Summary
Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways
and has impaired catalytic potential. These studies were initiated to obtain confirmation
that this molecule is multi-functionally defective due to the substitution of Ser
for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed
mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor
XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and
shown to have a molecular size identical to that of normal plasma factor X (pFX) by
gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages.
Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational
y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr
was over 85% and close to 100%, respectively, of expected levels. The specific activities
of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular
modeling suggested the involvement of a new H-bond between the side-chains of Ser-343
and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding
pocket of pFXFr. These results support the conclusion that the observed mutation in
pFXFr is responsible for its dysfunctional activation and catalytic potentials, and
that it accounts for the moderate bleeding tendency in the homozygous individuals
who possess this variant procoagulant.
