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. 1975 Apr;79(1):81–94.

Characterization of human platelets separated from blood by ADP-induced aggregation.

S F Mohammad, R L Reddick, R G Mason
PMCID: PMC1912647  PMID: 1124800

Abstract

Separation of platelets from plasma is achieved by adding ADP (final concentration 10-5 M) to platelet-rich plasma and allowing aggregates to form. Aggregates are removed quickly by brief, gentle centrifugation, washed two to three times with 0.9% NaCl (saline), and then incubated for 10 minutes in the presence of apyrase, albumin and calcium. Platelet aggregates deaggregate completely during this incubation period. The platelet suspension is then subjected to 1100g for 12 minutes, gently resuspended in a small volume of saline, and finally diluted with an appropriate medium to the desired concentration. The entire separation procedure requires approximately 30 minutes. Platelets obtained by this procedure are a) comparable in aggregability to the platelet preparations obtained by gel filtration, b) have normal intracellular amounts of ATP and ADP, and c) except for slight dilatation of the surface-connected canalicular system, have normal ultrastructural appearance. When suspended in an appropriate medium, these separated platelets take up serotonin 14-C and subsequently release it in nearly normal quantities when exposed to thrombin, collagen or ADP.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BORN G. V., CROSS M. J. Effect of adenosine diphosphate on the concentration of platelets in circulating blood. Nature. 1963 Mar 9;197:974–976. doi: 10.1038/197974a0. [DOI] [PubMed] [Google Scholar]
  2. BRINKHOUS K. M., LEROY E. C., CORNELL W. P., BROWN R. C., HAZLEHURST J. L., VENNART G. P. Macroscopic studies of platelet agglutination; nature of thrombocyte agglutinating activity of plasma. Proc Soc Exp Biol Med. 1958 Jun;98(2):379–383. doi: 10.3181/00379727-98-24050. [DOI] [PubMed] [Google Scholar]
  3. Behnke O. Microtubules in disk-shaped blood cells. Int Rev Exp Pathol. 1970;9:1–92. [PubMed] [Google Scholar]
  4. Corby D. G., Preston K. A., O'Barr T. P. Adverse effect of gel filtration on the function of human platelets. Proc Soc Exp Biol Med. 1974 May;146(1):96–98. doi: 10.3181/00379727-146-38050. [DOI] [PubMed] [Google Scholar]
  5. DAVEY M. G., LANDER H. EFFECT OF ADENOSINE DIPHOSPHATE ON CIRCULATING PLATELETS IN MAN. Nature. 1964 Mar 7;201:1037–1039. doi: 10.1038/2011037c0. [DOI] [PubMed] [Google Scholar]
  6. HOVIG T. AGGREGATION OF RABBIT BLOOD PLATELETS PRODUCED IN VITRO BY SALINE "EXTRACT" OF TENDONS. Thromb Diath Haemorrh. 1963 Jul 15;143:248–263. [PubMed] [Google Scholar]
  7. Holmsen H., Ostvold A. C., Day H. J. Behaviour of endogenous and newly absorbed serotonin in the platelet release reaction. Biochem Pharmacol. 1973 Oct 15;22(20):2599–2608. doi: 10.1016/0006-2952(73)90068-3. [DOI] [PubMed] [Google Scholar]
  8. Holmsen H., Storm E., Day H. J. Determination of ATP and ADP in blood platelets: a modification of the firefly luciferase assay for plasma. Anal Biochem. 1972 Apr;46(2):489–501. doi: 10.1016/0003-2697(72)90323-5. [DOI] [PubMed] [Google Scholar]
  9. Hutton R. A., Howard M. A., Deykin D., Hardisty R. M. Methods for the separation of platelets from plasma. A comparison of functional and morphological integrity. Thromb Diath Haemorrh. 1974 Mar 15;31(1):119–132. [PubMed] [Google Scholar]
  10. Lundblad R. L. A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme wtih phenylmethylsulfonyl fluoride. Biochemistry. 1971 Jun 22;10(13):2501–2506. doi: 10.1021/bi00789a012. [DOI] [PubMed] [Google Scholar]
  11. Mason R. G., Read M. S., Shermer R. W. Comparison of certain functions of human platelets separated from blood by various means. Am J Pathol. 1974 Aug;76(2):323–332. [PMC free article] [PubMed] [Google Scholar]
  12. Mustard J. F., Perry D. W., Ardlie N. G., Packham M. A. Preparation of suspensions of washed platelets from humans. Br J Haematol. 1972 Feb;22(2):193–204. doi: 10.1111/j.1365-2141.1972.tb08800.x. [DOI] [PubMed] [Google Scholar]
  13. Mustard J. F., Rowsell H. C., Lotz F., Hegardt B., Murphy E. A. The effect of adenine nucleotides on thrombus formation, platelet count, and blood coagulation. Exp Mol Pathol. 1966 Feb;5(1):43–60. doi: 10.1016/0014-4800(66)90004-9. [DOI] [PubMed] [Google Scholar]
  14. NORDOEY A., CHANDLER A. B. PLATELET THROMBOSIS INDUCED BY ADENOSINE DIPHOSPHATE IN THE RAT. Scand J Haematol. 1964;1:16–25. doi: 10.1111/j.1600-0609.1964.tb00002.x. [DOI] [PubMed] [Google Scholar]
  15. Nicholls D. G., Hampton J. R. Density gradient separation of human platelets from plasma and the role of plasma in adenosine diphosphate induced platelet electrophoretic mobility changes. Thromb Diath Haemorrh. 1972 Jul 31;27(3):425–433. [PubMed] [Google Scholar]
  16. RODMAN N. F., Jr, MASON R. G., BRINKHOUS K. M. SOME PATHOGENETIC MECHANISMS OF WHITE THROMBUS FORMATION: AGGLUTINATION AND SELF-DESTRUCTION OF THE PLATELET. Fed Proc. 1963 Nov-Dec;22:1356–1365. [PubMed] [Google Scholar]
  17. Reddick R. L., Mason R. G. Freeze-etch observations on the plasma membrane and other structures of normal and abnormal platelets. Am J Pathol. 1973 Mar;70(3):473–488. [PMC free article] [PubMed] [Google Scholar]
  18. Rodman N. F., Mason R. G. Platelet-platelet interaction: relationship to hemostasis and thrombosis. Fed Proc. 1967 Jan-Feb;26(1):95–105. [PubMed] [Google Scholar]
  19. Rossi E. C. A soluble, magnesium-dependent, calcium-inhibited ATPase in human platelets. J Lab Clin Med. 1971 Sep;78(3):483–498. [PubMed] [Google Scholar]
  20. Tangen O., Berman H. J., Marfey P. Gel filtration. A new technique for separation of blood platelets from plasma. Thromb Diath Haemorrh. 1971 Jun 30;25(2):268–278. [PubMed] [Google Scholar]
  21. Walsh P. N. Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities. Br J Haematol. 1972 Feb;22(2):205–217. doi: 10.1111/j.1365-2141.1972.tb08801.x. [DOI] [PubMed] [Google Scholar]
  22. White J. G. Fine structural alterations induced in platelets by adenosine diphosphate. Blood. 1968 May;31(5):604–622. [PubMed] [Google Scholar]

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