Normal prion protein is expressed on exosomes isolated from human plasma (2013)

Four probable cases of blood products spreading variant Creutzfeldt-Jakob Disease (vCJD) have been reported, with two additional cases linked through possible receipt of blood products from a single donor (Chohan et al, 2010). In the UK, leucodepletion and donor-exclusion have been employed to minimize the risk of blood product-mediated transmission. However uncertainty regarding the sources and risks of vCJD transmission mean asymptomatic carriers may inadvertently cause transmission of vCJD through blood donation. The causative agent of vCJD is the abnormally folded form of the prion protein (PrPSc), and this protein is also known to cause other transmissible spongiform encephalopathies (TSEs). The prion protein normally exists in a native, cellular form (PrPC). The paucity of samples from vCJD-infected humans, plus difficulties in distinguishing PrPC and PrPSc, mean that the experimental focus of human in vivo studies has been on PrPC, while animals models and in vitro work has been able to explore both. Animal models have clearly shown that TSEs can be transmitted by blood transfusion (Houston et al, 2000; Hunter et al, 2002; Siso et al, 2006; McCutcheon et al, 2011). Exosomes are the smallest recognized subset of microvesicles, being under 100 nm in diameter, and are released through the fusion of multivesicular bodies with the plasma membrane. They have a distinctive protein composition and can be detected in human plasma (Caby et al, 2005; Ren et al, 2011). Work by others has shown that both PrPC and PrPSc can be detected in association with exosomes generated in vitro by animal cell lines, and that such exosomes can spread TSEs (Fevrier et al, 2004; Vella et al, 2007; Alais et al, 2008). These findings have highlighted exosomes as a potential source of transmission for TSEs, but it is not known whether such concerns are valid for circulating exosomes in human blood. Here, we confirm that PrPC is indeed associated with exosomes isolated from human plasma. These findings are consistent with the potential for exosomes in human plasma to spread vCJD. Plasma units from healthy donors were obtained from National Health Service Blood and Transplant (NHSBT, Bristol, UK), mixed 1:1·5 with phosphate-buffered saline, and vesicles isolated following the technique of Caby et al (2005). Electron microscopy revealed a heterogeneous population of membrane-bound vesicles ranging in size from 30 to 250 nm, with the majority of vesicles in the 50 to 100 nm range (Fig 1A), demonstrating that vesicles that have the morphological characteristics of exosomes make up the bulk of the vesicle preparation. Western blotting revealed the presence of the exosome-associated tetraspanins CD9 and CD63; the endoplasmic reticulum marker calnexin was absent (Fig 1B). Flow cytometric analysis of exosomes bound to anti-CD63 coated beads confirmed that the isolated vesicles expressed the typical surface-marker profile of exosomes: they strongly expressed the exosome-associated tetraspanin molecules CD9, CD63, and CD81, as well as HLAI (Fig 1C). To explore the cellular source/s of these exosomes, markers previously associated with exosomes from various cell types were analysed. There was no detectable staining for human leucocyte antigen (HLA) Class II (antigen presenting cells), CD3 (T cells), CD11c (myeloid dendritic cells), CD20 (B cells), or CD86 (dendritic cells and B cells), while there was strong staining for CD41a (platelets and megakaryocytes) (Fig 1C). This suggests that the majority of exosomes isolated were derived from platelets and/or their precursors. This finding is consistent with the only previously published report on CD41a and human plasma exosomes (Caby et al, 2005), although the absence of HLA Class II on human plasma exosomes was contrary to the findings of two previous studies (Caby et al, 2005; Ren et al, 2011). It remains possible that other cell types, including antigen-presenting cells, produce exosomes in vivo, but these may be at lower levels than platelet-derived exosomes, be sequestered in compartments other than plasma, and/or have a short half-life in plasma. When analysed by flow cytometry, anti-CD63 beads coated with exosomes from eight individual plasma samples were shown to express PrPC (Fig 2A). Analysis of the vesicle pellet from four different plasma samples by Western blotting also consistently showed the presence of PrPC in this fraction (Fig 2B). Thus PrPC was detected in association with human plasma exosomes isolated from 12 different donors, using two detection techniques. It remains to be determined whether or not PrPSc is also associated with human plasma exosomes and an absence of samples means this may not be possible in the foreseeable future. Our data coupled with animal models showing exosomes can spread TSEs suggest it would be prudent to consider exosomes as one potential source of PrPSc in human blood products. We would like to thank Richard Kascak for reagents. This work was supported by the National Health Service Blood and Transplant (NHSBT) Research and Development (BS03/3/RB40). The authors declare no competing financial interests. AJR designed experiments, performed research, analysed data and prepared the manuscript, DMC performed research, DJPF performed electron microscopy, JB was responsible for the study concept, DJR was responsible for the study concept and designed experiments.