Influence of short-term refrigeration on collagen-dependent signalling mechanisms in stored platelets

Abstract

Platelet concentrates (PC) are used to treat patients with thrombocytopenia and hemorrhage, but there is still the demand to find the optimal strategy for temperature-dependent storage of PC. Recently, we could show that cold storage for 1 h (short-term refrigeration) is sufficient to induce enhanced platelet responsiveness. The aim of this study was to investigate effects of cold storage on collagen-dependent activating signalling pathways in platelets from apheresis-derived PC (APC). APC on day 1 or day 2 of storage, were either continuously kept at room temperature (RT, 22 °C), or for comparison, additionally kept at cold temperature (CT, 4 °C) for 1 h. CD62P expression was determined by flow cytometry. Western Blot technique was used to analyze collagen-induced phosphorylation of p38, ERK1/2 or Akt/PKB and its inhibition by prostaglandin E1 (PGE1) or nitric monoxide donor. Adhesion of platelets on collagen-coated surfaces and intracellular phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was visualized by immune fluorescence microscopy. CD62P expression was increased after short-term refrigeration. CT exposition for 1 h induced an elevation of basal ERK1/2 phosphorylation and an alleviation of PGE1- or DEA/NO-suppressed ERK1/2 phosphorylation in APC on day 1 and 2 of storage. Similar, but more moderate effects were observable for p38 phosphorylation. Akt/PKB phosphorylation was increased only in APC on day 2. Refrigeration for 1 h promoted platelet adhesion and reduced basal VASP phosphorylation in adherent platelets. The attenuation of inhibitory signalling in short-term refrigerated stored platelets is associated with enhanced reactivity of activating signalling pathways, especially ERK1/2. Functionally, these processes correlate with increased adhesion of refrigerated platelets on collagen-coated surfaces. The results help to further optimize temperature-dependent strategies for platelet storage.

Keywords: Activating signalling; ERK1/2; Inhibitory signalling; Platelet concentrate; Refrigeration; Vasodilator-stimulated phosphoprotein.