(Received for publication, May 24, 1995)
From the
Hypercoagulability of blood, monocytic infiltration, and changes
in pericellular and extracellular matrix glycosaminoglycans
(GAGs) are observed in atherosclerosis, inflammation, and
neoplasia. In the present studies, monocyte procoagulants and different
GAGs including chondroitin sulfate (CS) A, CSB, CSC, CSD, CSE, and
heparan sulfate, were tested either in clotting assays with whole
plasma or in chromogenic assays with purified coagulation proteases.
Procoagulant activity in plasma was inhibited by three of the seven
GAGs, including heparan sulfate, CSE, and CSB. In contrast, activity of
purified coagulation protease was inhibited only by CSE, and the
inhibition was observed with intrinsic (factor VIIIa/IXa) but not
extrinsic (tissue factor/factor VII) components. Reciprocal titration
experiments with enzyme and substrate and Scatchard type analyses were
consistent with concentration-dependent inhibitory interactions between
CSE and sites on both factor VIIIa and IXa. On purified phospholipids,
CSE concentration resulting in half-maximal inhibition (K
) was 5 ng/ml for interaction with
factor IXa and >500 ng/ml for interaction with factor VIIIa. The K
values were lower for reactions on
purified lipid than for reactions on monocyte surfaces and for
reactions on resting than on endotoxin-stimulated monocytes.
Experiments with CSE oligosaccharides of defined size indicated that
the smallest CSE fragment capable of inhibitory activity was composed
of 12-18 monosaccharide units. Collectively, these results
indicate that factor X-activating reactions are inhibited by GAGs
expressed on monocyte membranes. Inhibition is specific with respect to
the structure of both the GAG and the activating protease. Lack of
inhibition by added CSA, CSB, and CSC in contrast to CSE strongly
suggests a direct role of 4,6-di-O-sulfated N-acetylgalactosamine GAG structures in the inhibition of
intrinsic pathway protease. These findings also suggest potential
pharmacologic use of CSE as specific anticoagulant in the management of
prothrombotic states mediated by intrinsic pathway coagulation
reactions.
Mononuclear phagocytes constitute one of the major components in
the cellular infiltrates that characterize atherosclerotic, neoplastic,
and chronic inflammatory lesions (Ross, 1993; McGee et al.,
1978; Rickles et al., 1988). These lesions are also associated
with localized blood coagulation reactions and both qualitative and
quantitative changes in GAGs ()content (Wagner and
Salisbury, 1989; McGee et al., 1990; Wilcox et al.,
1989; Dietrich et al., 1993).
Monocytes and macrophages can
accelerate membrane-dependent coagulation reactions via expression of
TF (tissue factor) and membrane assembly sites for coagulation protease
complexes. Membrane TF interacts with plasma factor VII with high
affinity (K, approximately
10
M), forming functional protease
complexes that cleave plasma coagulation factors IX and X and generate
the serine esterases, factors IXa and Xa (Bach et al., 1986).
Factor IXa in turn assembles on acidic phospholipids with coagulation
factor VIIIa (K
, approximately
10
) to form the ``intrinsic pathway''
protease that also generates factor Xa (Ahmad et al., 1989).
Thus, this protease complex constitutes a seemingly redundant
amplifying loop for activation of factor X to Xa. However, both the
extrinsic and intrinsic coagulation proteases are essential for normal
hemostasis, as evidenced by the coagulation deficiencies exhibited by
patients with single genetic defects in either plasma factor VII, VIII,
or IX. (Brigg and Nossel, 1961; Malar et al., 1982).
Chondroitin sulfates are the predominant GAGs comprising the proteoglycans produced by monocyte/macrophages (Owens and Wagner, 1992; Kolset et al., 1983; McQuillan et al., 1989; Uhlin-Hansen et al., 1989; Edwards et al. 1995). Transition from monocyte to macrophage upon exposure to differentiating stimuli is often accompanied by the appearance on the membrane and pericellular spaces of an oversulfated chondroitin sulfate containing 4,6-di-O-sulfated N-acetylgalactosamine residues (i.e. CSE). Whereas previous studies address the importance of CSE during cell differentiation, no information is available on the effects of this unique GAG on procoagulant activity. In the present study we demonstrate that CSE specifically modulates the rate of factor X-activating reaction on monocyte/macrophage membranes.
The various GAGs were also incorporated in reaction mixtures with purified coagulation factors. Extrinsic pathway protease was measured in mixtures containing either cells (monocytes or THP-1) or human brain homogenates as a source of tissue factor. Intrinsic pathway protease was measured in mixtures with either cells or rabbit brain lipid extracts as a source of lipid co-factor for intrinsic complex assembly. Of the seven structurally different types of GAG tested, only CSE induced large changes in factor X activation rates as compared with reaction mixtures without GAGs (Fig. 1A).
Figure 1:
Differential modulation of intrinsic
and extrinsic protease by chondroitin 4,6-disulfate. Initial rates of
factor X activation were measured in reaction mixtures with cells
(0.8-1 10
/ml), factor IXa (1 nM),
factor VIIIa (1 nM), or factor VIIa, (3 nM),
CaCl
in TRIS buffer, pH 7.4, 0.15 N NaCl, and 0.5
mg/ml bovine serum albumin. Glycosaminoglycans, CSA, B, C, D, and E,
and HS were added at concentrations indicated on the abscissa.
Rates with all GAGs except CSE were within 10% of values measured in
the absence of GAGs. Only rates with CSE are shown in the figure for
clarity. Cells are THP-1 cells cultured for 48 h in the presence of
endotoxin. Similar results were obtained using human brain homogenates
as a source of procoagulant membranes.
Chondroitin sulfate E induced a modest but significant increase in the rate of factor X activation by extrinsic pathway protease. In contrast, the rate of factor X activation by the intrinsic pathway protease was markedly reduced by this GAG (Fig. 1B).
Figure 2:
Inhibition of intrinsic pathway protease
on cells and purified lipid surfaces. Initial rates of factor X
activation were measured in reaction mixtures without GAGs and mixtures
with 6 µg/ml CSE. Concentration phospholipid membranes supplied
either as rabbit brain lipid extract or as intact THP-1 cells was as
indicated on the abscissa. Intrinsic pathway components and
other reagents were as indicated in the legend to Fig. 1.
Inhibition, (I) was calculated as 1 - R/R, where R
and R are the initial rates measured in mixtures
with and without GAG, respectively. Apparent inhibition decreased with
cell membrane concentration (
) but not with the sugar-free,
protein-free lipid extract concentration
(
).
Further evidence for inhibitory GAGs expressed on the monocyte membrane was obtained by removing surface proteoglycans using mild trypsinization (0.05% Trypsin and 0.53 mM EDTA solution, Life Technologies, Inc.). Intrinsic pathway protease complexes were assembled on cells, either after a limited, 0.5-min trypsin treatment or after a 10-min trypsin treatment. Cells in both preparations were subjected to the same extent of physical manipulation except for the length of incubation in the presence of trypsin. As previously reported, trypsinization effectively removes CSE- and HS-containing proteoglycans from the monocyte surface. (Edwards et al., 1995). Rates of factor X activation on cell trypsinized for 10 min were 9.8 ± 0.8 nM factor Xa/min and significantly faster than rates on cells subjected to limited trypsinization (4.0 ± 0.2 nM factor Xa/min). This result is also consistent with membrane expression of inhibitory GAGs by the monocyte.
Monocytes and monocytoid cell lines are known to increase synthesis and expression of CSE upon incubation in vitro in the presence of inflammatory stimuli. (Kolset et al., 1983; Uhlin-Hansen et al., 1989; Uhlin-Hansen et al., 1993; Edwards et al., 1995). To determine if this incubation-dependent increase in CSE levels is reflected on intrinsic pathway protease activity, inhibition of factor X activation with CSE was measured on either human monocytes or THP-1 incubated with endotoxin for 48 h at either 37 or 4-8 °C. The concentrations of CSE required to achieve half-maximal inhibition were significantly (p < 0.05) increased (2.7-fold for three independent experiments) on cells incubated at 37 °C as compared with cells incubated at 4-8 °C. Again, results are consistent with baseline regulation of intrinsic protease by membrane GAGs on macrophages.
Figure 3:
Chondroitin sulfate E was fractionated in
Sepharose 6B. Fractions were analyzed with respect to uronic acid
content () and inhibitory activity (
). The inhibition
levels of factor X activation reactions with CSE were calculated as
indicated in the legend to Fig. 2for unfractionated CSE.
Elution of markers for void volume (Vo) total included volume (Vt) and peak elution of fraction with 12-18
monosaccharide units (OS) are
indicated.
Figure 4:
Parameters of intrinsic protease
inhibition on purified lipid extracts; Scatchard analyses. Rates of
factor X activation were measured in mixtures with and without CSE at
concentrations ranging from 8 to 1.6 10
ng/ml.
Other reagents and determination of inhibition were as indicated in the
legend to Fig. 2. The ratios between inhibition (I) and
CSE concentration ([CSE]) are plotted versus
I. The slopes of the two extreme linear segments calculated by
linear regression correspond to K
= 5 ng/ml and K
= 1000 ng/ml. The results are consistent with two
independent sites n
and n
resulting in 0.57 and
0.43 fractional inhibition, respectively.
Based on these results,
further investigations of GAG-protein interactions were approached
using multiple titration experiments with reaction components. For
these experiments, each one of the reactants was alternatively used as
a titrating component, whereas the inhibitor and the rest of the
reactants were maintained at constant concentrations. The rationale of
this approach is that the component containing binding sites for CSE
should be displaced by the inhibitor from the functional protease
complex and that the empirical value of the kinetic parameter (K), that is, concentration of component
resulting in half-maximal rates, will increase in reactions with CSE as
compared with reactions without the inhibitor. Similarly, the value of V
, that is, the rate at saturating concentration
of competing component, should be the same in reactions with and
without CSE. To avoid possible interference from cell-derived GAGs,
kinetic parameters were determined using lipid extracts from rabbit
brain as a source of protein-free and sugar-free lipid surface.
The
concentration ranges for the titrating component were: factor X,
0.1-300 nM; factor IXa, 0.05-8 nM; and
factor VIII 0.05-6 nM. The results (see Table 4)
from titrations with substrate are compatible with a noncompetitive
type inhibition. The observed decrease in K for
factor X with CSE is as expected for a diffusion-limited reaction
measured at two levels of enzyme. For diffusion-limited reactions, a
decrease in enzyme density decreases the extent of diffusion control,
and the value of K
tends to approach that of the
intrinsic K
(McGee et al., 1992).
Therefore, this result indicates that inhibition observed with CSE is
mediated by a decrease in the effective density of the enzyme without
direct interference with substrate binding and suggests that CSE binds
to epitopes on the protease components differently from substrate
binding sites. However, neither one of the two intrinsic protease
complex component(s) added in excess to the other were able to overcome
the inhibition. Because increasing either protease component increases
the concentration of functional VIIIa-IXa complex, these results
suggest that inhibition by CSE can be mediated by interaction with
sites on factor VIIIa and IXa independently of VIIIa/IXa complex
formation. When concentrations of VIIIa and IXa were increased
simultaneously maintaining equimolar concentrations of each, the
inhibitory effect of CSE was abolished and maximal rates were not
significantly different in mixtures with CSE as compared with mixtures
without CSE. However, IXa and VIIIa concentrations resulting in
half-maximal rates increased 5-fold (Table 3), indicating that
the diffusional limit was achieved at lower IXa and VIIIa concentration
in reaction mixtures without CSE.
The difference in results obtained
with equimolar increments of VIIIa and IXa as compared with results
obtained with one component added in excess to the other are consistent
with predictions based on simple probability considerations. Assuming
that both factor IXa and VIIIa bind CSE and that the IXa-CSE and
VIIIa-CSE species bind to free and bound VIIIa and IXa, respectively,
then when either protease component is in molar excess, the probability
of nonfunctional complexes is expected to increase with the
concentration of the component that is in excess. This is seen more
clearly when considering the inhibition as a function of the varied
component concentration. The inhibition increased only when either
factor IXa or factor VIII were increased but not when both were
increased. (Fig. 5, A, B, and C). To
further test the hypothesis and to determine the component with the
high affinity site for CSE, titrations were repeated at CSE
concentrations below the K estimated for the low
affinity site. In these experiments, increasing the concentration of
VIIIa while maintaining IXa constant had little effect on the
inhibition level, particularly at low concentrations of VIIIa. In
contrast, increasing factor IXa while maintaining factor VIIIa
concentration fixed resulted in a marked and progressive increase in
the inhibition. Therefore, the results exclude the possibility that CSE
inhibits the factor X-activating reaction via interaction with sites
expressed upon IXa-VIIIa complex formation and in their simplest
interpretation are consistent with at least two independent CSE binding
sites. One of the sites interact with high affinity and is located on
factor IXa; the other site(s) interacts with low affinity and is
located on factor VIII. Alternatively, the interaction with factor VIII
may also be high affinity but involves a polysaccharide size(s) present
at relatively low concentration in the CSE preparation. The results are
summarized in Table 4and Fig. 4, Fig. 5, and Fig. 6.
Figure 5: Inhibition as a function of protease component concentration; high inhibitor concentration regime. Rates of factor X activation on lipid membranes were measured in mixtures with and without CSE at 50 µg/ml. Concentration of IXa and VIIIa were increased as indicated on the abscissa either independently (A and B, respectively) or simultaneously at equimolar concentrations (C). Other reagents and determination of inhibition were as indicated in the legend to Fig. 2.
Figure 6: Inhibition as a function of protease component concentration; low inhibitor concentration regime. Rates of factor X activation of lipid membranes were measured in mixtures with and without CSE at 50 ng/ml. Concentrations of either VIIIa or IXa were increased as indicated on the abscissa of A and B, respectively.
Glycosaminoglycans are known to interact with and
modulate the activity of many biologically important proteins,
including coagulation inhibitors such as AT III (Marcum et
al., 1966) and heparin cofactor II (Pratt et al., 1989),
and growth factors such as fibroblast growth factor and hepatocyte
growth factor (Lyon and Gallagher, 1994). Available evidence indicates
that the interaction of GAGs with these proteins depends on the
sequence and size of their oligosaccharide units. For example,
chondroitin sulfates A, B, and C have common disaccharide units of
hexuronic acid (glucuronic acid in CSA and CSE; iduronic acid in CSB)
and N-acetylgalactosamine-O-sulfate. However, only
CSB is known to bind to AT III and heparin cofactor II (Maimone and
Tollefsen, 1990). Similarly, commercial heparin and heparan sulfates
have common disaccharide units of hexuronic acid (glucuronic
acid/iduronic acid) and N-acetyl (or N-sulfated)
glucosamine O-sulfates, but their protein-binding
characteristics are different. In this regard, the binding sites for
proteins have been mapped to specific regions comprising a few
oligosaccharide units (Gallager et al., 1992). The consensus
from these studies is that the minimum oligosaccharide length required
for binding is 5-7 disaccharide units but that the size required
to elicit biological activity after binding is larger. Therefore, it is
not surprising that in the present functional studies oligosaccharides
longer than dodecasaccharides were required for activity. Heparin has
been reported to inhibit intrinsic but not extrinsic or common
proteases (Barrow et al., 1994). The saccharide sequence(s)
required to mediate this inhibition was/were not determined. Because
heparin sequences are heterogeneous with respect to sugar composition
and sulfate substitutions, it is possible that the inhibition observed
was mediated by O-sulfate groups as shown for CSE in the
present studies. Alternatively, intrinsic protease components may have
sites for interaction both with CSE and with N-sulfated
sequences in commercial heparin. Whereas heparan sulfate GAGs are
present in most tissues, heparin sequences such as those in commercial
anticoagulant preparations are almost exclusively found in mast cell
granules. Anticoagulant effects of CSE mediated primarily by
acceleration of heparin cofactor II interaction with thrombin have been
reported (Scully et al., 1986). The apparent K for the interaction was in the µg/ml range, approximately 2
orders of magnitude higher than the values obtained in the present
studies for the interaction between CSE and the intrinsic pathway
protease.
Considered together, the variety of different molecular interactions increasingly being described in the regulation of blood coagulation by GAGs (Bourin and Lindahl, 1993) points to complex physiochemical mechanisms and underline the importance of considering structural requirements for each interaction independently rather than generalizing from studies with heparin.
Intense efforts in coagulation research are currently being directed to identify specific inhibitors and modulators of blood coagulation that may control hypercoagulability while maintaining the vital hemostatic function of the coagulation system. Because of the existence of alternate routes for factor Xa generation, it is reasonable to expect that specific inhibition of intrinsic pathway protease would result in less hemorrhagic complications than inhibition of the initiating complex, TF/FVIIa, or the prothrombinase complex. In this regard, studying the response of monocyte procoagulants to GAGs is of importance not only for characterization of coagulation reactions in a biologically relevant microenvironment but also for identification of inhibitors with potential use in pharmacologic control of hypercoagulable states associated with atherosclerosis, inflammation, and cancer.
The results of the present studies indicate specific modulation of the intrinsic pathway protease but not of extrinsic protease by chondroitin sulfate E. The results are consistent with modulation of baseline procoagulant activity by GAGs expressed on monocytes. Added GAGs in the extracellular medium further inhibited reactions in a manner that was strictly dependent on the spatial distribution of charges on the polysaccharide. Multiple functional titrations and Scatchard type analysis provided kinetic evidence consistent with a hypothetical model of inhibition mediated by interaction between CSE and at least two independent sites: one on factor IXa and another on VIIIa. In addition, results suggest that this GAG may provide a useful anticoagulant to manage prothrombotic states associated with activity of the intrinsic pathway amplifying loop.