Download PDF
Thromb Haemost 2000; 83(01): 157-164
DOI: 10.1055/s-0037-1613772
Commentary
Schattauer GmbH

Platelets Release their Lysosomal Content In Vivo in Humans upon Activation

Silvia Ciferri
1   From the Institute of Internal and Vascular Medicine, University of Perugia, Italy
2   Department of Cellular and Molecular Biology, University of Perugia, Italy
,
Carla Emiliani
2   Department of Cellular and Molecular Biology, University of Perugia, Italy
,
Giuseppe Guglielmini
1   From the Institute of Internal and Vascular Medicine, University of Perugia, Italy
,
Antonio Orlacchio   3
1   From the Institute of Internal and Vascular Medicine, University of Perugia, Italy
,
Giuseppe G. Nenci
1   From the Institute of Internal and Vascular Medicine, University of Perugia, Italy
,
Paolo Gresele
1   From the Institute of Internal and Vascular Medicine, University of Perugia, Italy
› Author Affiliations
Further Information

Publication History

Received 31 December 1998

Accepted after resubmission 27 August 1999

Publication Date:
06 December 2017 (online)

    Permissions and Reprints

Summary

Platelets contain, besides α- and δ-granules, lysosomes which store glycohydrolases able to degrade glycoproteins, glycolipids and glycosaminoglycans. While several studies have shown that α- and δ-granule secretion takes place “in vivo” in humans upon platelet activation, no data are available on the “in vivo” release of lysosomes. We have studied the release of platelet lysosomal contents “in vivo” in healthy volunteers at a localized site of platelet activation by measuring markers of lysosomal secretion in the blood oozing from a skin wound inflicted for the measurement of the bleeding-time. The levels of β-N-acetylhexosaminidase (Hex) were 13.1 ± 0.85 mU/ml in bleedingtime blood and 10.2 ± 0.66 mU/ml in plasma (p <0.001). Hex in serum was 16.4 ± 0.72 mU/ml. The levels of β-galactosidase were also higher in bleeding-time blood than in plasma (0.85 ± 0.07 mU/ml vs 0.4 ± 0.05 mU/ml, p <0.001). In bleeding-time blood collected at one minute intervals, Hex rose progressively consistent with ongoing platelet activation and flow-cytometry showed a progressive increase of the expression of LIMP and LAMP-2, two lysosomal associated proteins. In conclusion, our data demonstrate that platelet lysosomal glycohydrolases are released “in vivo” in humans upon platelet activation.

Key words

Bleeding time - flow cytometry - LIMP and LAMP2 - platelet secretion - platelet vessel wall interaction

3 Present address: Centre for Research in Neurodegenerative Diseases, University of Toronto, Ontario (Canada).


Presented in part at the XVIth Congress of the International Society on Thrombosis and Haemostasis, Florence, Italy, June 1997 and at the XVth Congress of the Italian Society on Haemostasis and Thrombosis, Naples, Italy, September 1998.


 

  • References

  • 1 White JG. Platelet secretory granules and associated proteins. Lab Invest 1993; 68: 497-8.
    PubMedGoogle Scholar
  • 2 Holmsen H. Platelet secretion and energy metabolism. In: Hemostasis and Thrombosis. Basic Principles and Clinical Practice. Colman RW, Hirsh J, Marder VJ, Salzman EW. eds. Philadelphia: JB Lippincott; 1994: 524-45.
    Google Scholar
  • 3 Holmsen H, Day HJ. The selectivity of the thrombin-induced platelet release reaction: subcellular localization of released and retained constituents. J Lab Clin Med 1970; 75: 841-55.
    PubMedGoogle Scholar
  • 4 Metzelaar M, Clevers HC. Lysosomal membrane glycoproteins in platelets. Thromb Haemost 1992; 68: 378-82.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 5 Gresele P, Arnout J, Deckmyn H, Huybrechts E, Pieters GP, Vermylen J. Role of proaggregatory and antiaggregatory prostaglandins in hemostasis. J Clin Invest 1987; 80: 1435-45.
    CrossrefPubMedGoogle Scholar
  • 6 Weiss HJ, Lages B. Evidence for tissue factor dependent activation of the classic extrinsic coagulation mechanism in blood obtained from bleeding time wounds. Blood 1988; 71: 629-35.
    PubMedGoogle Scholar
  • 7 Kyrle PA, Stockenhuber F, Brenner B, Gossinger H, Korninger C, Pabinger I, Sunder-Plassman G, Balcke P, Lechner K. Evidence for an increased generation of prostacyclin in microvasculature and an impairment of the platelet α-granule release in chronic renal failure. Thromb Haemost 1988; 60: 205-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 1990; 75: 128-38.
    PubMedGoogle Scholar
  • 9 Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF, Kestin AS. Downregulation of platelet surface glycoprotein Ib-IX in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound. Blood 1991; 77: 770-9.
    PubMedGoogle Scholar
  • 10 Holtzman E. Lysosomes. New York: Plenum; 1989
  • 11 Barret AJ. Proteolytic and other metabolic pathways in lysosomes. Biochem Soc Transact 1984; 12: 899-902.
    CrossrefPubMedGoogle Scholar
  • 12 Mahuran DJ, Novak A, Lowden JA. The lysosomal hexosaminidase isoenzymes. In: Isoenzymes, Current Topics in Biological and Medical Research. New York: Alan Liss Inc; 1985: 229-88.
    Google Scholar
  • 13 Thompson NJ, Stoolmiller AC, Matalon R, Dorfman A. N-Acetyl β-hexosaminidase: role in the degradation of glycosaminoglycans. Science 1973; 181: 866-7.
    CrossrefPubMedGoogle Scholar
  • 14 Koike K, Holmsen H. Requirement for thrombin receptor occupancy during platelet secretion under aggregating and non-aggregating conditions. Thromb Haemost 1987; 58: 1053-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Gresele P, Dottorini M, Selli ML, Iannacci L, Canino S, Todisco T, Romano S, Crook P, Page CP, Nenci GG. Altered platelet function associated with the bronchial hyperresponsiveness accompanying nocturnal asthma. J Allergy Clin Immunol 1993; 91: 894-902.
    CrossrefPubMedGoogle Scholar
  • 16 Nieuwenhuis HK, van Oosterhout JJG, Rozemuller E, van Iwaarden F, Sixma JJ. Studies with a monoclonal antibody against activated platelets: evidence that secreted 53,000-molecular weight lysosome-like granule protein is exposed on surface of activated platelets in the circulation. Blood 1987; 70: 838-45.
    PubMedGoogle Scholar
  • 17 Mc Ever RP. Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium. Blood Cells 1990; 16: 73-83.
    PubMedGoogle Scholar
  • 18 Israels SJ, McMillan EM, Robertson C, Singhroy S, McNicol A. The lysosomal granule membrane protein, LAMP-2 is also present in platelet dense granule membrane. Thromb Haemost 1996; 04: 623-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 McGregor JL, Brochier J, Wild F, Follea G, Trzeciak MC, James E, Dechavanne M, McGregor L, Clemetson K. Monoclonal antibodies against platelet membrane glycoproteins. Eur J Biochem 1983; 131: 427-36.
    CrossrefPubMedGoogle Scholar
  • 20 Emiliani C, Martino S, Orlacchio A, Vezza R, Nenci GG, Gresele P. Platelet glycohydrolase activities: characterization and release. Cell Biochem Funct 1995; 13: 31-9.
    CrossrefPubMedGoogle Scholar
  • 21 Emiliani C, Beccari T, Stirling JL, Sciarra R, Orlacchio A. New biospecific chromatographies for β-Hexosaminidase. In: Macromolecular Biorecognition. Chaiken I, Chicone E, Fontana A, Neri P. eds. Clifton: The Humana Press; 1988: 309-19.
    Google Scholar
  • 22 Kaplan KL, Owen J. Plasma levels of β-thromboglobulin and platelet factor 4 as indices of platelet activation “ in vivo”. Blood 1981; 57: 199-202.
    PubMedGoogle Scholar
  • 23 Laudano AP, Doolittle RF. Synthetic peptide derivatives that bind fibrinogen and prevent the polymerization of fibrin monomers. Proc Natl Acad Sci USA 1978; 75: 3085-9.
    CrossrefPubMedGoogle Scholar
  • 24 Knöfler R, Weissbach G, Khlisc E. Critical evaluation of ATP release reaction in whole blood. Thromb Res 1996; 84: 157-65.
    CrossrefPubMedGoogle Scholar
  • 25 Vezza R, Roberti R, Nenci GG, Gresele P. Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C. Blood 1993; 82: 2704-13.
    PubMedGoogle Scholar
  • 26 Gresele P, Arnout J, Coene MC, Deckmyn H, Vermylen J. Leukotriene B4 production by stimulated whole blood: comparative studies with isolated polymorphonuclear cells. Biochem Biophys Res Commun 1986; 137: 334-42.
    CrossrefPubMedGoogle Scholar
  • 27 Gresele P, Ribaldi E, Mezzasoma AM, Quero E, Stasi M, Prosdocimi M, Goracci G, Nenci GG. Cloricromene inhibits leukotriene formation by human polymorphonuclear leucocytes by suppressing arachidonate release from membrane phospholipids. Biochem Pharmacol 1993; 45: 123-30.
    PubMedGoogle Scholar
  • 28 Martino S, Emiliani C, Tabilio A, Falzetti F, Stirling JL, Orlacchio A. Distribution of active alpha- and beta-subunits of beta-N-acetylhexosaminidase as function of leukaemic cell type. Biochim Biophys Acta 1997; 1335: 5-15.
    CrossrefPubMedGoogle Scholar
  • 29 Gerli R, Paolucci C, Gresele P, Bistoni O, Fiorucci S, Muscat C, Belia S, Bertotto A, Costantini V. Salicylates inhibit adhesion and transmigration of T lymphocytes by preventing integrin activation induced by contact with endothelial cells. Blood 1998; 92: 2389-98.
    PubMedGoogle Scholar
  • 30 Beccari T, Emiliani C, Hosseini R, Orlacchio A, Stirling JL. Intermediate forms of beta-N-acetylhexosaminidase lack activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate. Biochem J 1987; 244: 801-4.
    CrossrefPubMedGoogle Scholar
  • 31 Murphy S. The oxidation of exogenously added organic anions by platelets facilitates maintainance of pH during their storage for transfusion at 22 degrees C. Blood 1995; 85: 1929-35.
    PubMedGoogle Scholar
  • 32 Leoncini G, Balestrero F, Maresca M. Lysosomal enzymes in human platelets. Cell Biochem Funct 1985; 03: 121-5.
    CrossrefPubMedGoogle Scholar
  • 33 Holmsen H, Setkowsky CA, Lages B, Day HJ, Weiss HJ, Scrutton MC. Content and thrombin-induced release of acid hydrolases in gel-filtered platelets from patients with storage pool disease. Blood 1975; 46: 131-42.
    PubMedGoogle Scholar
  • 34 Gresele P, Catalano M, Giammarresi C, Volpato R, Termini R, Ciabattoni G, Nenci GG, Davì G. Platelet activation markers in patients with peripheral arterial disease. A prospective comparison of different platelet function tests. Thromb Haemost 1997; 78: 1434-7.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 35 Nguyen A, Gemmell CH, Yeo EL, Packham MA, Rand ML. Ethanol inhibits thrombin-induced secretion of content of human platelet dense and α-granules and lysosomes. Thromb Haemost 1998; 80: 662-7.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 36 Macey MG. The protease-activated receptors and their cellular expression and function in blood-related cells. Br J Haematol 1998; 101: 1-9.
    CrossrefPubMedGoogle Scholar
  • 37 Senior RM, Hinek A, Griffin GL, Pipoly DJ, Crouch EC, Mecham RP. Neutrophils show chemotaxis to type IV collagen and its 7s domain and contain a 67 KD type IV collagen binding protein with lectin properties. Am J Cell Mol Biol 1989; 01: 479-87.
    PubMedGoogle Scholar
  • 38 Senger DR, Claffey KP, Detmar M. Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins. Proc Natl Acad Sci Usa 1997; 94: 13612-7.
    CrossrefPubMedGoogle Scholar
  • 39 Silverstein RL, Febbraio M. Identification of lysosomal-associated membrane protein-2 as an activation-dependent platelet surface glycoprotein. Blood 1992; 80: 1470-5.
    PubMedGoogle Scholar
  • 40 Kannan K, Divers SG, Lurie AA, Chervenak R, Fukuda M, Holcombe RF. Cell surface expression of lysosome-associated membrane protein-2 (LAMP-2) and CD 63 as markers of “in vivo” platelet activation in malignancy. Eur J Haematol 1995; 55: 145-51.
    PubMedGoogle Scholar
  • 41 Nishibori M, Cham B, McNicol A, Shalev A, Jain N, Gerrard JM. The protein CD63 is in platelet dense granules, is deficient in patients with Hermansky-Pudlak syndrome, and appears identical to granulophysin. J Clin Invest 1993; 91: 1775-82.
    CrossrefPubMedGoogle Scholar
  • 42 Janes SJ, Wilson DJ, Cox AD, Chronos NAF, Goodall AH. ADP causes partial degranulation of platelets in the absence of aggregation. Brit J Haematol 1994; 86: 568-73.
    CrossrefPubMedGoogle Scholar
  • 43 Doebber TW, Wu MS, Shen TY. Platelet activating factor intravenous infusion in rats stimulates lysosomal hydrolase secretion independent of blood neutrophils. Biochem Biophys Res Commun 1984; 125: 980-7.
    CrossrefPubMedGoogle Scholar
  • 44 Baron R, Neff L, Louvard D, Courtoy PJ. Cell-mediated extracellular acidification and bone resorption: evidence for low pH in resorbing lacunae and localization of a 100-KD lysosomal membrane protein at the osteoclast ruffled border. J Cell Biol 1985; 10: 2210-22.
    PubMedGoogle Scholar
  • 45 Montcourrier P, Mangeat PH, Salarar G, Morriset M, Sahuquet A, Rochefort H. Cathepsin D in breast cancer cells can digest extracellular matrix in large acidic vesicles. Cancer Res 1990; 50: 6045-54.
    PubMedGoogle Scholar
  • 46 Miller D, Gong X, Decker G, Shur BD. Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy. J Cell Biol 1993; 123: 1431-40.
    CrossrefPubMedGoogle Scholar
  • 47 Movat H, Wasi S. Severe microvascular injury induced by lysosomal releasates of human polymorphonuclear leukocytes. Increase in vasopermeability, hemorrhage, and microthrombosis due to degradation of subendothelial and perivascular matrices. Am J Pathol 1985; 12: 404-17.
    PubMedGoogle Scholar
  • 48 Muir EM, Bowyer DE. Inhibition of pinocytosis and induction of release of lysosomal contents by lysosomal overload of arterial smooth muscle cells “in vitro”. Atherosclerosis 1984; 50: 85-92.
    CrossrefPubMedGoogle Scholar
  • 49 Sasahara M, Hazamana F, Amano S, Hayase Y, Yukioka N, Kawai J, Kataoka H. Effects of hypertension on lysosomal enzymes activities in aortic endothelial cells. Atherosclerosis 1988; 24: 433-6.
    PubMedGoogle Scholar
  • 50 Hultberg B, Isaksson A, Nilsson JA, Lindgarde F. Serum beta-hexosaminidase isoenzymes are related to risk factors for atherosclerosis in a large population of postmenopausal women. Clin Chim Acta 1994; 227: 59-68.
    CrossrefPubMedGoogle Scholar