Download PDF
Thromb Haemost 2004; 92(04): 829-837
DOI: 10.1160/TH04-04-0263
Platelets and Blood Cells
Schattauer GmbH

Platelets from Munc18c heterozygous mice exhibit normal stimulus-induced release

Todd D. Schraw
1   Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA
,
Garland L. Crawford
1   Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA
,
Qiansheng Ren
1   Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA
,
Wangsun Choi
1   Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA
,
C. Thurmond Debbie
2   Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana, USA
,
Jeffery Pessin
3   The Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York, USA
,
Sidney W. Whiteheart
1   Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA
› Author AffiliationsFinancial support: Supported by grants from the Ohio Valley Affiliate of the American Heart Association (0215130B) (T.D.S.) and the National Institutes of Health (HL56652) (S.W.W.).
Further Information

Publication History

Received 28 April 2004

Accepted after resubmission 12 July 2004

Publication Date:
06 December 2017 (online)

    Permissions and Reprints

Summary

A critical aspect of hemostasis is the release of clot-forming components from the three intra-platelet stores: dense core granules, α-granules and lysosomes. Exocytosis from these granules is mediated by soluble (SNAPs and NSF) and integralmembrane proteins (v- and t-SNAREs).Three SM (Sec1/Munc18) proteins are present in mouse platelets (Munc18a, 18b and 18c) and each potentially regulates exocytosis via modulation of their cognate syntaxin binding partner. To define the molecular machinery required for platelet exocytosis, we analyzed platelets from Munc18c heterozygous knockout mice. These platelets show a decrease in Munc18c but no apparent reduction in other secretory machinery components. No differences in the rates of aggregation or of secretion of [3H]-5HT (dense core granules), platelet factor 4 (α-granules), or hexosaminidase (lysosomes) were detected between platelets from Munc18c heterozygous knockout or wild-type mice. The platelets also show normal morphology. Contrary to a predicted requirement for Munc18c in platelet secretion, data reported here show that reducing Munc18c levels does not substantially alter platelet function. These data show that despite Munc18c’s role in platelet secretion, the lack of a secretion defect may be attributed to compensation by other Munc18 isoforms or that one allele is sufficient to maintain secretion under standard conditions.

Keywords

Munc18 - syntaxin - platelets - SNARE - secretion
 

  • References

  • 1 Reed GL. Platelet Secretion. In: Platelets. Michelson AD. editor Academic Press; 2002: 181-195.
    Google Scholar
  • 2 Schraw TD, Rutledge TW, Crawford GL. et al. Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulusinduced release. Blood 2003; 102 (05) 1716-22.
    CrossrefPubMedGoogle Scholar
  • 3 Polgar J, Chung SH, Reed GL. Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion. Blood 2002; 100 (03) 1081-3.
    CrossrefPubMedGoogle Scholar
  • 4 Bernstein AM, Whiteheart SW. Identification of a cellubrevin/vesicle associated membrane protein 3 homologue in human platelets. Blood 1999; 93 (02) 571-9.
    PubMedGoogle Scholar
  • 5 Feng D, Crane K, Rozenvayn N. et al. Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2. Blood 2002; 99 (11) 4006-14.
    CrossrefPubMedGoogle Scholar
  • 6 Chen D, Bernstein AM, Lemons PP. et al. Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release. Blood 2000; 95 (03) 921-9.
    PubMedGoogle Scholar
  • 7 Chen D, Lemons PP, Schraw T. et al. Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 and 4 in lysosome release. Blood 2000; 96 (05) 1782-8.
    PubMedGoogle Scholar
  • 8 Flaumenhaft R, Croce K, Chen E. et al. Proteins of the exocytotic core complex mediate platelet alpha-granule secretion. Roles of vesicle-associated membrane protein, SNAP-23, and syntaxin 4. J Biol Chem 1999; 274 (04) 2492-501.
    CrossrefPubMedGoogle Scholar
  • 9 Garcia A, Prabhakar S, Brock CJ. et al. Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry. Proteomics 2004; 04 (03) 656-68.
    CrossrefPubMedGoogle Scholar
  • 10 Lemons PP, Chen D, Bernstein AM. et al. Regulated secretion in platelets: identification of elements of the platelet exocytosis machinery. Blood 1997; 90 (04) 1490-500.
    PubMedGoogle Scholar
  • 11 Weber T, Zemelman BV, McNew JA. et al. SNAREpins: minimal machinery for membrane fusion. Cell 1998; 92 (06) 759-72.
    CrossrefPubMedGoogle Scholar
  • 12 Jahn R. Sec1/Munc18 proteins: mediators of membrane fusion moving to center stage. Neuron 2000; 27 (02) 201-4.
    CrossrefPubMedGoogle Scholar
  • 13 Verhage M, Maia AS, Plomp JJ. et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 2000; 287 5454 864-9.
    CrossrefPubMedGoogle Scholar
  • 14 Garcia EP, Gatti E, Butler M. et al. A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin. Proc Natl Acad Sci USA 1994; 91 (06) 2003-7.
    CrossrefPubMedGoogle Scholar
  • 15 Pevsner J, Hsu SC, Scheller RH. n-Sec1: a neural-specific syntaxin-binding protein. Proc Natl Acad Sci USA 1994; 91 (04) 1445-9.
    CrossrefPubMedGoogle Scholar
  • 16 Riento K, Jantti J, Jansson S. et al. A sec1-related vesicle-transport protein that is expressed predominantly in epithelial cells. Eur J Biochem 1996; 239 (03) 638-46.
    CrossrefPubMedGoogle Scholar
  • 17 Thurmond DC, Kanzaki M, Khan AH. et al. Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles. Mol Cell Biol 2000; 20 (01) 379-88.
    CrossrefPubMedGoogle Scholar
  • 18 Thurmond DC, Pessin JE. Discrimination of GLUT4 vesicle trafficking from fusion using a temperature-sensitive Munc18c mutant. EMBO J 2000; 19 (14) 3565-75.
    CrossrefPubMedGoogle Scholar
  • 19 Voets T, Toonen RF, Brian EC. et al. Munc18-1 promotes large dense-core vesicle docking. Neuron 2001; 31 (04) 581-91.
    CrossrefPubMedGoogle Scholar
  • 20 Spurlin BA, Thomas RM, Nevins AK. et al. Insulin resistance in tetracycline-repressible Munc18c transgenic mice. Diabetes 2003; 52 (08) 1910-7.
    CrossrefPubMedGoogle Scholar
  • 21 Khan AH, Thurmond DC, Yang C. et al. Munc18c regulates insulin-stimulated GLUT4 translocation to the transverse tubules in skeletal muscle. J Biol Chem 2001; 276 (06) 4063-9.
    CrossrefPubMedGoogle Scholar
  • 22 Schulze KL, Littleton JT, Salzberg A. et al. rop, a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins, is a negative regulator of neurotransmitter release in vivo. Neuron 1994; 13 (05) 1099-108.
    CrossrefPubMedGoogle Scholar
  • 23 Schraw TD, Lemons PP, Dean WL. et al. A role for Sec1/Munc18 proteins in platelet exocytosis. Biochem J 2003; 374 (01) 207-17.
    CrossrefPubMedGoogle Scholar
  • 24 Houng A, Polgar J, Reed GL. Munc18-syntaxin complexes and exocytosis in human platelets. J Biol Chem 2003; 278 (22) 19627-33.
    CrossrefPubMedGoogle Scholar
  • 25 Richards-Smith B, Novak EK, Jang EK. et al. Analyses of proteins involved in vesicular trafficking in platelets of mouse models of Hermansky Pudlak syndrome. Mol Genet Metab 1999; 68 (01) 14-23.
    CrossrefPubMedGoogle Scholar
  • 26 Dresbach T, Burns ME, O’Connor V. et al. A neuronal Sec1 homolog regulates neurotransmitter release at the squid giant synapse. J Neurosci 1998; 18 (08) 2923-32.
    CrossrefPubMedGoogle Scholar
  • 27 Chen D, Minger SL, Honer WG. et al. Organization of the secretory machinery in the rodent brain: distribution of the t-SNAREs, SNAP-25 and SNAP-23. Brain Res 1999; 831 1-2 11-24.
    CrossrefPubMedGoogle Scholar
  • 28 Rutledge TW, Whiteheart SW. SNAP-23 is a target for calpain cleavage in activated platelets. J Biol Chem 2002; 277 (40) 37009-15.
    CrossrefPubMedGoogle Scholar
  • 29 Edelmann L, Hanson PI, Chapman ER. et al. Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine. EMBO J 1995; 14 (02) 224-31.
    PubMedGoogle Scholar
  • 30 Thurmond DC, Ceresa BP, Okada S. et al. Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes. J Biol Chem 1998; 273 (50) 33876-83.
    CrossrefPubMedGoogle Scholar
  • 31 Tagaya M, Wilson DW, Brunner M. et al. Domain structure of an N-ethylmaleimide-sensitive fusion protein involved in vesicular transport. J Biol Chem 1993; 268 (04) 2662-6.
    PubMedGoogle Scholar
  • 32 Whiteheart SW, Rossnagel K, Buhrow SA. et al. N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion. J Cell Biol 1994; 126 (04) 945-54.
    CrossrefPubMedGoogle Scholar
  • 33 Mishell BB, Shiigi SM, Henry C. et al. Cell Counts with a Hemacytometer. In: Selected Methods in Cellular Immunology. Mishell BB, Shiigi SM. editors W. H. Freeman and Company; 1980: 14-16.
    Google Scholar
  • 34 Lemons PP, Chen D, Whiteheart SW. Molecular mechanisms of platelet exocytosis: requirements for alpha-granule release. Biochem Biophys Res Commun 2000; 267 (03) 875-80.
    CrossrefPubMedGoogle Scholar
  • 35 Huizing M, Boissy RE, Gahl WA. Hermansky-Pudlak syndrome: vesicle formation from yeast to man. Pigment Cell Res 2002; 15 (06) 405-19.
    CrossrefPubMedGoogle Scholar
  • 36 Yang C, Coker KJ, Kim JK. et al. Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance. J Clin Invest 2001; 107 (10) 1311-8.
    CrossrefPubMedGoogle Scholar
  • 37 Bryant NJ, James DE. Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation. EMBO J 2001; 20 (13) 3380-8.
    CrossrefPubMedGoogle Scholar
  • 38 Toonen RF, Verhage M. Vesicle trafficking: pleasure and pain from SM genes. Trends Cell Biol 2003; 13 (04) 177-86.
    CrossrefPubMedGoogle Scholar
  • 39 Suzuki T, Oiso N, Gautam R. et al. The mouse organellar biogenesis mutant buff results from a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation. Proc Natl Acad Sci USA 2003; 100 (03) 1146-50.
    CrossrefPubMedGoogle Scholar
  • 40 Gissen P, Johnson CA, Morgan NV. et al. Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome. Nat Genet 2004; 36 (04) 400-4.
    CrossrefPubMedGoogle Scholar