Changes in platelet morphology and function during 24 hours of storage

Issue title: Selected Presentations held at the 33th Annual Conference of the German Society for Clinical Microcirculation and Hemorheology, Villingen-Schwenningen, Germany, 14-15 November, 2014

Article type: Research Article

Authors: Braune, S.^; | Walter, M.^; | Schulze, F. | Lendlein, A.^; | Jung, F.

Affiliations: Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany | Institute of Chemistry, University of Potsdam, Potsdam, Germany

Note: [] These authors contributed equally to this work.

Note: [] These authors contributed equally to this work.

Note: [] Corresponding author: Dr. F. Jung, Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Kantstraße 55, 14513 Teltow, Germany. Tel.: +49 3328 352 269; Fax: +49 3328 352 452; E-mail: [email protected]

Abstract: For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated whole blood (WB) using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5′-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL – 40 fL) decreased over time. Immediately after blood collection, no debris or platelet aggregates could be visualized microscopically. After four hours, first debris and very small aggregates occurred. After 24 hours, platelet aggregates and also debris progressively increased. In accordance to this, the CASY system revealed an increase of platelet aggregates (up to 90 μm diameter) with increasing storage time. The percentage of CD62P positive platelets and PF4 increased significantly with storage time in resting PRP. When soluble ADP was added to stored PRP samples, the number of activatable platelets decreased significantly over storage time. The present study reveals the importance of a consequent standardization in the preparation of WB and PRP. Platelet morphology and function, particularly platelet reactivity to adherent or soluble agonists in their surrounding milieu, changed rapidly outside the vascular system. This knowledge is of crucial interest, particularly in the field of biomaterial development for cardiovascular applications, and may help to define common standards in the in vitro hemocompatibility testing of biomaterials.

Keywords: Platelet, platelet function, platelet rich plasma, whole blood, platelet aging, platelet storage, hemocompatibility, biomaterials

DOI: 10.3233/CH-141876

Journal: Clinical Hemorheology and Microcirculation, vol. 58, no. 1, pp. 159-170, 2014