32
Views
2
CrossRef citations to date
0
Altmetric
Research Article

Comparative Analysis of Platelet Isolation Techniques for the In VivoStudy of the Microcirculation

, , , &
Pages 143-152 | Published online: 10 Jul 2009
 

Abstract

Objective:In vitroand in vivostudies using isolated platelets require that the cells used for testing are not activated by the isolation procedure. This ensures that the effects measured by the test are the result of the environment or the applied stimulus, but is not an artifact resulting from activation by cell isolation. Methods:Herein, we analyzed two different platelet isolation procedures (i.e., a Sepharose column versus density gradient centrifugation) with special emphasis on cell activation, including flow cytometric analysis of P-selectin expression, functional quantification of mechanical platelet retention, light microscopic assessment of platelet aggregation, and fluorescence microscopic determination of in vivorat liver platelet–endothelium cell interaction. Results:Under resting conditions, Sepharose column-isolated platelets showed a negligible fraction of only 2.7 ± 3.3% cells (mean ± SEM) with P-selectin expression, and an appropriate response (i.e., a 33-fold increase) upon activation with thrombin receptor-activating peptide (TRAP). In contrast, density gradient centrifugation resulted in P-selectin expression under resting conditions of ∼50% of the isolated cells and only a 1.6-fold increase on further TRAP stimulation. In addition, density gradient-isolated platelets, but not Sepharose column-isolated platelets, showed increased mechanical retention and agglutination/aggregation in vitro, as well as pronounced adhesion to hepatic venular endothelium in vivo. Interestingly, density gradient-isolated platelets additionally induced in vivoan increase of colocalization of platelets with adherent leukocytes, indicating a generalized microvascular inflammatory response that is comparable to that observed after a 60-minute ischemia/30-minute reperfusion insult. Conclusion:Density gradient centrifugation-isolated platelets, but not Sepharose column-isolated platelets, are activated already under resting conditions and induce in vivoa platelet–leukocyte–endothelial cell-associated inflammatory response. Thus, we propose that the method of platelet isolation using the Sepharose column is superior to the density gradient centrifugation technique and might therefore be preferred for in vitroand in vivoassays to study platelet function. Microcirculation(2003) 10,143–152. doi:10.1038/sj.mn.7800179

Reprints and Corporate Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

To request a reprint or corporate permissions for this article, please click on the relevant link below:

Academic Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

Obtain permissions instantly via Rightslink by clicking on the button below:

If you are unable to obtain permissions via Rightslink, please complete and submit this Permissions form. For more information, please visit our Permissions help page.