(Received for publication, October 6, 1994; and in revised form, January 18, 1995)
From the
Factor IX and factor X have sialic acid in O-linked and N-linked oligosaccharides on their activation peptides, and a terminal sialic acid is found on a recently described O-linked tetrasaccharide at Ser-61 in the light chain of human factor IXa. In studies presented here, the potential role of sialic acid residues in mediating activity of human coagulation factors IX and X was tested after enzymatic removal of sialic acid residues. In contrast to previous reports, treatment of factor IX or factor IXa with recombinant sialidase did not decrease the rate of factor IX activation or proteolytic properties of human factor IXa. The activation rates of factor IX and desialated factor IX were indistinguishable when treated with factor XIa, with factor VIIa/tissue factor complex, and with the factor X activating enzyme from Russell's viper venom. Desialated human factor IXa showed full activity in the non-activated partial thromboplastin time assay and retained full ``tenase'' activity in a coupled amidolytic assay. Similar experiments with human factor X showed no detectable loss of clotting activity in the prothrombin time assay after desialation. Additionally, desialated human factor X was cleaved by the factor X activating enzyme from Russell's viper venom and intrinsic tenase at the same rate as untreated factor X when analyzed by SDS-polyacrylamide gel electrophoresis. These studies have shown that factor IX and factor X clotting activity are not dependent on sialic acid content. Further studies are needed to determine whether desialated factor IX binds to endothelial cells, and whether factors IX and X are more rapidly cleared from circulation or have altered susceptibility to proteolysis after enzymatic removal of sialic acid.
Human coagulation factor IX is a vitamin K-dependent coagulation
factor which is essential for normal hemostasis(1) . This
glycoprotein undergoes several post-translational modifications
including -carboxylation of 12 N-terminal glutamic acid residues,
-hydroxylation of aspartic acid at residue 64, and incorporation
of both N-linked and O-linked oligosaccharides. Four
sites for attachment of O-linked oligosaccharides are present
in factor IX, including Ser-53 and Ser-61 within the first epidermal
growth factor (EGF) (
)domain, which are uniformly
glycosylated(2) , and Thr-159 and Thr-169 in the activation
peptide (residues 145-180), which are partially
glycosylated(3) . Threonine residues at positions 159 and 169
in the activation peptide of factor IX (residues 145-180) are
partially modified with O-linked oligosaccharides(3) .
There are also two sites for attachment of N-linked
oligosaccharides in the activation peptide of factor IX at asparagine
residues 157 and 167(4) . The trisaccharide linked to Ser-53
does not contain sialic acid, while the O-linked
oligosaccharide at the other three sites could contain as many as 3
sialic acids. The two N-linked oligosaccharides on the
activation peptide region of the molecule probably have the remainder
of the 8-10 sialic acid residues found on factor IX(5) .
Although
-carboxylation is critical for function of vitamin
K-dependent coagulation factors, there are little definitive data on
the importance of glycosylation and other post-translational
modifications in factor IX.
The structure of the fucose-linked
tetrasaccharide with a terminal sialic acid at Ser-61 in the first EGF
domain of human factor IX has been recently
described(2, 6) . The proposed consensus sequence
(Cys-X-X-Gly-Gly-Thr/Ser-Cys) for incorporation of
fucosyl modifications in EGF domains is found in human factor IX, t-PA,
urokinase, factor VII, and factor XII but not in factor X or bovine
factor IX(7) . Enzymatic removal of sialic acid has been
reported to result in the loss of factor IX clotting activity without
affecting the rate at which factor IX is cleaved by the factor X
activating enzyme from Russell's viper venom (RVV-X)(8) .
Loss of factor IXa activity (a partially activated factor IX
species with Arg-180/Val-181 cleavage only) following sialidase
treatment suggests that sialic acids in either the EGF or activation
peptide regions of factor IX are important for enzymatic activity. In
contrast, factor X, which lacks the consensus site for a fucose-linked
oligosaccharide in its light chain, did not lose clotting activity
following enzymatic removal of sialic acid(8) . Others,
however, have shown that sialidase treatment of human or bovine factor
X (9) and combined sialidase and O-glycanase treatment
of bovine factor X reduces the K
/K
for RVV-X or
intrinsic ``tenase'' (10) without any apparent effect
on factor Xa clotting activity.
Since there are conflicting reports on the effects of enzymatic removal of sialic acid from factor X and no experiments with factor IXa, purified preparations of human factor IX, factor IXa, and factor X were each treated with either recombinant sialidase expressed in Escherichia coli or sialidase from Clostridium perfringens in the present study. Desialated factor IX, IXa, and X were found to retain full enzymatic activity in plasma and purified systems.
Figure 1: SDS-PAGE of factor IX and X (control and desialated). Each lane contains 8 µg of protein. Samples were not reduced. Lane 1 contains molecular weight markers. Lane2 is untreated factor IX. Lane3 is factor IX after sialidase treatment. Lane4 is untreated factor X. Lane5 is desialated factor X.
Figure 2: Activation of factor IX and desialated factor IX by factor XIa. Each lane contains 8 µg of protein. Samples are not reduced. Lanes 1-5 contain control factor IX, and lanes6-10 contain desialated factor IX. Incubation times are as follows; lanes5 and 6 are at 0 h, lanes1 and 10 are at 0.5 h, lanes2 and 9 are at 1 h of incubation, lanes 3 and 8 are at 2 h, and lanes4 and 7 are at 3 h of incubation.
Figure 3: Activation of factor IX and desialated factor IX by RVV-X. Each lane contains 7-8 µg of protein. Samples are reduced. Lanes 2-5 contain desialated factor IX, and lanes 6-9 contain control factor IX. Incubation times are as follows: lanes5 and 6 are at 0 h, lanes4 and 7 are at 0.5 h, lanes3 and 8 are at 1 h, lanes2 and 9 are at 2 h, lanes1 and 10 are molecular weight markers.
Figure 4:
Activation of factor IX and desialated
factor IX by factor VIIa/tissue factor gel. Inset, each lane
contains 7-8 µg of protein. Samples are not reduced. Lanes 1-5 contain control factor IX, and lanes
6-10 contain desialated factor IX. Incubation times of
factor IX samples are as follows; lanes 5 and 6 are
at 0 min, lanes4 and 7 are at 0.5 min, lanes3 and 8 are at 1 min; lanes2 and 9 are at 5 min; lanes1 and 10 are at 30 min. The factor IX cleavage site that
gives a band at the apparent kDa of 55 is not known. Graph shows factor
IX clotting activity; the + symbol is control factor IX activity
in the NAPTT assay, and the symbol is desialated factor IX
activity in the same assay. Samples were diluted 1:10,000 prior to
assay after activation by tissue factor and factor
VIIa.
Figure 5:
Factor
IXa activity and factor IXa sialic acid content. A, factor IXa
(both control and desialated) clotting activity and sialic acid
content. B, factor IXa (both control and desialated) tenase
activity in a coupled amidolytic assay and sialic acid content. Symbols
used are as follows; is control factor IXa activity,
is
desialated factor IXa activity, and
is factor IXa sialic acid
content.
Figure 6: Binding of antithrombin III with factor IXa and desialated factor IXa. Each lane contains 7-8 µg of protein. Samples were not reduced. Lanes1 and 7 are molecular weight markers; lane2 is AT-III; lane3 is control factor IXa-AT-III complex; lane4 is control factor IXa; lane5 is desialated factor IXa; lane6 is AT-III-desialated factor IXa complex.
Figure 7: Activation of factor X and desialated factor X by RVV-X. Factor X samples were not reduced. Samples were activated with soluble RVV-X. All lanes contain 5 µg of protein. Lanes 1-5 contain control factor X, and lanes 6-10 contain desialated factor X. Incubation times of factor X samples are as follows; lanes1 and 10 are at 30 min, lanes2 and 9 are at 15 min, lanes3 and 8 are at 5 min, lanes4 and 7 are at 1 min, lanes5 and 6 are at 0 min.
Figure 8:
Activation of factor X and desialated
factor X by factor IXa/factor VIIIa/phospholipid
vesicles/CaCl. Each lane contains 7-8 µg of
protein. Samples were not reduced. Lanes1-5 contain desialated factor X, and lanes6-10 contain control factor X. Incubation time of factor X samples are
as follows; lanes1 and 10 are at 30 min, lanes2 and 9 are at 15 min, lanes3 and 8 are at 5 min, lanes4 and 7 are at 1 min, and lanes5 and 6 are at 0 min.
These experiments have shown that enzymatic removal of sialic
acid from human factor IX did not affect its activation or its
proteolytic activity against factor X. Additionally, treatment of
factor X with recombinant C. perfringens sialidase did not
affect activation of factor X or its enzymatic activity. The specific
activity of desialated factor IX was preserved in the PTT assay, which
depends on activation of the proenzyme and its ability to activate
factor X in the presence of cofactors. High values of specific activity
were probably due to minor activation of the factor IX samples after
incubation with sialidase. By SDS-PAGE analysis, factor XIa, the
complex of factor VIIa and tissue factor, and RVV-X cleaved desialated
factor IX and native factor IX at the same rate. Desialated factor IXa
retained full activity on the NAPTT assay and tenase activity measured
in a coupled amidolytic assay. Experiments with human factor X showed
no detectable loss of clotting activity of desialated factor X in the
PT assay, reflecting both activation and enzymatic activity. Factor X
and desialated factor X were cleaved by RVV-X, and by the complex of
factor IXa, factor VIIIa, phospholipid vesicles, and CaCl at the same rate when analyzed by SDS-PAGE. Additionally,
desialated factor Xa hydrolyzed a small peptide substrate (S-2765) at
the same rate as control factor Xa.
Others have reported decreased
enzymatic activity of partially activated human factor IX and either no
effect on human factor X activity or slowed activation of the proenzyme
after treatment with commercial
sialidase(8, 9, 10) . Chavin and Weidner (8) found that activation of factor IX by RVV-X was not
affected after removal of sialic acid but the partially activated
factor IXa (cleaved at Arg-180/Val-181) had reduced activity in
the PTT assay. In experiments with human factor X, Chavin and Weidner
demonstrated that even 6 h of treatment of human factor X with
sialidase did not change PTT activity despite removal of 54% of its
sialic acid(8) . Two reports, however, have demonstrated slow
activation of factor X after removal of sialic
acid(9, 10) . Sinha and Wolf (9) have reported
that both bovine and human factor X were slowly activated by either
intrinsic tenase or by the tissue factor-VIIa complex after short
treatment (3-4 h) with Vibrio cholerae sialidase. The
clotting activity of the desialated factor X was 2-12% of the
untreated factor X in PT or PTT assays. Similar results were obtained
using a chromogenic substrate to measure factor Xa generation.
Activation of both human and bovine factor X was less affected after N-glycosidase treatment but residual sialic acid was not
measured after treatment with either sialidase or N-glycosidase. Inoue and Morita (10) also showed that
the rate of activation of bovine factor X was decreased after treatment
with Arthrobacter ureafaciens sialidase and O-glycanase from Streptococcus pneumoniae, but not
after removal of N-linked carbohydrate. Sialidase alone was
not used in these experiments, and residual sialic acid was not
quantitated in this report. The activation peptide regions of human and
bovine factor X contain different numbers of sites for N- and O-linked oligosaccharides, so that results with bovine factor
X may not predict results with human factor X.
Preservation of clotting activity after sialidase treatment may be explained by differences in experimental approach. Use of recombinant sialidase instead of sialidase from bacterial lysates may have minimized proteolytic degradation of clotting factor preparations in the current report. In most experiments, sialidase-treated and control factor IX and X were reisolated using monoclonal antibody immunoaffinity chromatography prior to studies of enzymatic activity, further minimizing chances for protein degradation by trace amounts of proteolytic enzymes in the sialidase preparations.
Although sialic acid residues are concentrated in the activation peptide regions, they do not appear to be critical for activation of factors IX and X and their function remains speculative. The one sialic acid residue on the light chain of factor IXa is found in factor IX's EGF1 domain on an O-linked tetrasaccharide at Ser-61. This tetrasaccharide is not found on bovine factor IX or human factor X. Desialated factor IXa has full clotting activity in agreement with other observations, suggesting that the sialic acid on the EGF1 domain of factor IX is not essential for factor IXa interaction with its cofactor. Hybrid factor IX containing factor X EGF1 in place of the factor IX EGF1 domain lacks the fucosyl modification consensus sequence (hence it must lack the Ser-61 tetrasaccharide), yet retains full clotting activity(18) . Since no hemophilia B mutations have been found at Ser-61 or another site for glycosylation, Ser-53, the codons for these residues may either be relatively stable to mutations, or perhaps missense mutations affecting sites for O-linked oligosaccharide attachment within EGF1 do not affect clotting activity and are not detected(19) .
The O-linked
oligosaccharides within the EGF1 domain may have a role in
receptor-ligand interaction(20) . O-Linked
oligosaccharides are important for C type lectin interaction in the
binding of t-PA to HepG2 cells since this calcium-dependent reaction is
not seen with -fucosidase treated t-PA(21) . O-Linked oligosaccharides are also important in other
receptor-ligand interactions such as binding of transferrin and its
receptor(22) . Although sialic acids are not required for
coagulant activity of factor IX and X in vitro, they may be
important for survival of factor IX and X in circulation or for binding
to cellular surfaces. Clearance of ceruloplasmin has been shown to be
accelerated after treatment with sialidase for example(23) .
Studies are in progress to see if sialic acids are involved in factor
IX binding to endothelial cells.