title: Biochemical characterization of a recombinant von Willebrand factor (VWF) with combined type 2B and type 1 defects in the VWF gene in two patients with a type 2A phenotype of von Willebrand disease.
abstract: BACKGROUND
  In a patient previously diagnosed with type 2A von Willebrand disease (VWD) [absence of high and intermediate molecular weight von Willebrand factor (VWF) multimers and markedly reduced ristocetin-induced platelet aggregation (RIPA)], an infusion test of desmopressin was followed by mild thrombocytopenia. This led to further laboratory investigations of his affected brother and of family members, who showed different phenotypic patterns compatible with type 1, 2A, 2B and an uncertain classification of VWD. The two brothers were compound heterozygotes (C275R/P1337L), whereas the others members of the family were heterozygous for C275R (a novel mutation in the D1 domain) or P1337L (a type 2B mutation in the A1 domain).
  
  
  OBJECTIVE
  To evaluate the role of the combined effect of the two mutations in the two brothers, C275R and P1337L recombinant (r) VWFs were transiently expressed in COS-7 cells.
  
  
  RESULTS
  Recombinant VWF levels secreted in cell media were similar for wild-type (WT), P1337L and hybrid P1337L/WT rVWFs, reduced for hybrids C275R/P1337L and C275R/WT rVWFs, and strongly reduced for C275R rVWF. All rVWFs had a full set of multimers except C275R rVWF, which had only dimers. P1337L rVWF and C275R/P1337L rVWF showed the highest degree of binding to glycoprotein (GP) Ibalpha and the lowest to collagen, followed by P1337L/WT rVWF (with an intermediate level of binding to both ligands), and by WT rVWF with the lowest level of binding to GPIbalpha and the highest to collagen.
  
  
  CONCLUSIONS
  These results suggest that the two compound heterozygous patients have a circulating VWF mainly mutated in the A1 domain (P1337L). This peculiar type 2B VWF variant showed a remarkably high affinity for the GPIbalpha platelet receptor, leading to the loss of high and intermediate molecular weight multimers and hence to decreased RIPA, as in type 2A VWD.
authors.affiliation: Department of Medicine and Medical Specialties, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation and University of Milan, Milan, Italy. luciano.baronciani@unimi.it
authors.forename: L
authors.initials: L
authors.lastname: Baronciani
authors.forename: A B
authors.initials: AB
authors.lastname: Federici
authors.forename: G
authors.initials: G
authors.lastname: Cozzi
authors.forename: M T
authors.initials: MT
authors.lastname: Canciani
authors.forename: P M
authors.initials: PM
authors.lastname: Mannucci
mesh_terms: D003094:Collagen / Q000378:metabolism; D019281:Dimerization; D005192:Family Health; D006801:Humans; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D010375:Pedigree; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics; D035781:Siblings; D014842:von Willebrand Diseases / Q000145:classification / Q000175:diagnosis / Q000235:genetics*; D014841:von Willebrand Factor / Q000235:genetics*
chemical_list: D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D003094:Collagen