Isolation and characterization of the factor X Friuli variant
DS Fair, DJ Revak, JG Hubbard and A Girolami
Department of Immunology, Research Institute of Scripps Clinic, La Jolla,
CA.
Factor X Friuli was isolated from plasma by immunoaffinity and ion exchange
chromatography and compared with normal factor X purified by the same
method. Similar molecular weights were observed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the intact or
activated factor X molecules including their respective heavy and light
chains. These data indicated that there were no gross structural
differences between the normal and variant proteins. Immunochemical assays
employing either polyclonal or 46 monoclonal antibodies (MoAbs) did not
reveal any structural deviations. Two- dimensional peptide maps indicated
that while the light chains of normal and Friuli factor X were very
similar, the heavy chains of the native and activated molecules contained a
limited number of differences. These data suggested that the defect in
factor X Friuli may be a point mutation which lies within the activated
heavy chain defined by the 195-424 amino acid sequence. Activation of
factor X Friuli in purified systems showed that Russell's viper venom
cleaved the molecule at 70% of the normal rate, while the rate of
proteolysis of the variant protein was reduced 98% and 75% when incubated
with the extrinsic and intrinsic activation complexes, respectively. These
data support the clinical laboratory findings and the hypothesis that the
defect associated with the Friuli variant may reflect an abnormal
interaction between factor X Friuli and the nonproteolytic cofactors of the
extrinsic and intrinsic factor X activation complexes. Fluorescence
polarization studies suggested that a bound dansylated inhibitor of factor
Xa was not oriented to the same extent within the active site of the
variant enzyme relative to normal factor Xa until the addition of
phospholipid and factor Va. Activated factor X Friuli generated thrombin
from prothrombin in a purified system, but at one third the normal rate
that was attributed to the Kcat suggesting a secondary effect of this
defect.
Volume 73,
Issue 8,
pp. 2108-2116,
06/01/1989
Copyright © 1989 by The American Society of Hematology
