title: Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets.
abstract: BACKGROUND
  Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs.
  
  
  METHODS
  CPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test.
  
  
  RESULTS
  SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing-thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a(+) PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent.
  
  
  CONCLUSIONS
  Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing-thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Tseday Z
authors.initials: TZ
authors.lastname: Tegegn
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Silvia H
authors.initials: SH
authors.lastname: De Paoli
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Martina
authors.initials: M
authors.lastname: Orecna
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Oumsalama K
authors.initials: OK
authors.lastname: Elhelu
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Woodle
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Ivan D
authors.initials: ID
authors.lastname: Tarandovskiy
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Ovanesov
authors.affiliation: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA; jan.simak@fda.hhs.gov.
authors.forename: Jan
authors.initials: J
authors.lastname: Simak
keywords: atomic force microscopy; blood products; electron microscopy; extracellular vesicles; flow cytometry; microparticles; nanoparticle tracking analysis; platelet physiology; thrombin; transfusion medicine