title: Thrombin-Mediated Platelet Activation of Lysed Whole Blood and Platelet-Rich Plasma: A Comparison Between Platelet Activation Markers and Ultrastructural Alterations.
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.
authors.affiliation: School of Anatomical Sciences, Faculty of Health Sciences,University of the Witwatersrand,Johannesburg,South Africa.
authors.forename: Tanya N
authors.initials: TN
authors.lastname: Augustine
authors.affiliation: School of Anatomical Sciences, Faculty of Health Sciences,University of the Witwatersrand,Johannesburg,South Africa.
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: van der Spuy
authors.affiliation: School of Anatomical Sciences, Faculty of Health Sciences,University of the Witwatersrand,Johannesburg,South Africa.
authors.forename: Lindsay L
authors.initials: LL
authors.lastname: Kaberry
authors.affiliation: School of Anatomical Sciences, Faculty of Health Sciences,University of the Witwatersrand,Johannesburg,South Africa.
authors.forename: Millicent
authors.initials: M
authors.lastname: Shayi
mesh_terms: D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure*; D005434:Flow Cytometry; D006801:Humans; D008855:Microscopy, Electron, Scanning; D015539:Platelet Activation / Q000502:physiology*; D053657:Platelet-Rich Plasma / Q000166:cytology*; D013917:Thrombin / Q000378:metabolism*
chemical_list: D013917:Thrombin
keywords: flow cytometry; platelet activation; platelets; thrombin; ultrastructure