No abstract

Summary The protein C pathway provides multiple important functions to maintain a regulated balance between haemostasis and host defence systems in response to vascular and inflammatory injury. The anticoagulant protein C pathway is designed to regulate coagulation, maintain the fluidity of blood within the vasculature, and prevent thrombosis, whereas the cytoprotective protein C pathway prevents vascular damage and stress. The cytoprotective activities of activated protein C (APC) include anti-apoptotic activity, anti-inflammatory activity, beneficial alterations of gene expression profiles, and endothelial barrier stabilisation. These cytoprotective activities of APC, which require the endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR1), have been a major research focus. Recent insights, such as non-canonical activation of PAR1 at Arg46 by APC and biased PAR1 signalling, provided better understanding of the molecular mechanisms by which APC elicits cytoprotective signalling through cleavage of PAR1. The discovery and development of anticoagulant-selective and cytoprotective-selective APC mutants provided unique opportunities for preclinical research that has been and may continue to be translated to clinical research. New mechanisms for the regulation of EPCR functionality, such as modulation of EPCR-bound lipids that affect APC’s cytoprotective activities, may provide new research directions to improve the efficacy of APC to convey its cytoprotective activities to cells. Moreover, emerging novel functions for EPCR expand the roles of EPCR beyond mediating protein C activation and APC-induced PAR1 cleavage. These discoveries increasingly develop our understanding of the protein C pathway, which will conceivably expand its physiological implications to many areas in the future.

Key Points Isolation of BOECs from multiple patients with VWD is feasible, and the study of BOECs helps explain the pathogenic complexity of VWD. Abnormalities in WPB biogenesis and exocytosis and defects in VWF string formation correlate with the phenotypic features of patients with VWD.

BackgroundGene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion. Design and MethodsHuman blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human Bdomain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy. ResultsBlood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1×10 6 cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6-to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15-22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies. ConclusionsWe conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.