Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets
Tegegn, Tseday Z.
De Paoli, Silvia H.
Elhelu, Oumsalama K.
Woodle, Samuel A.
Tarandovskiy, Ivan D.
Ovanesov, Mikhail V.
- Publisher: Co-Action Publishing
Journal of Extracellular Vesicles,
platelet physiology | blood products | extracellular vesicles | thrombin | nanoparticle tracking analysis | Original Research Article | microparticles | transfusion medicine | electron microscopy | Extracellular vesicles; Microparticles; Platelet physiology; Blood products; Thrombin; Transfusion medicine; Nanoparticle tracking analysis; Flow cytometry; Atomic force microscopy; Electron microscopy | flow cytometry | atomic force microscopy
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.Keywords: extracellular vesicles; microparticles; platelet physiology; blood products; thrombin; transfusion medicine; nanoparticle tracking analysis; flow cytometry; atomic force microscopy; electron microscopy(Published: 4 May 2016)Citation: Journal of Extracellular Vesicles 2016, 5: 30422 - http://dx.doi.org/10.3402/jev.v5.30422