Characterization of fibrinogen Milano I: Amino acid exchange ?330Asp ? Val impairs fibrin polymerization

Abstract

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called 'fibrinogen Milano I'. The time course of fibrinopeptide A and B release from fibrinogen Milano I was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective ?-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the ?-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano I, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region ?303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano I. This fragment spans from position ?311 to ?336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp ? Val in the ?-chain at position 330.