#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS)


Autoři: Signe E. Cremer aff001;  James L. Catalfamo aff002;  Robert Goggs aff003;  Stefan E. Seemann aff004;  Annemarie T. Kristensen aff001;  Marjory B. Brooks aff002
Působiště autorů: University of Copenhagen, Department of Veterinary Clinical Sciences, Copenhagen, Denmark aff001;  Cornell University, Department of Population Medicine and Diagnostic Sciences, Ithaca, New York, United States of America aff002;  Cornell University, Department Clinical Sciences, Ithaca, New York, United States of America aff003;  University of Copenhagen, Department of Veterinary and Animal Sciences, Copenhagen, Denmark aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0224891

Souhrn

Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome (CAPS), and to compare the CAPS proteins to human and murine platelet proteomic data. Washed platelets were isolated from healthy dogs, and stimulated with saline (control) or gamma-thrombin (releasate). Proteins were separated by SDS-page, trypsin-digested and analyzed by liquid chromatography and tandem mass spectrometry (MS). CAPS proteins were defined as those with a MS1-abundance ratio of two or more for releasate vs. unstimulated saline control. A total of 1,918 proteins were identified, with 908 proteins common to all dogs and 693 characterized as CAPS proteins. CAPS proteins were similar to human and murine platelet secretomes and were highly represented in hemostatic pathways. Differences unique to CAPS included replacement of platelet factor 4 with other cleavage products of platelet basic protein (e.g. interleukin-8), novel proteins (e.g. C-C motif chemokine 14), and proteins in relatively high (e.g. protease nexin-1) or low (e.g. von Willebrand factor) abundance. This study establishes the first in-depth platelet releasate proteome from healthy dogs with a reference database of 693 CAPS proteins. Similarities between CAPS and the human secretome confirm the utility of dogs as translational models of human disease, but we also identify differences unique to canine platelets. Our findings provide a resource for further investigations into disease-related CAPS profiles, and for comparative pathway analyses of platelet activation among species.

Klíčová slova:

Dogs – Chemokines – Proteomes – Serum proteins – Proteomics – Platelets – Platelet activation – Fibrinogen


Zdroje

1. Gruba SM, Koseoglu S, Meyer AF, Meyer BM, Maurer-Jones MA, Haynes CL. Platelet membrane variations and their effects on delta-granule secretion kinetics and aggregation spreading among different species. Biochimica et biophysica acta. 2015;1848:1609–18. doi: 10.1016/j.bbamem.2015.04.006 25906946

2. Boudreaux MK, Catalfamo JL. Molecular and genetic basis for thrombasthenic thrombopathia in otterhounds. American journal of veterinary research. 2001;62:1797–804. doi: 10.2460/ajvr.2001.62.1797 11703027

3. Dodds WJ. Familial canine thrombocytopathy. Thrombosis et diathesis haemorrhagica Supplementum. 1967;26:241–8. 6064862

4. Raymond SL, Dodds WJ. Platelet membrane glycoproteins in normal dogs and dogs with hemostatic defects. The Journal of laboratory and clinical medicine. 1979;93:607–13. 311802

5. Brooks MB, Catalfamo JL, Brown AA, Ivanova P, Lovaglio J. A hereditary bleeding disorder of dogs caused by a lack of platelet procoagulant activity. Blood. 2002;99:2434–41. doi: 10.1182/blood.v99.7.2434 11895776

6. Brooks MB, Catalfamo JL, Friese P, Dale GL. Scott syndrome dogs have impaired coated-platelet formation and calcein-release but normal mitochondrial depolarization. Journal of thrombosis and haemostasis. 2007;5:1972–4. JTH2683 [pii]. doi: 10.1111/j.1538-7836.2007.02683.x 17723137

7. Callan MB, Bennett JS, Phillips DK, Haskins ME, Hayden JE, Anderson JG, et al. Inherited platelet delta-storage pool disease in dogs causing severe bleeding: an animal model for a specific ADP deficiency. Thrombosis and haemostasis. 1995;74:949–53. 8571327

8. Dodds WJ. Von Willebrand's disease in dogs. Mod Vet Pract. 1984;65:681–6. Epub 1984/09/01. 6332976

9. Goggs R, Mastrocco A, Brooks MB. Retrospective evaluation of 4 methods for outcome prediction in overt disseminated intravascular coagulation in dogs (2009–2014): 804 cases. Journal of veterinary emergency and critical care. 2018;28:541–50. Epub 2018/10/12. doi: 10.1111/vec.12777 30302935

10. Simpson K, Chapman P, Klag A. Long-term outcome of primary immune-mediated thrombocytopenia in dogs. The Journal of small animal practice. 2018;59:674–80. Epub 2018/08/14. doi: 10.1111/jsap.12912 30102418

11. Borresen B, Hansen AE, Kjaer A, Andresen TL, Kristensen AT. Liposome-encapsulated chemotherapy: Current evidence for its use in companion animals. Veterinary and comparative oncology. 2018;16:E1–e15. Epub 2017/10/14. doi: 10.1111/vco.12342 29027350

12. Fenger JM, London CA, Kisseberth WC. Canine osteosarcoma: a naturally occurring disease to inform pediatric oncology. 2014;55:69–85. Epub 2014/06/18. doi: 10.1093/ilar/ilu009 24936031

13. Du LM, Nurden P, Nurden AT, Nichols TC, Bellinger DA, Jensen ES, et al. Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A. Nature communications. 2013;4:2773. doi: 10.1038/ncomms3773 24253479

14. Burkhart JM, Gambaryan S, Watson SP, Jurk K, Walter U, Sickmann A, et al. What can proteomics tell us about platelets? Circulation research. 2014;114:1204–19. doi: 10.1161/CIRCRESAHA.114.301598 24677239

15. Burkhart JM, Vaudel M, Gambaryan S, Radau S, Walter U, Martens L, et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood. 2012;120:e73–82. doi: 10.1182/blood-2012-04-416594 22869793

16. Hernandez-Ruiz L, Valverde F, Jimenez-Nunez MD, Ocana E, Saez-Benito A, Rodriguez-Martorell J, et al. Organellar proteomics of human platelet dense granules reveals that 14-3-3zeta is a granule protein related to atherosclerosis. Journal of proteome research. 2007;6:4449–57. doi: 10.1021/pr070380o 17918986

17. Maynard DM, Heijnen HF, Horne MK, White JG, Gahl WA. Proteomic analysis of platelet alpha-granules using mass spectrometry. Journal of thrombosis and haemostasis. 2007;5:1945–55. JTH2690 [pii]. doi: 10.1111/j.1538-7836.2007.02690.x 17723134

18. Maynard DM, Heijnen HFG, Gahl WA, Gunay-Aygun M. The alpha-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome. Journal of thrombosis and haemostasis. 2010;8:1786–96. doi: 10.1111/j.1538-7836.2010.03932.x 20524979

19. Zufferey A, Schvartz D, Nolli S, Reny J-L, Sanchez J-C, Fontana P. Characterization of the platelet granule proteome: evidence of the presence of MHC1 in alpha-granules. Journal of proteomics. 2014;101:130–40. doi: 10.1016/j.jprot.2014.02.008 24549006

20. Coppinger JA, Cagney G, Toomey S, Kislinger T, Belton O, McRedmond JP, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood. 2004;103:2096–104. doi: 10.1182/blood-2003-08-2804 14630798

21. Coppinger JA, O'Connor R, Wynne K, Flanagan M, Sullivan M, Maguire PB, et al. Moderation of the platelet releasate response by aspirin. Blood. 2007;109:4786–92. doi: 10.1182/blood-2006-07-038539 17303692

22. Della Corte A, Maugeri N, Pampuch A, Cerletti C, de Gaetano G, Rotilio D. Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome. Platelets. 2008;19:43–50. doi: 10.1080/09537100701609035 18231937

23. Di Michele M, Thys C, Waelkens E, Overbergh L, D'Hertog W, Mathieu C, et al. An integrated proteomics and genomics analysis to unravel a heterogeneous platelet secretion defect. Journal of proteomics. 2011;74:902–13. doi: 10.1016/j.jprot.2011.03.007 21406263

24. O'Connor R, Cryan LM, Wynne K, de Stefani A, Fitzgerald D, O'Brien C, et al. Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate. Journal of biomedicine & biotechnology. 2010;2010:107859. doi: 10.1155/2010/107859 20368775

25. Piersma SR, Broxterman HJ, Kapci M, de Haas RR, Hoekman K, Verheul HMW, et al. Proteomics of the TRAP-induced platelet releasate. Journal of proteomics. 2009;72:91–109. doi: 10.1016/j.jprot.2008.10.009 19049909

26. van Holten TC, Bleijerveld OB, Wijten P, de Groot PG, Heck AJR, Barendrecht AD, et al. Quantitative proteomics analysis reveals similar release profiles following specific PAR-1 or PAR-4 stimulation of platelets. Cardiovascular research. 2014;103:140–6. doi: 10.1093/cvr/cvu113 24776597

27. Wijten P, van Holten T, Woo LL, Bleijerveld OB, Roest M, Heck AJR, et al. High precision platelet releasate definition by quantitative reversed protein profiling—brief report. Arteriosclerosis, thrombosis, and vascular biology. 2013;33:1635–8. doi: 10.1161/ATVBAHA.113.301147 23640497

28. Nassa G, Giurato G, Cimmino G, Rizzo F, Ravo M, Salvati A, et al. Splicing of platelet resident pre-mRNAs upon activation by physiological stimuli results in functionally relevant proteome modifications. Scientific reports. 2018;8:498. doi: 10.1038/s41598-017-18985-5 29323256

29. Parsons MEM, Szklanna PB, Guererro JA, Wynne K, Dervin F, O'Connell K, et al. Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance Between Healthy Adults. Proteomics. 2018:e1800219. doi: 10.1002/pmic.201800219 29932309

30. Velez P, Izquierdo I, Rosa I, Garcia A. A 2D-DIGE-based proteomic analysis reveals differences in the platelet releasate composition when comparing thrombin and collagen stimulations. Scientific reports. 2015;5:8198. doi: 10.1038/srep08198 25645904

31. Lewandrowski U, Wortelkamp S, Lohrig K, Zahedi RP, Wolters DA, Walter U, et al. Platelet membrane proteomics: a novel repository for functional research. Blood. 2009;114:e10–9. doi: 10.1182/blood-2009-02-203828 19436052

32. Senis YA, Tomlinson MG, Garcia A, Dumon S, Heath VL, Herbert J, et al. A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel immunoreceptor tyrosine-based inhibitory motif protein. Molecular & cellular proteomics. 2007;6:548–64. doi: 10.1074/mcp.D600007-MCP200 17186946

33. Garcia BA, Smalley DM, Cho H, Shabanowitz J, Ley K, Hunt DF. The platelet microparticle proteome. Journal of proteome research. 2005;4:1516–21. doi: 10.1021/pr0500760 16212402

34. Capriotti AL, Caruso G, Cavaliere C, Piovesana S, Samperi R, Lagana A. Proteomic characterization of human platelet-derived microparticles. Analytica chimica acta. 2013;776:57–63. doi: 10.1016/j.aca.2013.03.023 23601281

35. De Paoli SH, Tegegn TZ, Elhelu OK, Strader MB, Patel M, Diduch LL, et al. Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome. Cellular and molecular life sciences: CMLS. 2018. doi: 10.1007/s00018-018-2771-6 29427073

36. Kasprzyk J, Stepien E, Piekoszewski W. Application of nano-LC-MALDI-TOF/TOF-MS for proteomic analysis of microvesicles. Clinical biochemistry. 2017;50:241–3. doi: 10.1016/j.clinbiochem.2016.11.013 27865782

37. Milioli M, Ibanez-Vea M, Sidoli S, Palmisano G, Careri M, Larsen MR. Quantitative proteomics analysis of platelet-derived microparticles reveals distinct protein signatures when stimulated by different physiological agonists. Journal of proteomics. 2015;121:56–66. doi: 10.1016/j.jprot.2015.03.013 25835965

38. Banfi C, Brioschi M, Marenzi G, De Metrio M, Camera M, Mussoni L, et al. Proteome of platelets in patients with coronary artery disease. Experimental hematology. 2010;38:341–50. doi: 10.1016/j.exphem.2010.03.001 20226836

39. Lopez-Farre AJ, Zamorano-Leon JJ, Azcona L, Modrego J, Mateos-Caceres PJ, Gonzalez-Armengol J, et al. Proteomic changes related to "bewildered" circulating platelets in the acute coronary syndrome. Proteomics. 2011;11:3335–48. doi: 10.1002/pmic.201000708 21751358

40. Parguina AF, Grigorian-Shamagian L, Agra RM, Lopez-Otero D, Rosa I, Alonso J, et al. Variations in platelet proteins associated with ST-elevation myocardial infarction: novel clues on pathways underlying platelet activation in acute coronary syndromes. Arteriosclerosis, thrombosis, and vascular biology. 2011;31:2957–64. doi: 10.1161/ATVBAHA.111.235713 21921262

41. Parguina AF, Grigorian-Shamajian L, Agra RM, Teijeira-Fernandez E, Rosa I, Alonso J, et al. Proteins involved in platelet signaling are differentially regulated in acute coronary syndrome: a proteomic study. PloS one. 2010;5:e13404. doi: 10.1371/journal.pone.0013404 20976234

42. Liu J, Li J, Deng X. Proteomic analysis of differential protein expression in platelets of septic patients. Molecular biology reports. 2014;41:3179–85. Epub 2014/02/25. doi: 10.1007/s11033-014-3177-7 24562620

43. Gonzalez-Sanchez M, Diaz T, Pascual C, Antequera D, Herrero-San Martin A, Llamas-Velasco S, et al. Platelet Proteomic Analysis Revealed Differential Pattern of Cytoskeletal- and Immune-Related Proteins at Early Stages of Alzheimer's Disease. Molecular neurobiology. 2018;55:8815–25. Epub 2018/04/01. doi: 10.1007/s12035-018-1039-3 29603091

44. Randriamboavonjy V, Isaak J, Elgheznawy A, Pistrosch F, Fromel T, Yin X, et al. Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes. Blood. 2012;120:415–23. Epub 2012/06/06. doi: 10.1182/blood-2011-12-399980 22665935

45. Walkowiak B, Kaminska M, Okroj W, Tanski W, Sobol A, Zbrog Z, et al. The blood platelet proteome is changed in UREMIC patients. Platelets. 2007;18:386–8. Epub 2007/07/27. doi: 10.1080/09537100601095871 17654309

46. Brooks MB, Catalfamo JL, MacNguyen R, Tim D, Fancher S, McCardle JA. A TMEM16F point mutation causes an absence of canine platelet TMEM16F and ineffective activation and death-induced phospholipid scrambling. Journal of thrombosis and haemostasis. 2015;13:2240–52. doi: 10.1111/jth.13157 26414452

47. Martin-Granado V, Ortiz-Rivero S, Carmona R, Gutierrez-Herrero S, Barrera M, San-Segundo L, et al. C3G promotes a selective release of angiogenic factors from activated mouse platelets to regulate angiogenesis and tumor metastasis. Oncotarget. 2017;8:110994–1011. doi: 10.18632/oncotarget.22339 29340032

48. Coppinger J, Fitzgerald DJ, Maguire PB. Isolation of the platelet releasate. Methods in molecular biology. 2007;357:307–11. doi: 10.1385/1-59745-214-9:307 17172696

49. Garcia A, Senis YA, Antrobus R, Hughes CE, Dwek RA, Watson SP, et al. A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter. Proteomics. 2006;6:5332–43. doi: 10.1002/pmic.200600299 16941570

50. Maguire PB, Wynne KJ, Harney DF, O'Donoghue NM, Stephens G, Fitzgerald DJ. Identification of the phosphotyrosine proteome from thrombin activated platelets. Proteomics. 2002;2:642–8. doi: 10.1002/1615-9861(200206)2:6<642::AID-PROT642>3.0.CO;2-I 12112843

51. Schuberth HJ, Kucinskiene G, Chu RM, Faldyna M. Reactivity of cross-reacting monoclonal antibodies with canine leukocytes, platelets and erythrocytes. Veterinary immunology and immunopathology. 2007;119:47–55. Epub 2007/07/24. doi: 10.1016/j.vetimm.2007.06.013 17643496

52. Sharpe KS, Center SA, Randolph JF, Brooks MB, Warner KL, Stokol T, et al. Influence of treatment with ultralow-dose aspirin on platelet aggregation as measured by whole blood impedance aggregometry and platelet P-selectin expression in clinically normal dogs. American journal of veterinary research. 2010;71:1294–304. doi: 10.2460/ajvr.71.11.1294 21034320

53. Yang Y, Thannhauser TW, Li L, Zhang S. Development of an integrated approach for evaluation of 2-D gel image analysis: impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow. Electrophoresis. 2007;28:2080–94. doi: 10.1002/elps.200600524 17486657

54. Yang Y, Anderson E, Zhang S. Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane. Electrophoresis. 2018;39:2332–9. doi: 10.1002/elps.201800042 29644703

55. Zougman A, Selby PJ, Banks RE. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics. 2014;14:1000–6. doi: 10.1002/pmic.201300553 24678027

56. Yang Y, Anderson E, Zhang S. Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane. Electrophoresis. 2018;39:2332–9. Epub 2018/04/13. doi: 10.1002/elps.201800042 29644703

57. Thomas CJ, Cleland TP, Zhang S, Gundberg CM, Vashishth D. Identification and characterization of glycation adducts on osteocalcin. Analytical biochemistry. 2017;525:46–53. doi: 10.1016/j.ab.2017.02.011 28237256

58. Weber RJM, Li E, Bruty J, He S, Viant MR. MaConDa: a publicly accessible mass spectrometry contaminants database. Bioinformatics. 2012;28:2856–7. doi: 10.1093/bioinformatics/bts527 22954629

59. Durinck S, Spellman PT, Birney E, Huber W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nature protocols. 2009;4:1184–91. doi: 10.1038/nprot.2009.97 19617889

60. Fabregat A, Jupe S, Matthews L, Sidiropoulos K, Gillespie M, Garapati P, et al. The Reactome Pathway Knowledgebase. Nucleic acids research. 2018;46:D649–D55. doi: 10.1093/nar/gkx1132 29145629

61. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic acids research. 2016;44:W90–7. doi: 10.1093/nar/gkw377 27141961

62. Poole J. Red cell antigens on band 3 and glycophorin A. Blood reviews. 2000;14:31–43. doi: 10.1054/blre.1999.0124 10805259

63. Gohring K, Wolff J, Doppl W, Schmidt KL, Fenchel K, Pralle H, et al. Neutrophil CD177 (NB1 gp, HNA-2a) expression is increased in severe bacterial infections and polycythaemia vera. British journal of haematology. 2004;126:252–4. doi: 10.1111/j.1365-2141.2004.05027.x 15238147

64. LeVine DN, Birkenheuer AJ, Brooks MB, Nordone SK, Bellinger DA, Jones SL, et al. A novel canine model of immune thrombocytopenia: has immune thrombocytopenia (ITP) gone to the dogs? British journal of haematology. 2014;167:110–20. doi: 10.1111/bjh.13005 25039744

65. Vakili J, Standker L, Detheux M, Vassart G, Forssmann WG, Parmentier M. Urokinase plasminogen activator and plasmin efficiently convert hemofiltrate CC chemokine 1 into its active. Journal of immunology. 2001;167:3406–13.

66. Munch J, Standker L, Pohlmann S, Baribaud F, Papkalla A, Rosorius O, et al. Hemofiltrate CC chemokine 1[9–74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages. Antimicrobial agents and chemotherapy. 2002;46:982–90. doi: 10.1128/AAC.46.4.982-990.2002 11897579

67. Schulz-Knappe P, Schrader M, Standker L, Richter R, Hess R, Jurgens M, et al. Peptide bank generated by large-scale preparation of circulating human peptides. Journal of chromatography A. 1997;776:125–32. doi: 10.1016/s0021-9673(97)00152-0 9286086

68. Boulaftali Y, Adam F, Venisse L, Ollivier V, Richard B, Taieb S, et al. Anticoagulant and antithrombotic properties of platelet protease nexin-1. Blood. 2010;115:97–106. doi: 10.1182/blood-2009-04-217240 19855083

69. Baker JB, Gronke RS. Protease nexins and cellular regulation. Seminars in thrombosis and hemostasis. 1986;12:216–20. doi: 10.1055/s-2007-1003554 3775388

70. Boulaftali Y, Ho-Tin-Noe B, Pena A, Loyau S, Venisse L, Francois D, et al. Platelet protease nexin-1, a serpin that strongly influences fibrinolysis and thrombolysis. Circulation. 2011;123:1326–34. doi: 10.1161/CIRCULATIONAHA.110.000885 21403095

71. Soslau G, Goldenberg SJ, Class R, Jameson B. Differential activation and inhibition of human platelet thrombin receptors by structurally distinct alpha-, beta- and gamma-thrombin. Platelets. 2004;15:155–66. Epub 2004/06/19. doi: 10.1080/0953710042000199848 15203717

72. Lewis SD, Lorand L, Fenton JW, Shafer JA 2nd. Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII. Biochemistry. 1987;26:7597–603. Epub 1987/12/01. doi: 10.1021/bi00398a010 3427095

73. Zufferey A, Fontana P, Reny JL, Nolli S, Sanchez JC. Platelet proteomics. Mass spectrometry reviews. 2012;31:331–51. doi: 10.1002/mas.20345 22009795

74. Deutsch EW, Csordas A, Sun Z, Jarnuczak A, Perez-Riverol Y, Ternent T, et al. The ProteomeXchange Consortium in 2017: supporting the cultural change in proteomics public data deposition. Nucleic Acids Research. 2017;45:D1100–D1106. doi: 10.1093/nar/gkw936 27924013

75. Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Research. 2019:47:D442–D450. doi: 10.1093/nar/gky1106 30395289

Štítky
Detská reumatológia Pediatria Reumatológia

Článok vyšiel v časopise

PLOS One


2019 Číslo 11
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#