Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity

Abstract

Objective: In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation.

Materials and methods: Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing.

Results: The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014).

Conclusion: Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

Figure 1. Isolation and quantitation of microparticles in freeze-thawed apheresis platelet concentrate samples. A) P1 is showing the gate of annexin-bound microparticles, which are selected in comparison with 1.0 µm latex beads (P2). B) Q1 is showing CD62P (+) and CD41a (-) platelet-derived microparticles, Q4 is showing CD62P (-) and CD41a (+) platelet-derived microparticles. C) Q4 is showing CD62P (-) and CD41a (+) and annexin V (+) platelet-derived microparticles.

Figure 2. Viability evaluation assays showing the 7-AAD (-) unstained freeze-thawed platelets.

Figure 3. Correlation analyses related to platelet-derived microparticles. A) Scatterplot with linear fit graph of correlation analysis between platelet-derived microparticle levels and endogenous thrombin potential levels of freeze-thawed apheresis platelet concentrates. B) Scatterplot with linear fit graph of correlation analysis between platelet-derived microparticle levels and time to peak thrombin levels of freeze-thawed apheresis platelet concentrates.