title: Determination of ATP and ADP Secretion from Human and Mouse Platelets by an HPLC Assay.
abstract: BACKGROUND
  Secretion of ADP and ATP is an essential prerequisite for platelet aggregation. Impaired nucleotide secretion can cause aggregation defects and increased bleeding risk. Quantitative determination of platelet nucleotide content and exocytosis is thus of importance for the characterization and diagnosis of bleeding phenotypes. For transgenic animal models with hemostatic defects analysis of potential secretion defects is as well imperative.
  
  
  METHODS
  Supernatants of washed platelets and platelet-rich plasma were analyzed by HPLC for ADP and ATP concentration. Calibration of the HPLC data was accomplished with an internal standard compensating for loss of analyte, detection sensitivity, and interference of the biomatrix.
  
  
  RESULTS
  HPLC analysis of nucleotide secretion was carried out with human and mouse platelets. Detection limits were determined for washed platelet and platelet-rich plasma samples. In the physiological concentration range linearity with respect to the peak area is maintained.
  
  
  CONCLUSIONS
  The method combines reasonable sensitivity with robustness. The internal standard ensures reliable quantification of nucleotide concentrations even in presence of otherwise interfering substances. The low sample consumption renders possible the application to analysis of small samples like in mouse experiments.
authors: Institute for Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Germany.
authors: Michael
authors: M
authors: von Papen
authors: Stepan
authors: S
authors: Gambaryan
authors: Claudia
authors: C
authors: Schütz
authors: Jörg
authors: J
authors: Geiger
keywords: Delta granule; Nucleotides; Platelet exocytosis; Secretion defects; Storage pool deficiency
references: Clin Chem Lab Med. 2012 Jul;50(7):1253-62
references: 22149738
references: J Thromb Haemost. 2011 Jul;9 Suppl 1:76-91
references: 21781244
references: Clin Chem. 2005 Jun;51(6):957-65
references: 15817818
references: Methods Mol Biol. 2012;788:341-66
references: 22130718
references: Stroke. 1991 Jan;22(1):17-21
references: 1987668
references: Blood Rev. 2009 Jul;23(4):177-89
references: 19450911
references: Platelets. 2012;23(7):552-63
references: 23020613
references: Life Sci. 1990;47(19):1751-9
references: 1701205
references: Hamostaseologie. 2010 Jan;30(1):29-38
references: 20162250
references: Circulation. 2009 Sep 1;120(9):785-91
references: 19687360
references: Br J Haematol. 1995 Feb;89(2):380-5
references: 7873389
references: Eur J Pediatr. 2012 Jan;171(1):1-10
references: 21800040
references: Eur J Pediatr. 2007 Dec;166(12):1203-10
references: 17619901
references: Methods Enzymol. 1989;169:205-10
references: 2654560
references: Thromb Haemost. 2008 Mar;99(3):466-72
references: 18327393
references: Haemophilia. 2012 Jul;18 Suppl 4:154-60
references: 22726100
references: Blood. 1984 Feb;63(2):427-33
references: 6692042
references: J Chromatogr B Biomed Appl. 1996 Apr 12;678(2):181-6
references: 8738020
references: Thromb Haemost. 2011 Nov;106(5):922-33
references: 21800013
references: Br J Haematol. 2004 Oct;127(1):34-9
references: 15384975
references: Thromb Haemost. 1985 Feb 18;53(1):65-9
references: 3992522
references: Anal Biochem. 2010 Dec 15;407(2):261-9
references: 20708596
references: J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Mar 15;864(1-2):43-51
references: 18282748
references: Semin Thromb Hemost. 2009 Mar;35(2):242-54
references: 19408197
references: Am J Physiol. 1982 Sep;243(3):R454-61
references: 7114301
references: Clin Chem Lab Med. 2004 May;42(5):475-86
references: 15202782
references: Eur J Pharmacol. 1998 Jun 19;351(2):235-46
references: 9687008
references: Stroke. 2003 Mar;34(3):764-9
references: 12624305
references: Curr Opin Hematol. 2008 Sep;15(5):537-41
references: 18695380
references: Biochem Med. 1974 Mar;9(3):225-32
references: 4275005
references: Front Biosci. 2006 May 01;11:1977-86
references: 16368572
references: Br J Haematol. 2013 Jan;160(1):5-11
references: 23025459
references: Exp Hematol. 2001 Nov;29(11):1295-302
references: 11698125
references: Blood. 1990 Mar 1;75(5):1081-6
references: 2137716
references: Blood. 1990 Nov 1;76(9):1884-92
references: 2224137
references: J Neurol Neurosurg Psychiatry. 1994 May;57(5):557-61
references: 8201323
references: Br J Haematol. 1990 May;75(1):118-21
references: 2375909
references: J Thromb Haemost. 2012 Nov;10(11):2313-4
references: 23006797
references: J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Mar 15;889-890:110-5
references: 22382093