title: Endothelial cell processing and alternatively spliced transcripts of factor VIII: potential implications for coagulation cascades and pulmonary hypertension.
abstract: BACKGROUND
  Coagulation factor VIII (FVIII) deficiency leads to haemophilia A. Conversely, elevated plasma levels are a strong predictor of recurrent venous thromboemboli and pulmonary hypertension phenotypes in which in situ thromboses are implicated. Extrahepatic sources of plasma FVIII are implicated, but have remained elusive.
  
  
  RESULTS
  Immunohistochemistry of normal human lung tissue, and confocal microscopy, flow cytometry, and ELISA quantification of conditioned media from normal primary endothelial cells were used to examine endothelial expression of FVIII and coexpression with von Willebrand Factor (vWF), which protects secreted FVIII heavy chain from rapid proteloysis. FVIII transcripts predicted from database mining were identified by RT-PCR and sequencing. FVIII mAb-reactive material was demonstrated in CD31+ endothelial cells in normal human lung tissue, and in primary pulmonary artery, pulmonary microvascular, and dermal microvascular endothelial cells. In pulmonary endothelial cells, this protein occasionally colocalized with vWF, centered on Weibel Palade bodies. Pulmonary artery and pulmonary microvascular endothelial cells secreted low levels of FVIII and vWF to conditioned media, and demonstrated cell surface expression of FVIII and vWF Ab-reacting proteins compared to an isotype control. Four endothelial splice isoforms were identified. Two utilize transcription start sites in alternate 5' exons within the int22h-1 repeat responsible for intron 22 inversions in 40% of severe haemophiliacs. A reciprocal relationship between the presence of short isoforms and full-length FVIII transcript suggested potential splice-switching mechanisms.
  
  
  CONCLUSIONS
  The pulmonary endothelium is confirmed as a site of FVIII secretion, with evidence of synthesis, cell surface expression, and coexpression with vWF. There is complex alternate transcription initiation from the FVIII gene. These findings provide a framework for future research on the regulation and perturbation of FVIII synthesis, and of potential relevance to haemophilia, thromboses, and pulmonary hypertensive states.
authors.affiliation: National Heart and Lung Institute Cardiovascular Sciences, Imperial College London, London, United Kingdom. c.shovlin@imperial.ac.uk
authors.forename: Claire L
authors.initials: CL
authors.lastname: Shovlin
authors.forename: Gillian
authors.initials: G
authors.lastname: Angus
authors.forename: Richard A
authors.initials: RA
authors.lastname: Manning
authors.forename: Grace N
authors.initials: GN
authors.lastname: Okoli
authors.forename: Fatima S
authors.initials: FS
authors.lastname: Govani
authors.forename: Kay
authors.initials: K
authors.lastname: Elderfield
authors.forename: Graeme M
authors.initials: GM
authors.lastname: Birdsey
authors.forename: Inês G
authors.initials: IG
authors.lastname: Mollet
authors.forename: Michael A
authors.initials: MA
authors.lastname: Laffan
authors.forename: Francesco A
authors.initials: FA
authors.lastname: Mauri
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D001777:Blood Coagulation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons / Q000235:genetics; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006976:Hypertension, Pulmonary / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008168:Lung / Q000098:blood supply / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000235:genetics*; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D014841:von Willebrand Factor; D005169:Factor VIII