Proteins of the Exocytotic Core Complex Mediate Platelet α-Granule Secretion

doi:10.1074/jbc.274.4.2492

Proteins of the Exocytotic Core Complex Mediate Platelet α-Granule Secretion

ROLES OF VESICLE-ASSOCIATED MEMBRANE PROTEIN, SNAP-23, AND SYNTAXIN 4*

From the ^‡Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, ^§Howard Hughes Medical Institute, and ^‖Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

Abstract

To understand the molecular basis of granule release from platelets, we examined the role of vesicle-associated membrane protein, SNAP-23, and syntaxin 4 in α-granule secretion. A vesicle-associated membrane protein, SNAP-23, and syntaxin 4 were detected in platelet lysate. These proteins form a SDS-resistant complex that disassembles upon platelet activation. To determine whether these proteins are involved in α-granule secretion, we developed a streptolysin O-permeabilized platelet model of α-granule secretion. Streptolysin O-permeabilized platelets released α-granules, as measured by surface expression of P-selectin, in response to Ca²⁺ up to 120 min after permeabilization. Incubation of streptolysinO-permeabilized platelets with an antibody directed against vesicle-associated membrane protein completely inhibited Ca²⁺-induced α-granule release. Tetanus toxin cleaved platelet vesicle-associated membrane protein and inhibited Ca²⁺-induced α-granule secretion from streptolysinO-permeabilized platelets. An antibody to syntaxin 4 also inhibited Ca²⁺-induced α-granule release by approximately 75% in this system. These results show that vesicle-associated membrane protein, SNAP-23, and syntaxin 4 form a heterotrimeric complex in platelets that disassembles with activation and demonstrate that α-granule release is dependent on vesicle SNAP receptor-target SNAP receptor (vSNARE-tSNARE) interactions.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL51926 and HL57140.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.
↵¶ A Howard Hughes Medical Institute Physician Postdoctoral Fellow. To whom correspondence should be addressed: RE 318, Research East, P. O. Box 15732, Boston, MA 02215. Tel.: 617-667-0627; Fax: 617-975-5505; E-mail: rflaumen{at}bidmc.harvard.edu.
↵2 Flaumenhaft, R., Furie, B., Furie, B. C.,J. Cell. Physiol., in press.
Abbreviations:

SNARE

SNAP receptor

SNAP-23

soluble (N-ethylmaleimide-sensitive fusion protein) attachment protein 23

VAMP

vesicle-associated membrane protein

SL-O

streptolysin O

PE

phycoerythrin: PIPES, 1,4-piperazinediethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

NSF

N-ethylmaleimide-sensitive fusion protein.
- Received September 2, 1998.
- Revision received October 26, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.