title: Homozygous F5 deep-intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet-rich plasma.
abstract: BACKGROUND
  Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet-rich plasma (PRP). Therefore, we hypothesized that FV-deficient patients with severe bleeding manifestations may lack platelet FV.
  
  
  OBJECTIVE
  To characterize a FV-deficient patient with a severe bleeding diathesis.
  
  
  METHODS
  We performed FV mutation screening and functional studies in a 31-year-old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage.
  
  
  RESULTS
  The proband was homozygous for a deep-intronic mutation (F5 IVS8 +268A→G) causing the inclusion of a pseudo-exon with an in-frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the proband's plasma or platelets with a prothrombinase-based assay. Moreover, no thrombin generation was observed in PRP triggered with 1-50 pm tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the proband's whole blood, as assessed by thromboelastometry, was markedly delayed but not abolished.
  
  
  CONCLUSIONS
  This is the first report of a pathogenic deep-intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands. e.castoldi@maastrichtuniversity.nl
authors.forename: E
authors.initials: E
authors.lastname: Castoldi
authors.forename: C
authors.initials: C
authors.lastname: Duckers
authors.forename: C
authors.initials: C
authors.lastname: Radu
authors.forename: L
authors.initials: L
authors.lastname: Spiezia
authors.forename: V
authors.initials: V
authors.lastname: Rossetto
authors.forename: G
authors.initials: G
authors.lastname: Tagariello
authors.forename: J
authors.initials: J
authors.lastname: Rosing
authors.forename: P
authors.initials: P
authors.lastname: Simioni
mesh_terms: D000328:Adult; D017398:Alternative Splicing; D001777:Blood Coagulation; D005165:Factor V / Q000235:genetics*; D005166:Factor V Deficiency / Q000235:genetics*; D006395:Hemarthrosis; D006406:Hematoma / Q000473:pathology; D006720:Homozygote; D006801:Humans; D047468:Immunoprecipitation; D007438:Introns; D008297:Male; D009154:Mutation; D053657:Platelet-Rich Plasma / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D005165:Factor V; D013925:Thromboplastin; D013917:Thrombin