Expression of cellular prion protein on vascular endothelial cells: more evidence than controversies

Recent data have shown that significant levels of cellular prion protein (PrPC) are found within human blood. However, although PrPC is expressed by PLTs, WBCs, and RBCs, the majority of PrPC is detected within the plasma fraction by dissociation-enhanced fluoroimmunoassay (DELFIA, Wallac, United Kingdom). 1 This is particularly relevant as PrPSC can also be found within the plasma fraction in animal models of the disease.2 In their recent TRANSFUSION article, Simak and coworkers3 reported that PrPC is expressed on endothelial cells and is released during apoptosis, on membrane microparticles that are also detectable in human plasma. We have performed similar experiments with human umbilical vein endothelial cells (HUVECs) and report comparable findings. In addition, we have also used human microvascular endothelial cells (HMEC-1). 4 Both cell types were found to express PrPC mRNA, and flow cytometric analysis of HUVECs demonstrated a similar quantity of cell surface PrPC as reported by Simak et al.3 The HMEC-1 cells were found to express double the amount of surface PrPC. Simak and colleagues3 measured the release of microparticles associated with PrPC from apoptotic endothelial cells by flow cytometry and demonstrated that endothelial-derived microparticles within both cell culture and blood plasma express PrPC, suggesting that they could contribute to the plasma pool of PrPC. To clarify the contribution of microparticles to the PrPC present in endothelial cell supernatants, we have measured PrPC by DELFIA before and after ultracentrifugation of HUVEC and HMEC-1 cell supernatants at 100,000 g for 2 hours. We then resuspended the microparticle precipitates (verified by flow cytometry with annexin V labeling) and measured the PrPC concentration by DELFIA. There was a slight decrease of signal in the ultracentrifuged supernatant, indicating that a small proportion of PrPC was associated with insoluble material (Fig. 1). There was no detectable PrPC within the resuspended microparticle precipitates. Similar results were obtained with normal human plasma before and after ultracentrifugation (Fig. 1). Ultracentrifugation removed both microparticles and a small proportion of the PrPC DELFIA signal, supporting the data of Simak et al.3 It has already been shown that blood plasma infectivity is not eliminated by high-speed centrifugation in a mouse model of CJD, indicating that the infectivity is present in the soluble plasma fraction.2 We conclude that it is likely that endothelial cell microparticles do express PrPC, but that most PrPC released by endothelial cells and within human blood plasma is in soluble form. Richard Starke, BSc Haemostasis Research Unit University College London 3rd Floor Jules Thorn Building 48 Riding House Street London W1N 8AA, UK e-mail: r.starke@ucl.ac. uk Paul Harrison, BSc, PhD, MRCPath Oxford Haemophilia Center Churchill Hospital Headington Oxford, UK Olive Drummond, GIBiol Ian Macgregor, BSc, PhD Products and Components R & D Group National Science Laboratory Scottish National Blood Transfusion Service Edinburgh, UK Ian Mackie, BSc, PhD, FIBMS, FRCPath Samuel Machin, MB ChB, FRCP, FRCPath Haemostasis Research Unit University College London 3rd Floor Jules Thorn Building 48 Riding House Street London W1N 8AA, UK