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

doi:10.1074/jbc.271.51.32986

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*

From the ^‡ Divisions of Experimental Medicine and Hematology-Oncology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School and
^∥ Division of Signal Transduction, Department of Medicine, Beth Israel Hospital and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

^§To whom correspondence should be addressed. Tel.: 617-278-0323; Fax: 617-734-2248.

↵^¶ Present address: Division of Hematology, Centre Hospitalo-Universitaire Vaudois, Lausanne, Switzerland.

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 μM 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 μM) 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.

Footnotes

↵** Supported by the Medical Foundation Inc., Boston, MA.
↵* This work was supported by United States Public Health Service Grants GM 41890 (to L. C. C.), AR 38910 (to P. J.), HL 19429 (to T. S.), DK 38452 (to J. H. H.), HL 54145 (to J. H. H.), and HL56949 (to J. H. H.) and by grants from the American Cancer Society and the Edwin S. Webster Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GP

glycoprotein

PMA

phorbol 12-myristate 13-acetate

DAG

1,2-dioctanoyl-sn -glycerol

PtdIns

phosphatidylinositol

PtdIns-3-P

phosphatidylinositol 3-phosphate

PtdIns-4-P

phosphatidylinositol 4-phosphate

PtdIns-3,4-P₂

phosphatidylinositol 3,4-bisphosphate

PtdIns-4,5-P₂

phosphatidylinositol 4,5-bisphosphate

PtdIns-3,4,5-P₃

phosphatidylinositol 3,4,5-trisphosphate

PKC

protein kinase C

PI-3-K

phosphoinositide 3-kinase

TRAP

thrombin receptor-activating peptide

TRAF

thrombin receptor-activating fragment peptide

FITC

fluorescein isothiocyanate

LIBS

ligand-induced binding site

TRITC

tetramethylrhodamine B isothiocyanate.
↵² A. Michelson, personal communication.
- Received April 4, 1996.
- Revision received September 18, 1996.