Abstract
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.
Highlights
A n inheriteddysfunctionalfibrinogenvariant,dethus uncovering of the amino-terminalpolymerization sites in noted as fibrinogen Milano[111], was found in a 13-year- the fibrinogen molecule by thrombin
The struc- properties have been described in which the structural defect
The follow-up was complicated by two additional episodes of severe hemolytic anemia necessitating treatment with azathioprine and prednisone, and by pulmonary embolism
Summary
Coagulation Studies-Activated partial thromboplastin time, prothrombin time, thrombin time, and Reptilase time were assayed by conventional methods. 0.15 M ammonium acetate buffer, pH 8.5, were incubated with thrombin (0.04NIH units/ml) at 37 "C for 16 h.Following incubation, the samples were boiled for 3 min to precipitate any residual fibrinogen. After SDS-PAGE (22) of reduced fibrinogen chains on 8% gels, proteins were either visualized by silver staining (25), or they were subjected to electroblotting for 2 hat 26 V. For asymmetric amplification the PCR was performed in thepresence of 0.1p~ sense and 0.5PM antisense primer, 200 p of each deoxynucleotide,[200] ng of the purified doublestranded PCR fragment and 4 uniotsf Tag DNApolymerase in thesame PCR buffer as described above, in a final volume of 100 pl. Radiolabeled bands were visualized by autoradiography using 3M Trimax medical imaging film
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