Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, A alpha R16C and gamma G165R

Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrin ogen levels in functional assays were considerably lower than levels in imm unologic assays. Polymerization of purified fibrinogen was strongly impaire d in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A alpha R16 C and gamma G165R, As seen previously with other heterozygous Aa R16C varia nts, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Addi tionally, the release of fibrinopeptide B was delayed. Immunoblotting analy sis with antibodies to human serum albumin indicated that albumin is bound to A alpha 16 C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the pres ence of calcium or EDTA showed both normal and novel D1 and D3 fragments. F urther digestion of abnormal D3 fragments by chymotrypsin resulted in degra dation products' of the same size as the fragments derived;from normal fibr inogen, SDS-PAGE analysis under reducing conditions showed no difference be tween normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments, Circular dichroism analysis revealed a shift in the mean residua l ellipticity and a significant reduction of the or-helix content in the va riant D3 fragment. It is concluded that the A alpha -chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitut ion in the gamma -chain induced a conformational change in the D3 fragment. (C) 2001 by The American Society of Hematology.