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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 555-560
The Low-Density Lipoprotein Receptor-Related Protein (LRP) Mediates
Clearance of Coagulation Factor Xa In Vivo
By
Masaaki Narita,
Amy E. Rudolph,
Joseph P. Miletich, and
Alan L. Schwartz
From the Edward Mallinckrodt Departments of Pediatrics, Molecular
Biology, and Pharmacology, and the Departments of Medicine and
Pathology at the Washington University School of Medicine, St Louis,
MO.
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ABSTRACT |
Blood coagulation factor X plays a pivotal role in the clotting
cascade. When administered intravenously to mice, the majority of
activated factor X (factor Xa) binds to  2-macroglobulin
(2M) and is rapidly cleared from the circulation into
liver. We show here that the low-density lipoprotein receptor-related
protein (LRP) is responsible for factor Xa catabolism in vivo. Mice
overexpressing a 39-kD receptor-associated protein that binds to LRP
and inhibits its ligand binding activity displayed dramatically
prolonged plasma clearance of 125I-factor Xa.
Preadministration of 2M-proteinase complexes
(2M*) also diminished the plasma clearance of
125I-factor Xa in a dose-dependent fashion. The clearance
of preformed complexes of 125I-factor Xa and
2M was similar to that of 125I-factor Xa
alone and was also inhibited by mice overexpressing a 39-kD
receptor-associated protein. These results thus suggest that, in vivo,
factor Xa is metabolized via LRP after complex formation with
2M.
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INTRODUCTION |
FACTOR X, A PLASMA glycoprotein involved
in the blood coagulation cascade, can be converted to its active form
(factor Xa) by both the intrinsic pathway (via factors IXa and VIIIa)
and extrinsic pathway (via factor VIIa together with tissue
factor).1 Activated factor X generated from either pathway
participates in the prothrombinase complex in which factors Xa and Va
activate prothrombin to thrombin in the presence of calcium and
phospholipid. Thus, control of factor Xa levels by plasma protease
inhibitors may be pivotal in the regulation of the coagulation process.
Although little is known regarding factor Xa catabolism, a number of
plasma serine protease inhibitors are thought to inhibit factor Xa
activity. These include 1-protease inhibitor
(1PI),2 2-macroglobulin (2M),3 antithrombin III
(AT-III),4 and tissue factor pathway inhibitor
(TFPI).5 In vitro, factor Xa is mainly inactivated by
AT-III and 1-PI, as observed in a purified system and in
plasma.6-8 Although 2M accounts for only
10% to 15% of the factor Xa inactivation in vitro, Fuchs and
Pizzo7 showed that, by 2 minutes after injection of
125I-factor Xa (125I-Xa) into mice, 90% of the
radioactivity was bound to 2M. This finding suggests
that 2M is a major inhibitor of factor Xa in vivo. After
intravenous administration, 125I-Xa is cleared rapidly into
the liver, with a plasma half-life of approximately 2 minutes in
rabbits.7 However, the biology underlying this clearance
process has not been elucidated.
The low-density lipoprotein receptor-related protein (LRP), initially
identified by its structural homology with the low-density lipoprotein
(LDL) receptor,9 is a large multifunctional endocytosis receptor10 having several structurally and functionally
distinct ligands. Ligands of LRP include 2M-protease
complexes (2M*),11 tissue-type
plasminogen activator (t-PA),12,13 and TFPI.14 A unique ligand, the 39-kD protein, which copurifies with LRP, inhibits all known ligand interactions with LRP.15,16
In vivo, the role of LRP in the clearance of the circulating ligands
such as t-PA and TFPI has been shown after overexpression of the 39-kD
protein in mice using an adenoviral mediated gene transfer
technique.17-19 Because several protease-inhibitor (eg, 2M) complexes are endocytosed and degraded via LRP, we
examined the role of LRP in the catabolism of factor Xa in vivo. Our
results show that LRP mediates the plasma clearance of factor Xa
after complex formation with protease inhibitors and thus suggest
strategies for regulation of its catabolism.
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MATERIALS AND METHODS |
Reagents.
N-Succinimidyl 3-(4-hydroxy-5-[125I] iodophenyl)
propionate (Bolten and Hunter reagent) was purchased from Amersham
(Arlington Heights, IL). Human 2M was
purified and activated (2M*) as described previously.20 Balb/c mice were obtained from Jackson
Laboratories (Bar Harbor, ME). AT-III was purchased from Kabi Pharmacia
Diagnostics (Piscataway, NJ). Factor VII- and X-deficient plasma and
phospholipid (rabbit brain cephalin) were obtained from Sigma (St
Louis, MO). Rabbit brain thromboplastin was from Ortho (Raritan, NJ).
The chromogenic substrate Spectrozyme FXa
(methoxycarbonyl-D-hexahydrotyrosyl-L-alanyl-L-arginine-p-nitroanilide-diacetate) was obtained from American Diagnostica Inc (Greenwich, CT). Factor X-deficient plasma was from George King Bio-Medicals Inc (Overland Park, KS).
Coagulation factors.
Recombinant factor X was prepared and purified as described by Miletich
et al,21 with minor modifications.22 The cDNA encoding human factor X was obtained by polymerase chain reaction amplification from first-strand cDNA template from a human hepatocyte library, ligated into the mammalian expression vector ZMB3, and stably
transfected into human kidney 293 cells. Factor X was isolated from the
culture media by absorption to barium citrate, followed by resuspension
in 32% saturated (NH4)2SO4 in the
presence of 5 mmol/L diisopropyl fluorophosphate and incubation for
hour at 4°C. The precipitate was collected by centrifugation and
the resulting supernatant was dialyzed into 10 mmol/L HEPES, pH 7.0, 100 mmol/L NaCl, and 1 mmol/L benzamidine. The dialyzed supernatant was
concentrated and immunoaffinity purified using antibody 3698.1A8.10,
which is a calcium-dependent monoclonal antibody directed against the Gla domain of factor X. Factor X was further purified using a Pharmacia
Mono Q FPLC column. Plasma (human)-derived factor X was isolated by the
same procedure. In some experiments, factor X was activated using
factor X coagulant protein (XCP) from Russell's viper venom in a
factor X:XCP ratio (wt/wt) of 100:1 in 10 mmol/L HEPES, pH 7.4, 100 mmol/L NaCl, 5 mmol/L CaCl2. XCP was purified from crude
viper venom as described previously.23 Because native factor X is composed of two polypeptide chains joined by a disulfide bond, the gel pattern in the presence of  -mercaptoethanol is particularly useful to confirm successful processing from the 1-chain
form to the 2-chain native form. On nonreducing sodium dodecyl sulfate
(SDS)-gels (Fig 1, lanes 1 and 2), the
apparent molecular weight of both plasma-derived and recombinant factor Xs was approximately 68 kD (noted by arrow). On the other hand, under
reducing conditions (lanes 3 and 4), the apparent molecular weight of
both factor Xs was 49 kD for the heavy chain (solid arrowhead) and 23 kD for the light chain (open arrowhead). Plasma-derived (lanes 1 and 3)
and recombinant (lanes 2 and 4) factor X migrated identically on both
reducing and nonreducing conditions, indicating that they have the same
apparent molecular size. Furthermore, no difference in functional
activity between plasma-derived and recombinant factor X was
detected.24
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| Fig 1.
SDS-PAGE of plasma-derived 125I-X and
recombinant 125I-X in 15% polyacrylamide gels.
Plasma-derived 125I-X (lanes 1 and 3) and recombinant
125I-X (lanes 2 and 4) were subjected to 15% SDS-PAGE and
autoradiography, under nonreducing conditions (lanes 1 and 2) and
reducing conditions (lanes 3 and 4) (68-kD single chain; 47-kD heavy
chain; 23-kD light chain).
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Mutagenesis of factor X.
The S379A factor X mutant was constructed as follows. The mutation was
engineered into the cDNA of factor X using a Transformer Site-Directed
Mutagenesis Kit from Clontech (Palo Alto, CA). The codon encoding the
active site serine at position 379 was mutated to alanine:
(GAC)(AGC)(GGG) (GAC) (GCT)(GGG). The DNA sequence of
the constructs was verified by the dideoxy chain termination technique.
Mutated constructs were shuttled into the ZMB3 mammalian expression
vector, transfected into the 293 cells for expression, and purified as
described above.
Protein iodination.
Factor X was iodinated using the Bolton and Hunter reagent according to
the manufacturer's instructions. Specific radioactivities were 5 to 10 µCi/µg of protein (0.13 to 0.26 mol 125I/mol protein).
Functional activity of recombinant 125I-labeled and
unlabeled factor X was evaluated using the modified prothrombin time
(PT) as follows. Various dilutions of factor X samples were mixed with
factor X-deficient plasma and incubated at 37°C for 5 minutes in a
fibrometer. Clotting was initiated by the addition of thromboplastin
and 15 mmol/L CaCl2. No difference was seen in the
coagulant activity between labeled and unlabeled protein. Enzymatic
activity of recombinant 125I-labeled and unlabeled factor X
was also determined by measurement of factor Xa activity against the
chromogenic peptide substrate, Spectrozyme FXa. No difference was seen
in the enzymatic activity between labeled and unlabeled protein. These
results show that iodination using Bolten and Hunter reagent does not
affect functional and enzymatic activity of factor X and Xa.
Adenovirus purification.
Recombinant adenovirus containing the full-length 39-kD protein cDNA
(Ad-39-kD) or the Escherichia coli -galactosidase cDNA (Ad--Gal) were prepared and titered as described
previously.17,18 The virus titer was approximately 100 virus particles/plaque-forming unit.
In vivo viral delivery.
In vivo viral delivery was performed via intravenous administration as
described previously.18 Various viral particle doses were
examined. Optimal expression was achieved after the administration of 4 × 1011 particles (4 × 109
plaque-forming units) of either Ad-39-kD or Ad--Gal. All experiments were performed on day 5 after viral delivery, because optimal expression of the 39-kD protein was at day 4 or 5 after virus administration.
In vivo plasma clearance in mice.
Twelve- to 16-week-old BALB/c mice (weighing 20 to 25 g) were
anesthetized with sodium pentobarbital (1 mg/ 20 g body) during the
course of the experiment. The indicated radiolabeled protein (~10
pmol of factor 125I-X, 125I-Xa, or
125I-S379A Xa) in sterile saline (total volume, 100 µL)
was injected into a tail vein over 30 seconds. At the indicated times,
40 to 50 µL of blood was collected by periorbital bleeding. The
amount of radiolabeled protein in the plasma samples was determined as described previously.18 After 20 minutes, the animals were
killed, and the liver, spleen, kidneys, and lungs were removed and
weighed. 125I-radioactivity was determined in a gamma
counter from Packard Instrument Co (Meriden, CT). Studies
were performed in 2 to 7 animals for each condition described.
Recombinant factor Xa was used in all experiments in Figs 2-5.
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| Fig 2.
Effect of Ad-39-kD on the plasma clearance of
125I-Xa and 125I-X. As described in the
Materials and Methods, mice were injected with 10 pmol of
plasma-derived 125I-Xa (A) or recombinant
125I-Xa (B) or 125I-X (C) with ( ) or without
( ) preinjection of 4 × 1011 particles of Ad-39-kD 5 days previously. Blood samples were collected at the indicated times
and trichloroacetic acid-insoluble radioactivity was determined.
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Reaction of factor Xa with purified plasma proteinase inhibitors.
Complexes of 125I-Xa with human 2M and
AT-III were prepared by reacting 125I-Xa with increasing
concentrations of each inhibitor. The optimal AT-III:factor Xa binding
ratio was determined by quantitation of the residual factor Xa activity
against the chromogenic peptide substrate, Spectrozyme FXa. Factor Xa
inhibition by 2M could not be determined by this method,
because the complex still retains partial activity toward peptide
substrates. Therefore, the optimal 2M:factor Xa ratio
was determined by the clotting method of Fujikawa et al,25
using factor VII- and X-deficient plasma in the presence of
CaCl2 and phospholipid. Inhibition reactions were performed at room temperature for 30 minutes in the buffer containing 10 mmol/L
HEPES, 100 mmol/L NaCl, 5 mmol/L CaCl2, 1 mg/mL PEG 8000, 1 mg/mL bovine serum albumin, pH 7.0. The initial factor Xa concentration was kept constant at 1.0 µmol/L, and the concentration of the inhibitors varied to obtain the desired molar ratio. After incubation, aliquots were removed and tested for residual factor Xa activity. Residual factor Xa activity decreased linearly with increasing inhibitor concentration. An AT-III:factor Xa ratio of 1:1.8 yielded complete inhibition of factor Xa. Complete inhibition of factor Xa by
2M was obtained at a 1:1 molar ratio.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
autoradiography.
SDS-PAGE was performed according to Laemmli.26 Dried gels
were exposed to Kodak XAR film (Eastman Kodak, Rochester,
NY) at  80°C for autoradiography.
| |
RESULTS |
125I-factor Xa and 125I-factor X clearance in
normal mice and Ad-39-kD-injected mice.
The plasma clearance of plasma-derived 125I-Xa after
intravenous administration of 10 pmol of 125I-Xa in normal
mice and Ad-39-kD-injected mice is shown in
Fig 2A. The initial plasma half-life of
125I-Xa was approximately 3 minutes, with 10% of the
administered dose remaining in the circulation at 20 minutes. Figure 2B
shows the plasma clearance of 125I-recombinant Xa. The
plasma clearance curves for 125I-plasma-derived Xa and
125I-recombinant Xa are identical. The plasma clearance of
125I-X was substantially slower than that of
125I-Xa, with a half-life of approximately 10 minutes (Fig
2C). The plasma clearance curve for 125I-Xa seen in Fig 2A
was similar to that reported earlier by Fuchs and Pizzo.7
Approximately 60% of the injected 125I-X and
125I-Xa was associated with the liver at 20 minutes
(Table 1 and data not shown, see below). These data show
rapid hepatic clearance of 125I-Xa and are consistent with
a receptor-mediated endocytosis mechanism. Among those candidate
receptors is LRP, which mediates the endocytosis of several
protease-protease inhibitor complexes such as
2M-complexes, uPA:PAI-1, t-PA:PAI-1, and protease
nexin:uPA. To examine the role of LRP in 125I-Xa and
125I-X clearance in vivo, we took advantage of an
adenoviral delivery vector to express an LRP-antagonist, the 39-kD
protein, in mouse liver. Overexpression of the 39-kD protein in liver
results in plasma accumulation of the 39-kD protein. Previously, we
showed that mice receiving 4 × 1011 particles of
Ad-39-kD expressed sufficient 39-kD protein in plasma to completely
inhibit LRP, and we also showed that viral infection induced no gross
or microscopic morphologic changes in the liver.18,19 This
dose of Ad-39-kD was administered intravenously to mice via tail vein.
Five days after administration, plasma clearance studies of recombinant
125I-Xa and 125I-X were performed. As seen in
Fig 2, administration of Ad-39-kD prolonged the plasma clearance of
125I-Xa significantly (Fig 2A and B), whereas
administration of Ad-39-kD did not alter the plasma clearance of
125I-X (Fig 2C). To confirm that the viral infection did
not induce adverse effects on the clearance of 125I-Xa,
studies were performed in mice after administration of Ad--Gal. The
plasma clearance of 125I-Xa in Ad--Gal-injected mice
was essentially the same as that of the noninfected mice (data not
shown). These results indicate that the rapid hepatic clearance of
125I-Xa, but not of 125I-X, is mediated by LRP.
In vivo association of 125I-Xa among proteinase
inhibitors.
Previous studies have shown that, 2 minutes after the injection of
125I-Xa into mice, 90% of that bound within the plasma was
associated with 2M.7 To confirm and extend
this observation, plasma samples obtained 30 seconds after the
injection of 125I-Xa or the injection of the preformed
complex of 2M and 125I-Xa
(2M:125I-Xa) were subjected to SDS-PAGE
(Fig 3). Standards containing 2M:125I-Xa, AT-III:125I-Xa,
125I-Xa, and 125I-X were also analyzed. In both
plasma samples, complexes of 2M with 125I-Xa
were observed at the identical size as the
2M:125I-Xa standard. These results show that
the vast majority of exogenously administered 125I-Xa is
complexed to 2M.
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| Fig 3.
Plasma species of 125I-Xa and
2M:125I-Xa after injection to mice. As
described in the text, plasma samples (3 µL) obtained 30 seconds after the injection of 125I-Xa or
2M:125I-Xa were subjected to 7.5% SDS-PAGE
and autoradiography. Standards containing
2M:125I-Xa, AT-III:125I-Xa,
125I-Xa, and 125I-X were also analyzed as
indicated. Molecular weight markers in kilodaltons are indicated on the
left.
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Effect of preadministration of 2M* or
2M on the clearance of 125I-Xa in mice.
Because in vivo the vast majority of factor Xa is bound to
2M (Fuchs and Pizzo7 and Fig 3) and because
2M:protease complexes and activated 2M
(2M*) but not free or uncomplexed 2M are
metabolized via LRP,11 we next examined whether
preadministration of 2M* (which competes for the
2M* binding site on LRP) can influence on the plasma
clearance of 125I-Xa. As seen in Fig 4,
preinjection of various amount of 2M* (0.5, 1, and 2.5 mg) altered the plasma half-life of 125I-Xa from
approximately 3 minutes to >>20 minutes in a dose-dependent manner.
On the other hand, preinjection of various amount of 2M (which does not bind to LRP) essentially did not change the plasma half-life of 125I-Xa. These results further support the
hypothesis that factor Xa complexed to 2M is cleared via
LRP.
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| Fig 4.
Effect of preadministration of 2M* and
2M on the plasma clearance of 125I-Xa.
Various doses (0.5, 1.0, and 2.5 mg) of 2M*
() or 2M () were preadministered 1 minute
before injection of 125I-Xa. Plasma 125I-Xa
radioactivities were determined at the indicated times, as described in
Fig 2.
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Clearance of 2M:125I-Xa and
AT-III:125I-Xa.
To further characterize the proteinase inhibitor-dependent phase of
125I-Xa clearance, complexes of 125I-Xa with
2M (2M:125I-Xa) were prepared
and examined in clearance studies
(Fig 5A). The clearance of
2M:125I-Xa in normal mice was rapid, with a
plasma half-life of approximately 5 minutes. On the other hand, the
clearance of 2M:125I-Xa in
Ad-39-kD-injected mice was extremely slow. The characteristics of
these clearance curves are essentially the same as those of 125I-Xa in normal mice and Ad-39-kD-injected mice, as
described above (Fig 2A and B). Next, the role of proteinase inhibitor,
AT-III, was examined. Complexes of 125I-Xa with AT-III
(AT-III:125I-Xa) were prepared and evaluated in clearance
studies (Fig 5B). Plasma clearance of AT-III:125I-Xa was
rapid, with a half-life of approximately 5 minutes. Clearance was also
prolonged in Ad-39-kD mice but not to the extent observed with
2M:125I-Xa. Fuchs et al27 have
previously reported that AT-III:protease complexes are rapidly cleared,
with a half-life of approximately 6 minutes, and that this rapid
clearance is independent of 2M*. Our data suggest that
2M:125I-Xa is metabolized solely via LRP
and, in addition, that LRP plays an important role in the metabolism of
AT-III:125I-Xa. This latter conclusion is consistent with
recent observations of Kounnas et al28 in studies of
AT-III-thrombin and LRP. However, our observation that, after the
injection of AT-III:125I-Xa, the clearance of approximately
75% of the 125I-Xa was prolonged, suggests that another
clearance receptor may well be involved. Future studies will address
this issue further.
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| Fig 5.
Effect of Ad-39-kD on the plasma clearance of
2M:125I-Xa or AT-III:125I-Xa.
Mice were injected with 2M:125I-Xa (A) or
AT-III:125I-X (B) with () or without () preinjection
of Ad-39-kD 5 days previously. Clearance studies were performed as
described in Fig 2.
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Clearance of a factor Xa mutant that cannot bind proteinase
inhibitors.
Previously, Fuchs and Pizzo7 showed that the clearance of
diisopropyl fluorophosphoryl (DFP)-inactivated factor Xa, which cannot
bind to the protease inhibitors, was more rapid than factor Xa alone,
suggesting that DFP-inactivated factor Xa has a binding site(s) that is
distinct from LRP. To further characterize the role of the proteinase
inhibitors in factor Xa clearance, we prepared mutant factor Xa in
which the active-site serine of factor Xa has been substituted by
alanine. The resultant mutant (S379A Xa) cannot bind to the proteinase
inhibitors (2M, AT-III, etc). Plasma samples obtained 30 seconds after intravenous administration of 125I-Xa and
125I-S379A Xa were subjected to SDS-PAGE and
autoradiography. The injected 125I-S379A Xa migrated at the
same molecular size as the uninjected standard, indicating that S379A
Xa does not form SDS-stable complexes with proteinase inhibitors in
vivo (data not shown). Clearance studies were then performed using this
mutant. The clearance of 125I-S379A Xa was extremely rapid,
with a plasma half-life of approximately 1 minute, which was
essentially the same as that of DFP-inactivated factor Xa (data not
shown). Ad-39-kD did not affect the clearance of 125I-S379A
Xa (data not shown). These data suggest that a pathway(s) distinct from
LRP is involved in the clearance of 125I-S379A Xa.
Organ distribution of 125I-Xa and 125I-S379A
Xa in normal mice.
The organ distributions of radioactivity at 20 minutes are summarized
in Table 1. 125I-Xa is found primarily in liver (56%).
However, recovery of 125I-S379A Xa in liver (32%) was
significantly less than that of 125I-Xa. Lung distribution
was also examined. The distribution of radioactivity was low in the
lung for both 125I-Xa (0.5%) and 125I-S379A Xa
(0.9%).
| |
DISCUSSION |
The present observations show that (1) inhibition of LRP in vivo by
gene transfer of a 39-kD protein prolongs the plasma half-life of
125I-Xa; (2) the plasma half-life of 125I-X is
slow and unaltered by the 39-kD protein; (3) the vast majority of
exogenously administered 125I-Xa is bound to
2M; (4) preadministration of 2M*, but not
2M, prolongs the plasma half-life of
125I-Xa; and (5) the clearance of preformed
2M:125I-Xa complexes is the same as that of
125I-Xa. Taken together, these results indicate that, in
vivo, LRP mediates the hepatic clearance of 125I-Xa after
complex formation with 2M.
Factor Xa plays a pivotal role in the clotting cascade, because it can
be activated by both the intrinsic and the extrinsic pathways. After
intravenous administration, 125I-Xa was rapidly cleared
from the circulation into liver, whereas clearance of
125I-X was much slower. The observation that the expression
of the 39-kD protein altered the clearance of 125I-Xa
indicates that the clearance of 125I-Xa, but not
125I-X, is mediated by LRP. The mechanism(s) responsible
for the clearance of 125I-X is currently unknown.
Previously, using adenoviral gene transfer of the 39-kD protein, we
showed that LRP mediates the clearance of both t-PA and TFPI in
vivo.18,19 TFPI is cleared as the free species, whereas
t-PA is cleared as both the free species as well as that complexed to
PAI-1.
LRP did not mediate the plasma clearance of 125I-S379A Xa,
an active-site serine factor Xa mutant that cannot bind serine-site proteinase inhibitors, including 2M, AT-III,
1PI, etc. This finding suggests that LRP does not
modulate the active coagulation process occurring in the periphery such
as the microvascular environment, but once circulating coagulation
factors are inactivated by protease inhibitors, these may be eliminated
from the circulation via LRP.
Regulation of the blood coagulation cascade is a complex,
multifactorial process. Several proteinase inhibitors are active participants in this process via inhibition of the activated
coagulation factors. In vitro, factor Xa is primarily inactivated by
AT-III and 1PI, as has been observed in both purified
systems as well as whole plasma.6-8 Although
2M only accounts for 10% to 15% of factor Xa
inactivation in vitro, Fuchs and Pizzo7 showed that, 2 minutes after injection of 125I-Xa into mice, 90%
of the radioactivity was associated with 2M. This
finding suggested that 2M is a major inhibitor of factor Xa in vivo. Our present observations are consistent with these findings.
The vascular endothelium also plays an important role in the
coagulation process. Activation of prothrombin to thrombin by factor Xa
and factor Va is believed to occur not only on platelets but also on
vascular endothelial cells. Lollar and Owen29 reported that
thrombin binds to active site-independent high-affinity binding sites
on the endothelial cell surface. This interaction catalyzes the
inactivation of thrombin by AT-III. Thereafter, thrombin-AT-III complexes are selectively removed by the liver. Because both thrombin and factor Xa are inactivated by AT-III on the vascular endothelial cell surface, factor Xa inactivation by 2M may also
occur on or in proximity to the vascular endothelial cell surface.
Numerous cellular binding sites/receptors have been implicated in the
recognition of factor Xa (reviewed in Altieri30). These
include those on the surface of platelets,31 endothelial cells,32 alveolar macrophages,33 leukocytes,
and hepatoma cells. Specific recognition (inhibitory) molecular
candidates include cell-associated AT-III, TFPI,34 protease
nexin-1,34 and effector cell protease-1
(EPR-1).35 Each of these binding sites has been implicated
in intravascular activation or inhibition of coagulation via
interaction with factor Xa. For example, platelets and vascular
endothelial cells provide cell surface sites for the generation of
prothrombin as described above. Recently, in in vitro studies in the
absence of other protease inhibitors, we have identified a new factor
Xa cellular binding/uptake pathway that requires TFPI and is
independent of LRP.36 However, in vivo, LRP appears to play
a distinctly different role, because it functions as the major pathway
responsible for elimination of inactivated Xa from the circulation.
| |
FOOTNOTES |
Submitted July 10, 1997;
accepted September 18, 1997.
Supported in part by National Institutes of Health Grants No. HL14147
(to J.P.M.) and HL 53280 and HL 52040 (to A.L.S.).
Address reprint requests to Masaaki Narita, MD, PhD, Department of
Pediatrics, Box 8116, Washington University School of Medicine, One
Children's Place, St Louis, MO 63110.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank J.S. Traush-Azar for preparing the virus. We also
thank Dave Wilson for critical reading of the manuscript.
| |
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Abstract
Introduction
Abstract