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

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 extracellular Ca²⁺ predominate in APU MP stimulated calcium flux. No significant [Ca²⁺]_i increase was achieved even with 3x concentrated MP from HVP. To test apoptotic activity (TUNEL), washed APU MP were incubated overnight with endothelial cells (HUVEC). About 25% of APU MP induced significant apoptotic activity which correlated with CD154⁺CD45⁻CD41a⁺MP (r=0.81; p=0.002) and CD45⁺MP (r=0.75; p=0.009) and was higher in day 6 vs. day 8 APU MP vs. HVP MP (p=0.03). In addition, cell cycle analysis showed that APU MP induced G1 arrest. In conclusion, APU MP exhibited various pathophysiological activities in vitro. Some individual units showed a high MP content, and correlations were found between certain MP phenotypes and APU MP activities. The 7-day storage of effectively leukoreduced APU should not have a dramatic impact on a potentially pathogenic MP content. Studies investigating an association of high MP counts in some APU with clinical adverse effects of APU transfusions are warranted. The views of the authors should not be construed as FDA policy.