Evaluation of Immunophenotypes and Activities of Cell Membrane Microparticles in Apheresis Platelets: Impact of Seven Day Storage

• Published in: Blood 2005-11, Vol.106 (11), p.1893-1893
• Main Authors: Gelderman-Fuhrmann, Monique P., Simakova, Olga, Carter, Laura B., Simak, Jan
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V106.11.1893.1893

Seven day storage of platelets has become a reality in transfusion management. Cell membrane microparticles (MP), 0.2–1.0 μm in size are released from stimulated platelets, blood- and endothelial cells. The pathogenic potential of MP has been documented. Since a portion of platelet transfusion adverse effects is likely not antibody related, MP are good candidates to study. We have previously demonstrated the presence of MP derived from platelets, blood-, and endothelial cells in apheresis platelet units (APU) on day 6 of storage. Here we investigate the pathophysiological activities of MP in APU and the impact of extended storage on MP immunophenotype and activity profiles. We analyzed MP in non-frozen APU supernatants (n=28) on day 6 and 8 of storage. A three-color flow cytometry assay of MP (Simak et al, British J Haematol, 2004) was used, and several functional assays were performed. Plasma from healthy volunteers (HVP) (n=38) was used for comparison. On day 6, the median platelet count in APU was 1514x103/μL (range: 1144x103–2248x103) and pH 7.28 (6.67–7.51). The pH decreased significantly to 6.97 (6.19–7.40) on day 8. All APU bacterial cultures were negative. When comparing APU on day 6 and 8 of storage, platelet MP (CD41+), endothelial MP (CD144+), red blood cell MP (CD235a+), and proinflammatory CD154+MP did not change significantly. In contrast, an increase of leukocyte MP, including leukocyte MP exposing tissue factor (TF, CD142) (CD142+CD45+MP; p=0.001) was found. Since both platelet CD41a+CD142+ and endothelial CD144+CD142+ MP tend to decrease during storage, the total CD142+MP (detected by V1C7 and HFT-1 Mab) showed only a slight increase. All studied MP phenotypes were significantly higher in APU on day 6 and 8 when compared to HVP. TF activity (using FVIIa, FX and FXa substrate) of washed MP was 619 nM TF (35–393) in APU on day 6 vs. 629 nM TF (38–2443) on day 8. MP associated TF activity correlated with counts of CD142+MP (r=0.76; p=0.02) and was in some individual APU over 10x the median value of HVP MP, 190 nM TF (20–440). The effect of APU MP on intracellular free Ca2+ concentration [Ca2+]i was also studied, using a ratio fluorometry in GT1-7 cells loaded with a Ca2+-sensitive probe FURA-2AM. Washed APU MP induced a rapid MP concentration dependent peak of [Ca2+]i, followed by a sustained slow increase of [Ca2+]i. The activity was significantly higher in day 6 vs. day 8 APU MP (p=0.001) and could be inhibited by EGTA, suggesting that extr