Supplementary Material for: Increased responsiveness of stored platelets after short-term refrigeration

Introduction: Refrigeration of platelets is considered to provide advantages in therapy of acute hemorrhage due to increased platelet responsiveness. The alleviation of inhibitory signaling caused by cold temperature (CT) has been identified as an important mechanism contributing to enhanced platelet reactivity, detectable in freshly prepared platelets within 1 h of cold storage. The aim of this study was to confirm the effects of short-term refrigeration in platelets from apheresis-derived platelet concentrates (APC). Methods: APC were stored under standardized conditions for 1 day or for 2 days at room temperature (RT) and then refrigerated for 1 h, followed by sampling of platelets for analysis. Platelet reactivity was measured by aggregation studies using threshold concentrations of different agonists and by detection of fibrinogen binding using flow cytometry. The exploration of inhibitory signaling comprised the detection of VASP phosphorylation using flow cytometry or Western Blot and the measurement of cyclic nucleotide levels. Results: Aggregation responses induced with ADP, collagen or TRAP-6 were increased in APC after cold storage for 1 h, associated with elevated TRAP-6-induced fibrinogen binding. VASP phosphorylation levels were decreased after cold exposition, detectable in 1-day and 2 days-stored APC with flow cytometry, and in 2 days-stored APC with Western Blot technique. Induced cGMP levels were lower after storage at CT in APC on day 1 and on day 2, whereas cAMP levels were reduced on 2 days-stored APC. Conclusion: Short-term refrigeration for 1 h is sufficient to induce an attenuation of inhibitory signaling, accompanied with increased aggregation responses in APC stored for up to two days. The “on demand”-refrigeration of PC may be a reasonable approach for the preparation of platelets with enhanced responsiveness to treat patients with hemorrhage more effectively, which should be further addressed in consecutive studies.