilized solids or from chloroform solutions dried in a nitrogen stream and sonicated at maximal power for three 20-s bursts as described previously [21]

RESULTS

Phosphoinositides and Outside-In Integrin Signaling

DISCUSSION