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Thrombin-Mediated Platelet Activation of Lysed Whole Blood and Platelet-Rich Plasma: A Comparison Between Platelet Activation Markers and Ultrastructural Alterations

Published online by Cambridge University Press:  22 June 2016

Tanya N. Augustine ,
Wendy J. van der Spuy ,
Lindsay L. Kaberry  and
Millicent Shayi
Tanya N. Augustine*
Affiliation:
School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
Wendy J. van der Spuy
Affiliation:
School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
Lindsay L. Kaberry
Affiliation:
School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
Millicent Shayi
Affiliation:
School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
*
*Corresponding author. tanya.augustine@wits.ac.za
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Abstract

Platelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool.

Keywords

plateletsultrastructureflow cytometrythrombinplatelet activation
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 22 , Issue 3 , June 2016 , pp. 630 - 639
Copyright
Copyright © Microscopy Society of America 2016

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