title: Functional analysis of two haplotypes of the human endothelial protein C receptor gene.
abstract: OBJECTIVE
  To confirm the effect of the endothelial protein receptor gene (PROCR) haplotypes H1 and H3 on venous thromboembolism (VTE), to study their effect on endothelial protein C receptor (EPCR) expression in human umbilical vein endothelial cells, and to investigate the functionality of H1 tagging single-nucleotide polymorphisms in an in vitro model.
  
  
  RESULTS
  Protein C (PC), activated PC, and soluble EPCR (sEPCR) levels were measured in 702 patients with VTE and 518 healthy individuals. All subjects were genotyped for PROCR H1 and H3. Human umbilical vein endothelial cells isolated from 111 umbilical cords were used to study the relation between PROCR haplotypes, PROCR mRNA, cellular distribution of EPCR, and rate of PC activation. Finally, the functionality of the intragenic PROCR H1 single-nucleotide polymorphisms was analyzed using a luciferase-based method. We confirmed that individuals carrying H1 have reduced VTE risk, increased plasma activated PC levels, and reduced plasma sEPCR levels and that individuals with the H3H3 genotype have an increased VTE risk and increased plasma sEPCR levels. In cultured human umbilical vein endothelial cells, H1 is associated with increased membrane-bound EPCR, increased rate of PC activation, and reduced sEPCR in conditioned medium, but does not significantly influence PROCR mRNA levels. In contrast, H3 is associated with reduced membrane-bound EPCR and increased sEPCR in human umbilical vein endothelial cell-conditioned medium, higher levels of a truncated mRNA isoform, and a lower rate of PC activation. Finally, we identified the g.2132T>C single-nucleotide polymorphism in intron 1 as an intragenic H1-specific functional single-nucleotide polymorphism.
  
  
  CONCLUSIONS
  These results support a protective role of PROCR H1 against VTE and an increased risk of VTE associated with the H3 haplotype.
authors.affiliation: From the Grupo de Investigación en Hemostasia, Trombosis, Arteriosclerosis y Biología Vascular, Centro de Investigación (P.M., S.N., E.B., L.M., A.E., F.E.) and Servicio de Análisis Clínicos (E.B.), Hospital Universitario y Politécnico La Fe, Valencia, Spain; and Department of Thrombosis and Haemostasis, Einthoven Laboratory, Leiden University Medical Centre, Leiden, The Netherlands (R.M.B., H.L.V.).
authors.forename: P
authors.initials: P
authors.lastname: Medina
authors.forename: S
authors.initials: S
authors.lastname: Navarro
authors.forename: E
authors.initials: E
authors.lastname: Bonet
authors.forename: L
authors.initials: L
authors.lastname: Martos
authors.forename: A
authors.initials: A
authors.lastname: Estellés
authors.forename: R M
authors.initials: RM
authors.lastname: Bertina
authors.forename: H L
authors.initials: HL
authors.lastname: Vos
authors.forename: F
authors.initials: F
authors.lastname: España
mesh_terms: D020016:Activated Protein C Resistance / Q000235:genetics; D000328:Adult; D015703:Antigens, CD / Q000235:genetics / Q000502:physiology*; D017077:Culture Media, Conditioned / Q000737:chemistry; D000074283:Endothelial Protein C Receptor; D004789:Enzyme Activation; D005165:Factor V / Q000235:genetics; D005260:Female; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006239:Haplotypes; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D011486:Protein C / Q000032:analysis; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D011516:Prothrombin / Q000235:genetics; D011655:Pulmonary Embolism / Q000453:epidemiology / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D012306:Risk; D013030:Spain / Q000453:epidemiology; D019851:Thrombophilia / Q000453:epidemiology / Q000235:genetics*; D054556:Venous Thromboembolism / Q000453:epidemiology / Q000235:genetics*
chemical_list: D015703:Antigens, CD; D017077:Culture Media, Conditioned; D000074283:Endothelial Protein C Receptor; D008565:Membrane Proteins; C478370:PROCR protein, human; D011486:Protein C; D020033:Protein Isoforms; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin
keywords: gene expression; human umbilical vein endothelial cells; receptors, cell surface; venous thromboembolism