From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
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
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 a2macroglobulin (a2M) 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 receptorassociated protein that binds to LRP and inhibits its ligand
binding activity displayed dramatically prolonged plasma
clearance of 125I-factor Xa. Preadministration of a2M-proteinase complexes (a2M*) also diminished the plasma clearance
of 125I-factor Xa in a dose-dependent fashion. The clearance
of preformed complexes of 125I-factor Xa and a2M 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 a2M.
r 1998 by The American Society of Hematology.
F
(Arlington Heights, IL). Human a2M was purified and activated (a2M*)
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
ACTOR 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 a1-protease inhibitor (a1PI),2 a2-macroglobulin
(a2M),3 antithrombin III (AT-III),4 and tissue factor pathway
inhibitor (TFPI).5 In vitro, factor Xa is mainly inactivated by
AT-III and a1-PI, as observed in a purified system and in
plasma.6-8 Although a2M 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 a2M. This finding
suggests that a2M 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 lowdensity lipoprotein (LDL) receptor,9 is a large multifunctional
endocytosis receptor10 having several structurally and functionally distinct ligands. Ligands of LRP include a2M-protease
complexes (a2M*),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, a2M) 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.
MATERIALS AND METHODS
Reagents. N-Succinimidyl 3-(4-hydroxy-5-[125I] iodophenyl) propionate (Bolten and Hunter reagent) was purchased from Amersham
Blood, Vol 91, No 2 (January 15), 1998: pp 555-560
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.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9102-0033$3.00/0
555
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
556
the presence of b-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). Plasmaderived (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
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)8(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
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).
NARITA ET AL
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
b-galactosidase cDNA (Ad-b-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 3 1011 particles (4 3 109 plaque-forming units)
of either Ad-39-kD or Ad-b-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 through 5.
Reaction of factor Xa with purified plasma proteinase inhibitors.
Complexes of 125I-Xa with human a2M 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 a2M could not be
determined by this method, because the complex still retains partial
activity toward peptide substrates. Therefore, the optimal a2M: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 a2M 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 280°C for autoradiography.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
LRP MEDIATES FACTOR Xa CLEARANCE
RESULTS
125I-factor
125I-factor
Xa and
X clearance in normal mice and
Ad-39-kD–injected mice. The plasma clearance of plasmaderived 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 a2M-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 3 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-b-Gal. The plasma clearance of 125I-Xa in Ad-b-Gal–
557
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 a2M.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 a M and
2
125I-Xa (a M:125I-Xa) were subjected to SDS-PAGE (Fig 3).
2
Standards containing a2M:125I-Xa, AT-III:125I-Xa, 125I-Xa, and
125I-X were also analyzed. In both plasma samples, complexes
of a2M with 125I-Xa were observed at the identical size as the
a2M:125I-Xa standard. These results show that the vast majority
of exogenously administered 125I-Xa is complexed to a2M.
Effect of preadministration of a2M* or a2M on the clearance
of 125I-Xa in mice. Because in vivo the vast majority of factor
Xa is bound to a2M (Fuchs and Pizzo7 and Fig 3) and because
a2M:protease complexes and activated a2M (a2M*) but not free
or uncomplexed a2M are metabolized via LRP,11 we next
examined whether preadministration of a2M* (which competes
for the a2M* binding site on LRP) can influence on the plasma
clearance of 125I-Xa. As seen in Fig 4, preinjection of various
amount of a2M* (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 a2M (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 a2M is cleared via LRP.
Clearance of a2M:125I-Xa and AT-III:125I-Xa. To further
characterize the proteinase inhibitor-dependent phase of 125I-Xa
clearance, complexes of 125I-Xa with a2M (a2M:125I-Xa) were
prepared and examined in clearance studies (Fig 5A). The
clearance of a2M:125I-Xa in normal mice was rapid, with a
plasma half-life of approximately 5 minutes. On the other hand,
the clearance of a2M: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
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 (X) or without (W) preinjection of 4 3 1011 particles of Ad-39-kD 5
days previously. Blood samples were collected at the indicated times and trichloroacetic acid-insoluble radioactivity was determined.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
558
NARITA ET AL
Table 1. Organ Distribution of 125I-Xa and 125I-S379A Xa
125I-Xa
Liver
Spleen
Kidney
Blood
Lung
56
3.0
3.0
11
0.5
125I-S379A
Xa
32
3.2
2.4
25
0.9
Values are the percentage of the injected dose. Mice were injected
with approximately 10 pmol of 125I-Xa and 125I-S379A Xa as described
in the Materials and Methods. After 20 minutes, the animals were
killed, organs and plasma were obtained, and their content of 125Iradioactivity was determined by gamma counting.
Fig 4. Effect of preadministration of a2M* and a2M on the plasma
clearance of 125I-Xa. Various doses (0.5, 1.0, and 2.5 mg) of a2M* (W) or
a2M (X) were preadministered 1 minute before injection of 125I-Xa.
Plasma 125I-Xa radioactivities were determined at the indicated times,
as described in Fig 2.
Fig 3. Plasma species of 125I-Xa and a2M:125I-Xa after injection to
mice. As described in the text, plasma samples (3 mL) obtained 30
seconds after the injection of 125I-Xa or a2M:125I-Xa were subjected to
7.5% SDS-PAGE and autoradiography. Standards containing a2M:125IXa, AT-III:125I-Xa, 125I-Xa, and 125I-X were also analyzed as indicated.
Molecular weight markers in kilodaltons are indicated on the left.
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 a2M:125IXa. 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 a2M*.
Our data suggest that a2M: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.
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 DFPinactivated 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 (a2M, 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
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
LRP MEDIATES FACTOR Xa CLEARANCE
559
Fig 5. Effect of Ad-39-kD on the plasma clearance
of a2M:125I-Xa or AT-III:125I-Xa. Mice were injected
with a2M:125I-Xa (A) or AT-III:125I-X (B) with (X) or
without (W) preinjection of Ad-39-kD 5 days previously. Clearance studies were performed as described in Fig 2.
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 DFPinactivated 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 a2M; (4) preadministration of a2M*, but not a2M, prolongs the plasma
half-life of 125I-Xa; and (5) the clearance of preformed a2M:
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 a2M.
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 serinesite proteinase inhibitors, including a2M, AT-III, a1PI, 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 a1PI, as has been observed in both purified
systems as well as whole plasma.6-8 Although a2M 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
a2M. This finding suggested that a2M 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 highaffinity 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 a2M 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
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
560
NARITA ET AL
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.
ACKNOWLEDGMENT
The authors thank J.S. Traush-Azar for preparing the virus. We also
thank Dave Wilson for critical reading of the manuscript.
REFERENCES
1. Hertzberg M: Biochemistry of factor X. Blood Rev 8:56, 1994
2. Colman RW, Marder VJ, Salzman EW, Hirsh J: Plasma coagulation factors, in Colman RW, Hirsh J, Marder VJ, Salzman EW (eds):
Hemostasis and Thrombosis, vol 3. Philadelphia, PA, Lippincott, 1987,
p3
3. Jackson CM, Nemerson Y: Blood coagulation. Annu Rev Biochem 49:765, 1980
4. Kurachi KK, Fujikawa K, Schmer G, Davie EW: Inhibition of
bovine factor IXa and factorab by antithrombin III. Biochemistry
15:373, 1976
5. Broze GJ Jr, Girard TJ, Novotny WF: Regulation of coagulation
by a multivalent kunitz-type inhibitor. Biochemistry 29:7539, 1990
6. Ellis V, Scully M, MacGregor I, Kakkar V: Inhibition of human
factor Xa by various plasma proteinase inhibitors. Biochim Biophys
Acta 701:24, 1982
7. Fuchs HE, Pizzo SV: The regulation of factor Xa in vitro in human
and mouse plasma and in vivo in mouse. The role of the endothelium
and the plasma proteinase inhibitors. J Clin Invest 72:2041, 1983
8. Gitel SN, Medina VM, Wessler S: Inhibition of human activated
factor X by antithrombin and a1-proteinase inhibitor in human plasma. J
Biol Chem 259:6890, 1984
9. Herz J, Hamann U, Rogne SK, Kyklebost O, Gausepohl H,
Stanley KK: Surface location and high affinity for calcium of a 500-kd
liver membrane protein closely related to the LDL-receptor suggest a
physiological role as lipoprotein receptor. EMBO J 7:4119, 1988
10. Krieger M, Herz J: Structures and functions of multiligand
lipoprotein receptors: Macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 63:601, 1994
11. Kristensen T, Moestrup SK, Gliemann J, Bendtsen L, Sand O,
Sottrup-Jensen L: Evidence that the newly cloned low-densitylipoprotein receptor related protein (LRP) is the a2-macroglobulin
receptor. FEBS Lett 276:151, 1990
12. Bu G, Williams S, Strickland DK, Schwartz AL: Low density
lipoprotein receptor-related protein/a2-macroglobulin receptor is a
hepatic receptor for tissue-type plasminogen activator. Proc Natl Acad
Sci USA 89:7427, 1992
13. Orth K, Madison EL, Gething M-J, Sambrook JF, Herz J:
Complexes of tissue-type plasminogen activator and its serpin inhibitor
plasminogen-activator inhibitor type 1 are internalized by means of the
low density lipoprotein receptor-related protein/a2-macroglobulin receptor. Proc Natl Acad Sci USA 89:7422, 1992
14. Warshawsky I, Broze GJ Jr, Schwartz AL: The low density
lipoprotein receptor-related protein mediates the cellular degradation of
tissue factor pathway inhibitor. Proc Natl Acad Sci USA 91:6664, 1994
15. Herz J, Goldstein JL, Strickland DK, Ho YK, Brown MS: 39kDa
protein modulates binding of ligands to low density lipoprotein
receptor-related protein/a2-macroglobulin receptor. J Biol Chem 266:
21232, 1991
16. Bu G, Morton PA, Schwartz AL: Identification and partial
characterization by chemical cross-linking of a binding protein for
tissue-type plasminogen activator (t-PA) on rat hepatoma cells. J Biol
Chem 267:15595, 1992
17. Willnow TE, Sheng Z, Ishibashi S, Herz J: Inhibition of hepatic
chylomicron remnant uptake by gene transfer of a receptor antagonist.
Science 264:1471, 1994
18. Narita M, Bu G, Herz J, Schwartz AL: Two receptor systems are
involved in the plasma clearance of tissue-type plasminogen activator
(t-PA) in vivo. J Clin Invest 96:1164, 1995
19. Narita M, Bu G, Olins GM, Higuchi DA, Herz J, Broze GJ Jr,
Schwartz AL: Two receptor systems are involved in the plasma
clearance of tissue factor pathway inhibitor in vivo. J Biol Chem
270:24800, 1995
20. Warshawsky I, Bu G, Schwartz AL: Identifications of domains
on the 39-kDa protein that inhibit the binding of ligands to the low
density lipoprotein receptor-related protein. J Biol Chem 268:22046,
1993
21. Miletich JP, Jackson CM, Majerus PW: Properties of the factor
Xa binding site on platelets. J Biol Chem 253:6908, 1978
22. Rudolph AE, Mullane MP, Porche-Sorbet R, Tsuda S, Miletich
JP: Factor X St Louis II: Identification of a glycine substitution at
residue 7 and characterization of the recombinant protein. J Biol Chem
271:28601, 1996
23. Esmon C: Function of factor V in prothrombin activation.
Doctoral thesis. Washington University, St Louis, MO, 1974
24. Rudolph AE, Mullane MP, Porche-Sorbet R, Miletich JP:
Expression, purification, and characterization of recombinant human
factor X. Protein Expression Purification 10:373, 1997
25. Fujikawa K, Legarz ME, Davie EW: Bovine factors X1 and X2
(Stuart factor). Isolation and characterization. Biochemistry 11:4882,
1972
26. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970
27. Fuchs HE, Shifman MA, Pizzo SV: In vivo catabolism of
a1-proteinase-trypsin, antithrombin III-thrombin and a2-macroglobulinmethylamine. Biochim Biophys Acta 716:151, 1982
28. Kounnas MZ, Church FC, Argraves WS, Strickland DK: Cellular
internalization and degradation of antithrombin III-thrombin, heparin
cofactor II-thrombin, and alpha1-antitrypsin-trypsin complexes is mediated by the low density lipoprotein receptor-related protein. J Biol
Chem 271:6523, 1996
29. Lollar P, Owen WG: Clearance of thrombin from circulation in
rabbits by high-affinity binding sites on endothelium. J Clin Invest
66:1222, 1980
30. Altieri DC: Xa receptor EPR-1. FASEB J 9:860, 1995
31. Tracy PB, Nesheim ME, Mann KG: Platelet factor Xa receptor.
Methods Enzymol 215:329, 1992
32. Rodgers GM, Shuman MA: Prothrombin is activated on vascular
endothelial cells by factor Xa and calcium. Proc Natl Acad Sci USA
80:7001, 1983
33. McGee MP, Rothberger H: Assembly of the prothrombin activator complex on rabbit alveolar macrophage high-affinity factor Xa
receptors. A kinetic study. J Exp Med 164:1902, 1986
34. Kazama Y, Komiyama Y, Kisiel W: Tissue factor pathway
inhibitor and protease nexin-1 are major factor Xa binding proteins on
HepG2 cell surface. Blood 81:676, 1993
35. Nicholson AC, Nachman RL, Altieri DC, Summers BD, Ruf W,
Edgington TS, Hajjar DP: Effector cell protease receptor-1 is a vascular
receptor for coagulation factor Xa. J Biol Chem 271:28407, 1996
36. Ho G, Toomy JR, Broze GJ Jr, Schwartz AL: Receptor-mediated
endocytosis of coagulation factor Xa requires cell surface-bound tissue
factor pathway inhibitor. J Biol Chem 271:9497, 1996
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1998 91: 555-560
The Low-Density Lipoprotein Receptor-Related Protein (LRP) Mediates
Clearance of Coagulation Factor Xa In Vivo
Masaaki Narita, Amy E. Rudolph, Joseph P. Miletich and Alan L. Schwartz
Updated information and services can be found at:
http://www.bloodjournal.org/content/91/2/555.full.html
Articles on similar topics can be found in the following Blood collections
Hemostasis, Thrombosis, and Vascular Biology (2485 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.