An inherited dysfunctional
fibrinogen variant, denoted as
fibrinogen Milano III, was found in a 13-year-old girl suffering from recurrent
venous thrombosis. Plasma of the patient exhibited prolonged thrombin time and Reptilase time. Polymerization of
fibrin monomers in the presence and absence of
calcium ions was strongly impaired. SDS-
polyacrylamide gel electrophoresis of reduced
fibrinogen showed normal B beta- and gamma-chains, whereas no normal A alpha-chain was detected in the proposita. Immunoblot analysis with the
monoclonal antibody Y18, detecting an
epitope within the stretch of
amino acids A alpha 1-51, revealed an A alpha-chain of about 50 kDa with an intact amino terminus. Immunoblotting with
antibodies directed against
serum albumin demonstrated the presence of
albumin covalently linked to
fibrinogen via a
disulfide bridge. The structural defect of
fibrinogen Milano III was determined by sequence analysis of a single-stranded fragment of genomic
DNA amplified by polymerase chain reaction. An insertion of a
thymine in the exon V of the A alpha-chain gene after the triplet ATT coding for IleA alpha 451 altered the reading frame and caused premature termination of the
protein synthesis (Trp452(TGG)-Ser453(TCC)-Stop454(TGA)). In both parents, normal and mutant alleles were established, leading to duplication of the sequence pattern after the
thymine insertion site, whereas the proposita is homozygous for the new mutation in the
fibrinogen A alpha-chain gene.