PDF herunterladen
Thromb Haemost 2001; 86(03): 887-893
DOI: 10.1055/s-0037-1616152
Review Articles
Schattauer GmbH

The 33-kDa Platelet α-granule Membrane Protein (GMP-33) Is an N-terminal Proteolytic Fragment of Thrombospondin

Conchi Damas
1   Department of Haematology and Graduate School for Biomembranes, University of Utrecht, The Netherlands
,
Tom Vink
1   Department of Haematology and Graduate School for Biomembranes, University of Utrecht, The Netherlands
,
Karel H. Nieuwenhuis
1   Department of Haematology and Graduate School for Biomembranes, University of Utrecht, The Netherlands
,
Jan J. Sixma
1   Department of Haematology and Graduate School for Biomembranes, University of Utrecht, The Netherlands
› InstitutsangabenThis work was financially supported by the Dutch Heart Foundation (NHS) (grant no. 43.051).
Weitere Informationen

Publikationsverlauf

Received 12. Dezember 2000

Accepted after revision 07. Mai 2001

Publikationsdatum:
14. Dezember 2017 (online)

Auch verfügbar auf
    Lizenzen und Reprints

Summary

GMP-33 is a platelet membrane associated protein that is recognised by RUU-SP 1.77, an antibody raised against activated platelets. GMP-33 is predominantly associated with the membrane of platelet α-granules and it is translocated to the plasma membrane upon platelet activation (Metzelaar et al. Blood 1992; 79: 372-9). In this study we have isolated the protein by immunoaffinity chromatography. The N-terminus was sequenced and was identical to the N-terminal sequence of human thrombospondin. The protein was N-glycosylated and bound to heparin as would be expected of the N-terminal part of thrombospondin. RUU-SP 1.77 reacted only with reduced thrombospondin. Plasmin and trypsin digestion of thrombospondin yielded fragments of approximately the same size as GMP 33 that reacted with RUU-SP 1.77 after reduction. No evidence for alternative splicing was found. We postulate that GMP 33 is an N-terminal proteolytic fragment of thrombospondin that is membrane associated.

Keywords

Thrombospondin - platelet α-granule - platelet activation - platelet membrane
 

  • References

  • 1 Metzelaar MJ, Heijnen HF, Sixma JJ, Nieuwenhuis HK. Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets. Blood 1992; 79: 372-9.
    PubMedGoogle Scholar
  • 2 Lawler J, Hynes RO. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol 1986; 103: 1635-48.
    CrossrefPubMedGoogle Scholar
  • 3 Gartner TK, Walz DA, Aiken M, Starr-Spires L, Ogilvie ML. Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation. Biochem Biophys Res Commun 1984; 124: 290-5.
    CrossrefPubMedGoogle Scholar
  • 4 Metzelaar MJ, Korteweg J, Sixma JJ, Nieuwenhuis HK. Comparison of platelet membrane markers for the detection of platelet activation in vitro and during platelet storage and cardiopulmonary bypass surgery. J Lab Clin Med 1993; 121: 579-87.
    PubMedGoogle Scholar
  • 5 Fauvel J, Chap H, Roques V, Levy-Toledano S, Douste-Blazy L. Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients. Biochim Biophys Acta 1986; 856: 155-64.
    CrossrefPubMedGoogle Scholar
  • 6 Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedGoogle Scholar
  • 7 Edman P, Begg G. A protein sequenator. Eur J Biochem 1967; 1: 80-91.
    CrossrefPubMedGoogle Scholar
  • 8 Aiken ML, Ginsberg MH, Plow EF. Divalent cation-dependent and independent surface expression of thrombospondin on thrombin-stimulated human platelets. Blood 1987; 69: 58-64.
    PubMedGoogle Scholar
  • 9 Morandi V, Edelman L, Legrand YJ, Legrand C. Characterization of a novel monoclonal antibody (V58A4) raised against a recombinant NH2-terminal heparin-binding fragment of human endothelial cell thrombospondin. FEBS Lett 1994; 346: 156-60.
    CrossrefPubMedGoogle Scholar
  • 10 Fugman DA, Witte DP, Jones CL, Aronow BJ, Lieberman MA. In vitro establishment and characterization of a human megakaryoblastic cell line. Blood 1990; 75: 1252-61.
    PubMedGoogle Scholar
  • 11 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9.
    PubMedGoogle Scholar
  • 12 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory; 1989
    Google Scholar
  • 13 Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: 6-13.
    CrossrefPubMedGoogle Scholar
  • 14 Frohman MA, Dush MK, Martin GR. Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 1988; 85: 8998-9002.
    CrossrefPubMedGoogle Scholar
  • 15 Schaefer BC. Revolutions in rapid amplification of cDNA ends: New strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem 1995; 227: 255-73.
    CrossrefPubMedGoogle Scholar
  • 16 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Bio. 1990; 215: 403-10.
    CrossrefPubMedGoogle Scholar
  • 17 Dixit VM, Grant GA, Santoro SA, Frazier WA. Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin. J Biol Chem 1984; 259: 10100-5.
    PubMedGoogle Scholar
  • 18 Lawler J. The structure and functional properties of thrombospondin. Blood 1986; 67: 1197-209.
    PubMedGoogle Scholar
  • 19 George JN, Lyons RM, Morgan RK. Membrane changes associated with platelet activation. Exposure of actin on the platelet surface after thrombin-induced secretion. J Clin Invest 1980; 66: 1-9.
    CrossrefPubMedGoogle Scholar
  • 20 Legrand C, Thibert V, Dubernard V, Begault B, Lawler J. Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation. Blood 1992; 79: 1995-2003.
    PubMedGoogle Scholar
  • 21 Bornstein P, Sage EH. Thrombospondins. Methods Enzymol 1994; 245: 62-85.
    PubMedGoogle Scholar
  • 22 Lawler J, Cohen AM, Chao FC, Moriarty DJ. Thrombospondin in essential thrombocythemia. Blood 1986; 67: 555-8.
    PubMedGoogle Scholar
  • 23 Bornstein P. Diversity of function is inherent in matricellular proteins: An appraisal of thrombospondin 1. J Cell Biol 1995; 130: 503-6.
    CrossrefPubMedGoogle Scholar
  • 24 Reed MJ, Iruela-Arispe L, O’Brien ER, Truong T, LaBell T, Bornstein P, Sage EH. Expression of thrombospondins by endothelial cells – Injury is correlated with TSP-1. Am J Pathol 1995; 147: 1068-80.
    PubMedGoogle Scholar
  • 25 Bornstein P, O’Rourke K, Wikstrom K, Wolf FW, Katz R, Li P, Dixit VM. A second, expressed thrombospondin gene (Thbs2) exists in the mouse genome. J Biol Chem 1991; 266: 12821-4.
    PubMedGoogle Scholar
  • 26 Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA, Bouck NP. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 1990; 87: 6624-8.
    CrossrefPubMedGoogle Scholar
  • 27 Rabhi Sabile S, Thibert V, Legrand C. Thrombospondin peptides inhibit the secretion-dependent phase of platelet aggregation. Blood Coag Fibrinolysis 1996; 7: 237-40.
    CrossrefPubMedGoogle Scholar
  • 28 Agbanyo FR, Sixma JJ, De Groot PG, Languino LR, Plow EF. Thrombospondin-platelet interactions. Role of divalent cations, wall shear rate, and platelet membrane glycoproteins. J Clin Invest 1993; 92: 288-96.
    CrossrefPubMedGoogle Scholar
  • 29 Gawaz M, Ott I, Reininger AJ, Heinzmann U, Neumann FJ. Agglutination of isolated platelet membranes. Arterioscler Thromb Vasc Biol 1996; 16: 621-7.
    CrossrefPubMedGoogle Scholar
  • 30 Murphy Ullrich JE, Gurusidappa S, Frazier WA, Hook M. Heparin binding peptides from thrombospondins 1 and 2 contain focal adhesion-labilizing activity. J Biol Chem 1993; 268: 26784-9.
    PubMedGoogle Scholar
  • 31 Legrand C, Morandi V, Mendelovitz S, Shaked H, Hartman JR, Panet A. Selective inhibition of platelet macroaggregate formation by a recombinant heparin-binding domain of human thrombospondin. Arterioscler Thromb 1994; 14: 1784-91.
    CrossrefPubMedGoogle Scholar