Blood, 15 July 2001, Vol. 98, No. 2, pp. 351-357
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Fibrinogen Milano XII: a dysfunctional variant containing 2 amino
acid substitutions, A
R16C and 
G165R
Bettina Bolliger-Stucki,
Susan T. Lord, and
Miha Furlan
From the Central Hematology Laboratory, Inselspital,
University Hospital, Bern, Switzerland; and the Department of Pathology
and Laboratory Medicine, University of North Carolina at Chapel Hill.
Fibrinogen Milano XII was detected in an asymptomatic Italian
woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous
structural defects were detected by DNA analysis: A
R16C and
G165R. As seen previously with other heterozygous A R16C
variants, thrombin-catalyzed release of fibrinopeptide A was 50% of
normal. Additionally, the release of fibrinopeptide B was delayed.
Immunoblotting analysis with antibodies to human serum albumin
indicated that albumin is bound to A 16 C. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of
plasmin digests of fibrinogen Milano XII in the presence of calcium or
EDTA showed both normal and novel D1 and D3 fragments. Further
digestion of abnormal D3 fragments by chymotrypsin resulted in
degradation products of the same size as the fragments derived from
normal fibrinogen. SDS-PAGE analysis under reducing conditions showed
no difference between normal fibrinogen and fibrinogen Milano XII or
between their plasmic fragments. Circular dichroism analysis revealed a
shift in the mean residual ellipticity and a significant reduction of
the -helix content in the variant D3 fragment. It is concluded that
the A-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the -chain induced a
conformational change in the D3 fragment.
