From the Research and Development Department, Canadian Blood Services and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1G 4J5, Canada
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ABSTRACT |
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Known anticoagulant pathways have been shown to
exclusively inhibit blood coagulation cofactors and enzymes. In the
current work, we first investigated the possibility of a novel
anticoagulant mechanism that functions at the level of zymogen
inactivation. Utilizing both clotting and chromogenic assays, the
fibrinolysis protease plasmin was found to irreversibly inhibit the
pivotal function of factor X (FX) in coagulation. This was due to
cleavage at several sites, the location of which were altered by
association of FX with procoagulant phospholipid (proPL). The final
products were ~28 and ~47 kDa for proPL-bound and unbound FX,
respectively, which did not have analogues when activated FX (FXa) was
cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that
plasmin exposed a binding site only on proPL-bound FX. The highest
apparent affinity was for the 28-kDa fragment, which was identified as
the light subunit disulfide linked to a small fragment of the heavy
subunit (Met-296 to ~Lys-330). After cleavage by plasmin, proPL-bound
FX furthermore was observed to accelerate plasmin generation by
tissue plasminogen activator. Thus, a feedback mechanism localized by
proPL is suggested in which plasmin simultaneously inhibits FX
clotting function and converts proPL-bound FX into a fibrinolysis
cofactor. These data also provide the first evidence for an
anticoagulant mechanism aimed directly at the zymogen FX.
Blood coagulation and fibrinolysis are opposing hemostatic
processes. Whereas the coagulation pathway produces insoluble fibrin clot to seal vascular leaks, the fibrinolysis pathway solubilizes the
clot to restore normal blood flow. It is obvious that for either of
these pathways to be effective, their amplification must be sequential.
Several coordinating mechanisms have been identified that each control
the availability of fibrinolysis cofactors. The best described of these
is the production of fibrin (1), which, unlike its precursor
fibrinogen, is a fibrinolysis cofactor that colocalizes the zymogen
plasminogen and an activating protease, tissue plasminogen activator
(tPA),1 for subsequent
plasmin generation. Plasmin directly mediates fibrin solubilization by
proteolysis. In order for plasminogen to assemble into the ternary
activation complex, the cofactor molecule must contain a COOH-terminal
Lys, which is an essential constituent of the plasminogen binding site
(2-10). Carboxypeptidases in plasma have been identified that excise
COOH-terminal Lys and thereby limit the generation of plasmin (11-13)
by restricting fibrinolysis cofactor function. In particular, an
important regulatory role has been demonstrated for one of these
carboxypeptidases, TAFI, which has been shown to first become activated
and then later inactivated by the final coagulation enzyme, thrombin
(12-14). Thus, thrombin orchestrates a lag between coagulation and the amplification of fibrinolysis to ensure sufficient clot production.
An additional mechanism of communication between coagulation and
fibrinolysis has recently been reported. This involves the enzyme
complex responsible for physiological thrombin production, prothrombinase (15, 16). Prothrombinase consists of the serine protease
factor Xa (FXa), and the non-enzymatic cofactor Va (FVa), which in the
presence of Ca2+ associate with each other and a
procoagulant phospholipid (proPL)-containing surface. Together the
constituents of prothrombinase enhance the FXa-mediated activation of
prothrombin to thrombin by 5 orders of magnitude (17). Previous studies
have shown that plasmin can inhibit the prothrombinase cofactor
function of FVa (18) and the procofactor V (19). We have extended these
findings to show that plasmin also proteolytically inhibits the
coagulation activity of FXa, and furthermore converts both FXa and FVa
into fibrinolysis cofactors (9, 20). Interestingly, the acquisition of
fibrinolysis function was dependent on the prior association of FXa or
FVa with proPL. This may provide a means to accelerate plasmin
generation specifically at sites of vascular damage, where the exposure
of proPL is selectively triggered.
The physiological generation of FXa requires recruitment of its
inactive precursor, factor X (FX), to sites of vascular damage through
an association with proPL. Therefore, following sufficient fibrin
formation to initiate fibrinolysis, it is conceivable that localized
plasmin production may modulate FX function. We now report that FX is a
substrate for plasmin, but the final proteolytic products differ from
that of FXa. The cleavage of proPL-bound FX by plasmin results in a
diametric functional change that inhibits participation in coagulation
and simultaneously induces the expression of fibrinolysis cofactor activity.
Chemicals and Reagents--
HEPES, EDTA, (Sigma),
2-guanidino-ethylmercaptosuccinic acid (GEMSA), aprotonin (Calbiochem),
N- Proteins--
Human coagulation factor X (FX) was purified from
fresh frozen plasma (21) or from prothrombin complex concentrate
diluted to 1 unit/ml factor X clotting activity (obtained as generous gifts from the Canadian Red Cross Society, London Collection Center, and from Bayer, Inc., respectively). For comparison to in-house preparations, FX was also purchased (Hematologic Technologies, Inc.).
Human FXa (22, 23) and Lys-plasminogen were produced, as described
(25). Lys-plasminogen was radioiodinated (50,000-150,000 dpm/µg)
using Iodogen (Pierce) and chromatographically desalted (Excellulose 5, Pierce) to remove unincorporated 125I. Purified human
plasmin was purchased (Hematologic Technologies, Inc.).
Proteolytic Time Courses--
FX (9 µM) was
treated with plasmin (0.1 µM) in 20 mM HEPES,
150 mM NaCl, pH 7.2 (HBS), in the presence of various
combinations of Ca2+ (2 mM), EDTA (5 mM), or proPL (300 µM) at 21 °C. Each
digest was sampled over time, subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) (12% acrylamide) (26) and silver-stained.
For electrophoretic comparison to the FXa fragments generated by
plasmin, at each time point, aprotonin (50 kalikrein inhibitory units
(KIU)/ml) was added to the plasmin-digested FX followed by treatment
with RVV (10 µg/ml) at 37 °C for 1 h in 10 mM
CaCl2.
FX Enzymatic Activity Assays--
The effect of plasmin
treatment of FX on the generation of FXa coagulation and amidolytic
activity was evaluated in plasmin digestion time courses identical to
those described above. At each time point, aprotonin (50 KIU/ml) was
added to stop further effects of plasmin. Clotting activity was
measured by thromboplastin clotting time assays using FX-deficient
plasma (Sigma) reconstituted with 5 units/ml purified FX treated with
plasmin as a substrate. The amount of FX activation was determined by
comparison to a standard curve obtained by titrating purified FXa in
the presence of precomplexed plasmin and aprotonin to duplicate the
conditions of the time course samples. The generation of FXa amidolytic
activity at 22 °C in HBS using S2765 (200 µM) as a
chromogenic substrate was followed after treatment of plasmin-digested
FX with RVV as described above. The rate of color development was
monitored in a kinetic multiwell plate reader
(Vmax, Molecular Devices).
Ligand Blots--
To identify FX-derived species that interact
with plasminogen, ligand blotting experiments were performed according
to methods published by several laboratories (5, 3, 2). In our studies, FX fragments were separated by SDS-PAGE and electrotansferred to PVDF
(27, 28). The PVDF was blocked overnight at 4 °C in bovine serum
albumin (Sigma, 10 mg/ml) and then incubated with 125I-Lys-plasminogen (0.1 µM) for 1 h at
22 °C in the presence of the protease inhibitors GEMSA (50 nM) and aprotonin (50 KIU/ml, Calbiochem). Following
extensive washing with HBS, the location of bound
125I-plasminogen was determined by autoradiography and
compared with the electrophoretic patterns made visible by staining the
PVDF with Coomassie Brilliant Blue R-250.
Effect of Plasmin-treated FX on tPA Activity--
The activation
of plasminogen (1 µM) to plasmin by single-chain tPA (10 nM) was followed as a time course utilizing the chromogenic substrate S2366. To determine whether plasmin-treated FX affects tPA
function, FX was treated with plasmin (or left untreated) exactly as
described above for 30 min. Just enough aprotonin (9.5 KIU/ml) was
added to inhibit the plasmin used in this step prior to the addition of
tPA and plasminogen. To control for the possible effects of added
aprotonin and plasmin, reactions that did not involve a plasmin
treatment step contained aprotonin and plasmin that had been preincubated.
Amino Acid Sequencing--
The subunit composition of
plasmin-digested FX products was investigated both by nonreducing
SDS-PAGE and by two-dimensional SDS-PAGE involving a first dimension
under nonreducing conditions and a second dimension under reducing
conditions (29, 20). To identify proteolytic cleavage sites,
electrophoretic patterns were transferred to PVDF, and bands were
excised for sequence analysis (27, 28).
Inactivation of FX Coagulation Activity by Plasmin--
To
determine whether FX coagulation activity is altered upon pretreatment
with plasmin, Fig. 1 shows
FXa-dependent thromboplastin clotting (panel A)
and RVV-initiated amidolytic (panel B) assays. Both methods
demonstrated that plasmin inhibits the coagulation enzyme function of
FX. This was observed regardless of whether binding of FX to proPL was
facilitated by Ca2+.
Cleavage of FX by Plasmin--
The demonstration that FX enzymatic
activity is lost due to plasmin pretreatment strongly indicates that FX
is cleaved by plasmin. To provide direct evidence, changes in
plasmin-treated FX electrophoretic mobility were followed using the
same digestion conditions and pretreatment times as in Fig. 1. As
demonstrated by the SDS-PAGE shown in Fig.
2, plasmin sequentially cleaves FX at
several sites. Interestingly, the proteolytic sites recognized by
plasmin depended on whether Ca2+ was present to facilitate
FX-proPL interactions. It should be noted that minor amounts of the
alternate proteolytic products were detected under both reaction
conditions, indicating that one pathway is favored over the other but
is not exclusive. The nomenclature adopted previously for Pn-mediated
FXa fragments (20) was used for the analogous FX fragments observed
here. However, two additional species were observed for FX that had apparent molecular masses of 28 kDa (Fig. 2A) and 47 kDa
(Fig. 2B) and resulted from cleavage by plasmin in the
presence of Ca2+ and EDTA, respectively.
To determine whether only binding of FX to Ca2+ was the
determinant in altering the cleavage sites recognized by plasmin, the 30-min time points shown in Fig. 2 were compared with identically treated FX in the presence of Ca2+ but no proPL. These data
are shown in Fig. 3 and demonstrate that
Ca2+ alone is insufficient to generate the fragmentation
profile that resulted in the presence of Ca2+ and proPL.
Thus, binding to proPL and not just to Ca2+ is suggested to
alter the cleavage of FX by plasmin.
To further compare the cleavage of FX and FXa by plasmin, the same
fragmentation time courses were conducted as in Fig. 2 except that
prior to electrophoresis, the FX-derived fragments generated by plasmin
were treated with RVV to remove the activation domain. The resulting
SDS-PAGE profiles are shown in Fig. 4,
with the mobility of plasmin-mediated species generated directly from purified FXa indicated. As shown in Fig. 4A, when
FX
In contrast to FX Activation of FX Fibrinolysis Function by Plasmin--
Because
plasmin liberates COOH-terminal basic amino acids and COOH-terminal Lys
is a requisite for plasminogen binding to known fibrinolysis cofactors,
we investigated whether the proteolysis of FX by plasmin generated
species capable of participating in fibrinolysis. As an initial step,
we determined whether the plasmin-mediated FX fragments were capable of
interacting with plasminogen. To do so, the SDS-PAGE patterns shown in
Fig. 2 were transferred to PVDF and probed with
125I-plasminogen. The resulting autoradiographs are shown
in Fig. 5. Binding of
125I-plasminogen to untreated FX was not detectable.
However, under conditions that facilitated proPL binding (Fig.
5A), plasmin induced the expression of a receptor site(s) in
FX
Under conditions that did not facilitate binding of FX to proPL during
the plasmin pretreatment step (Fig. 5B), we observed 125I-plasminogen binding only marginally to FX
Based on our ligand blotting experiments, the 28-kDa fragment in
particular was suggested to have potential as a fibrinolysis cofactor.
Therefore, we followed the effect of plasmin-cleaved FX on the
activation of plasminogen by tPA using a chromogenic assay for plasmin
(Fig. 6). As shown, a mixture of
FX Identity of FX-derived Fibrinolysis Cofactor(s)--
Because the
cleavage sites recognized by plasmin to produce the 28-kDa fragment are
unique to FX-bound to proPL, and because this species was found to be a
good tPA accelerator and plasminogen-binder, we conducted amino acid
sequence analyses to locate the specific cleavage sites. Table
I summarizes these data. When the
nonreduced FX
When the nonreduced 28-kDa species was excised from PVDF and sequenced,
three equimolar residues per cycle were observed. These corresponded to
the NH2 terminus of the heavy and light subunits, in
addition to an internal heavy chain sequence beginning at Met-296. To
further investigate this mixture of fragments, two-dimensional SDS-PAGE
was conducted in which the 28-kDa species was first electrophoresed
nonreduced and then subjected to a second dimension under reducing
conditions (not shown). Two species were identified after transfer to
PVDF and were sequenced. The mobility of the first was not affected by
reduction and yielded a sequence identical to the NH2
terminus of the FX heavy subunit. The second reduced fragment had an
apparent molecular mass of approximately 20 kDa and migrated exactly to
the position of the native FX light subunit. NH2-terminal
amino acid sequence analysis confirmed its identity as the light
subunit. These data cumulatively suggested that the nonreduced 28-kDa
band was a co-migrating mixture of an NH2 terminus-derived
heavy subunit fragment ending at Arg-295 and a heterodimer consisting
of the intact light subunit disulfide-linked to a small heavy chain
fragment. Under reducing conditions, the small heavy chain fragment was
not detectable after transfer to PVDF, and consequently only the amino
acid sequence under nonreducing conditions was obtained.
Prothrombinase is the final enzyme complex generated within the
coagulation cascade and functions directly to generate thrombin. Consequently, numerous modes of regulation of prothrombinase assembly and activity have evolved to enable fine-tuning of thrombin production (31). These include anticoagulant mechanisms that exclusively target
the prothrombinase constituent cofactor FVa (32) and enzyme FXa (33),
as well as the upstream tenase complexes, which are responsible for
activating FXa from its inactive precursor (32, 34). In the current
study, we investigated whether the fibrinolysis effector protease
plasmin can modulate the properties of the zymogen FX. Utilizing
functional assays initiated by tissue factor or RVV FX activator, we
show that plasmin is able to function as an anticoagulant through
proteolytic inhibition of FX. To our knowledge this is the first
evidence for an anticoagulant mechanism that operates directly at the
level of FX inactivation.
Because proteolysis by plasmin is capable of producing COOH-terminal
Lys, which is an essential feature of known fibrinolysis cofactors
(2-10), the possibility that plasmin-cleaved FX acquires fibrinolytic
activity was investigated. We found that in addition to having an
anticoagulant effect on FX, plasmin converted the FX into a
fibrinolysis cofactor. This conclusion is based on experiments that
showed plasmin induces the expression of a plasminogen binding site on
FX and the ability to accelerate the rate of tPA-dependent plasmin generation by approximately 30-fold. The latter effect was
observed below the circulating concentration of FX. This supports the
hypothesis that at sites of clot formation, where proPL increases the
local concentration of coagulation proteins (15), the in vitro effects of Pn-cleaved FX on tPA reported here may have a physiological function in concert with other tPA accelerators, notably
fibrin. Because the expression of FX-derived tPA cofactor activity was
observed only under conditions that facilitated FX-proPL interactions,
the proposed pathway would be advantageously localized to sites of clot formation.
To understand the structural basis by which FX function is altered by
plasmin, Fig. 7 shows a fragmentation map
of FX bound to proPL. The first plasmin-mediated cleavage produces
FX
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Z-D-Arg-Gly-Arg-p-nitroanalide (S2765), and
benzoyl-Ile-Glu-(piperidyl)-Gly-Arg-p-nitroanalide (S2366)
(Helena) were purchased. As a source of proPL, small unilamellar phospholipid vesicles consisting of 75% phosphatidylcholine and 25%
of the procoagulant lipid, phosphatidylserine, were prepared and
quantified as described (16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of plasmin on FX enzymatic
activity. FX (5 µM) was subjected to cleavage by
plasmin (0.1 µM) in the presence of proPL (300 µM) and Ca2+ (2 mM) ( ) or
proPL (300 µM) and EDTA (5 mM) (
).
Incubation times (min) at 22 °C are indicated. FX activation
and subsequent enzymatic activity were evaluated in a
thromboplastin-based clotting assay (A) or in a
chromogenic assay initiated by RVV (B). (n = 2; mean ± S.E.)
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Fig. 2.
Time course of FX cleavage by plasmin.
Silver-stained nonreduced SDS-PAGE (12% acrylamide) showing the time
course of FX (5 µM) digested with plasmin (0.1 µM) in the presence of proPL and Ca2+ (2 mM) (A) or in the presence of proPL and EDTA (5 mM) (B). Incubation times (min) at 22 °C are
as follows: lane 1, 0; lane 2, 2; lane
3, 5; lane 4, 7; lane 5, 10; lane
6, 15; lane 7, 20; lane 8, 30.
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Fig. 3.
Dependence of FX cleavage sites on proPL and
Ca2+. Silver-stained SDS-PAGE showing FX treated for
30 min with plasmin under the indicated conditions. Concentrations used
are the same as in Fig. 2.
Pn, Ca and FX
Pn, Ca were treated with
RVV under conditions that favored binding to proPL, the resulting
species had electrophoretic migrations identical to FXa
Pn,
Ca and FXa
Pn, Ca, respectively. This suggests that plasmin cleaves the same sites in FX and FXa to generate these
fragments. However, the 28-kDa product was unique to FX bound to proPL,
and RVV had no effect on the mobility of this fragment.
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Fig. 4.
Comparison of plasmin-mediated FX and FXa
cleavage products. Time courses identical to those shown in Fig. 2
were conducted, except that at each time point, aprotonin (50 KIU/ml)
was added followed by treatment with RVV (10 µg/ml) at 37 °C for
1 h in 10 mM CaCl2 to excise the FX
activation fragment. The electrophoretic mobility of
plasmin-mediated species generated directly from purified FXa is
indicated. Plasmin incubation times (min) at 22 °C are as follows:
lane 1 0; lane 2, 2; lane 3, 5;
lane 4, 7; lane 5, 10; lane 6, 15;
lane 7, 20; lane 8, 30.
Pn, Ca, but similar to the further
proteolyzed 28-kDa species, the data presented in Fig. 4B
show that RVV does not cleave FX
Pn, EDTA over the
indicated time. This is additional evidence that two pathways of FX
proteolysis by plasmin are followed, depending on whether the prior
interaction with proPL is facilitated.
Pn, Ca, FX
Pn, Ca, the 28-kDa fragment,
and the ~13-kDa fragment. Using this ligand blotting method, the
species clearly having the highest apparent affinity for
125I-plasminogen was the 28-kDa fragment for as yet unknown
reasons. These observations suggested that plasmin liberates a
COOH-terminal Lys on each of these FX fragments.
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Fig. 5.
Plasminogen binding to plasmin-cleaved
FX. A, autoradiograph of ligand blot showing
125I-plasminogen (0.1 µM) binding to FX
species formed in the presence of plasmin, proPL (300 µM), and Ca2+ (2 mM) (corresponds
to stained gel in Fig. 2A); B, autoradiograph of
ligand blot showing 125I-plasminogen (0.1 µM)
binding to FX species formed in the presence of plasmin, proPL (300 µM), and EDTA (5 mM) (corresponds to stained
gel in Fig. 2B).
Pn,
EDTA and a species that migrated with the dye front. The former
appeared to be present as a minor constituent of our starting FX
preparation and is likely independent of plasmin pretreatment. No
interaction was detectable between 125I-plasminogen and the
other major cleavage products, FX
Pn, EDTA and the 47-kDa
fragment, suggesting that a COOH-terminal Lys is not made available by
proteolysis unless binding to proPL is facilitated .
Pn, Ca and the 28-kDa species produced by pretreatment
of proPL-bound FX with plasmin for 30 min (final concentration, 0.5 µM FX) resulted in a 27-fold enhancement of tPA activity
(Fig. 6A). In contrast, when either native FX or FX
pretreated with plasmin to produce FX
Pn, EDTA and the
47-kDa fragment (Fig. 6B) were used, insignificant effects
on tPA activity were observed. As shown in Fig. 6C,
acceleration of tPA by the FX
Pn, Ca and the 28-kDa
species mixture was titratable to levels below the circulating
concentration of FX (0.17 µM).
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Fig. 6.
Effect of plasmin-cleaved FX on tissue
plasminogen activator function. A and B, the
activation of plasminogen (1 µM) to plasmin by tPA (10 nM) was followed as a time course utilizing the chromogenic
substrate, S2366, in the absence ( ) or presence (
) of FX (final
concentration, 0.5 µM) or the same amount of
plasmin-treated FX (
). The FX was pretreated with plasmin exactly as
described in Fig. 2 for 30 min. Just enough aprotonin (9.5 KIU/ml) was
added to inhibit the plasmin used in this step prior to the addition of
tPA and plasminogen. A, conducted in the presence of
Ca2+ (2 mM) and proPL (300 µM),
as in Fig. 2A. B, conducted in the presence of
EDTA (5 mM) and proPL (300 µM), as in Fig.
2B. C, FX was plasmin-pretreated as in
A for 30 min, and the concentration dependence of tPA
activity was monitored. The data were corrected for endogenous tPA
activity. (n = 3; mean ± S.D.).
Pn, Ca band was analyzed, two equimolar
sequences were revealed that corresponded to the NH2
terminus of the heavy and light subunits of FX. Determination of the
~13-kDa species amino acid sequence, which appeared concomitant with
FX
Pn, Ca, revealed that FX
Pn, Ca is
generated due to cleavage of the heavy subunit at Gly-331 (based on
amino acid numbering adopted previously (30)).
Amino acid sequence of plasmin-mediated FX fragments
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pn, Ca, which has the same electrophoretic mobility as
previously documented FX
generated by treatment of FX with FXa (35).
The location of the cleavage site that excises the
-peptide is
inferred and is based partly on the supposition that FX
Pn,
Ca must have a newly exposed COOH-terminal Lys to account for
the observed plasminogen binding. Autolytic cleavages at Lys-435 and
Lys-433 have been reported for FXa, with the latter representing a
minor product (36). Thus, we have positioned the first plasmin-mediated
cleavage of FX to be consistent with the former.
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Fig. 7.
FX cleavage by plasmin. A scale model
based on the number of amino acids shows the plasmin-mediated
fragmentation pattern of FX bound to proPL. Species observed to bind
plasminogen (Pg) and accelerate tPA are indicated.
Plasminogen binding sites have been inferred by predicted creation of
COOH-terminal Lys due to plasmin cleavage. Act., FX
activation peptide; protease, protease domain; ,
-domain or
-peptide.
, NH2-terminal amino acid
sequences derived in the current work (Table I). *, inferred cleavage
position based on plasminogen binding and previously reported sequence
analysis due to cleavage by FXa (36).
The second cleavage of proPL-bound FX by plasmin produced FXPn,
Ca and a 13-kDa species. Amino acid sequence analysis of the
former under nonreducing conditions produced two sequences corresponding to the NH2 terminus of the FX heavy and light
subunits. Sequence analysis of the latter indicated cleavage of the
heavy subunit within the "autolysis loop" between Lys-330 and
Gly-331, which is the same as that previously reported for FXa cleaved under identical conditions (20). Unlike the recently reported proteolysis of FX by FXa (37), cleavage in the autolysis loop by
plasmin was not observed by us to be protected by Ca2+. The
exposure of a new COOH-terminal Lys at position 330 is consistent with
our finding that plasminogen interacts with FX
Pn, Ca in ligand blotting experiments. Furthermore, cleavage at this site explains the loss of FX coagulation function because disruption of the
protease domain results.
Continued incubation of proPL-bound FX with plasmin resulted in a third sequential cleavage that produced a 28-kDa band under nonreducing conditions. Amino acid sequencing identified a heterodimer consisting of the light subunit disulfide linked to a small piece of the heavy subunit starting at Met-296 and presumably extending to Lys-330. The small heavy subunit fragment encompasses Cys-302, which is known to link the FX subunits (38). The end position at Lys-330 is inferred from the prior cleavage known to expose Gly-331 as the NH2 terminus of the 13-kDa fragment. Immunoblot analysis utilizing the well studied monoclonal antibody H11 (39) specific for the Gla-containing domains of several proteins, including residues 4-9 of FX, confirmed that the light chain is a constituent of the 28-kDa heterodimer (not shown). Interestingly, cleavage at Met-296 was not observed after an identical incubation of FXa with plasmin (20), suggesting that the activation domain in FX influences the accessibility of cleavage sites.
Comigrating with the 28-kDa heterodimer was the expected fragment corresponding to the NH2-terminal portion of the heavy subunit produced by cleavage at Met-296. Reduction changed the electrophoretic mobility of the 28-kDa heterodimer containing the light subunit, but not the mobility of the fragment corresponding to the NH2-terminal heavy subunit fragment. Compared with the other FX fragments generated by plasmin, the apparent affinity observed for plasminogen was clearly strongest for the nonreduced 28-kDa species. The prediction that a COOH-terminal Lys is available in the heterodimer (Lys-330) but not in the single chain heavy subunit fragment (predicted COOH terminus at Arg-295) strongly suggests that the former is responsible for the observed plasminogen binding. Consistent with this conclusion is the lack of plasminogen binding to the 28-kDa monomer under reducing conditions (not shown). The basis for comparatively strong plasminogen binding to the 28-kDa heterodimer is under investigation.
When conditions were used to prevent the interaction of FX with proPL
(i.e. in the presence of EDTA), the electrophoretic pattern
due to plasmin cleavage was altered. At present, the specific cleavage
sites have not been determined by amino acid sequencing. However,
immunoblot analysis revealed that upon production of FXPn,
EDTA, antigenic recognition by the Gla domain-specific antibody,
H11, is lost (not shown). Therefore, unlike our findings for
proPL-bound FX, cleavage of FX in the absence of proPL binding resulted
in rapid cleavage of the light chain. This is consistent with our
previous report (20) showing that the light subunit of FXa is cleaved
by plasmin in the presence of EDTA. Subsequent cleavage of
FX
Pn, EDTA was observed to result in the appearance of a
47-kDa species that does not have an analogous plasmin-mediated fragment when FXa rather than FX is the substrate (20). Cleavage of the
FX light subunit by plasmin was protected only in the presence of both
Ca2+ and proPL, indicating that proPL binding and not just
divalent cation binding was necessary. The Gla domain has been shown to be involved in activation of FX by tissue factor/FVIIa or RVV FX
activator (24), which provides an explanation for inhibition of FX
coagulation activity by plasmin under conditions that did not
facilitate proPL binding. Further evidence for differences in the sites
recognized by plasmin due to binding of FX to proPL comes from
plasminogen binding and tPA activity studies. Whereas plasmin cleavage
was found to convert proPL-bound FX into a plasminogen receptor and tPA
accelerator, neither activity was detectable when FX was cleaved by
plasmin in the absence of proPL and/or Ca2+.
The FX data presented here, together with our earlier reports that
showed that plasmin has similar modulatory effects on proPL-bound FXa
and FVa (9, 20), support a model in which plasmin functions to create a
profibrinolytic milieu through direct conversion of specific proteins
into tPA cofactors. We have found that neither FX, FXa, nor FVa
expresses tPA cofactor function when not bound to proPL. Although the
plasmin-mediated cleavage patterns differ, both proPL-bound and unbound
FX, FXa, and FVa are inhibited from participating in any further clot
formation. In this way, plasmin may participate in a feedback amplified
mechanism by producing tPA cofactors localized by the selective
accessibility of cellular proPL trapped in the fibrin network. We are
currently investigating the relative contribution of fibrin to
proPL-bound tPA cofactors within a clot.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Alex Kurosky and Steve Smith (Protein Chemistry Laboratory, University of Texas at Galveston, Medical Branch) for expert amino acid sequence analysis. We also thank Dr. Réal Lemieux, Tina Raynor, and Abed Zeibdawi for helpful suggestions regarding manuscript preparation.
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FOOTNOTES |
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* This work was supported by the Canadian Blood Services Research and Development Grant HO1O 928.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Research and
Development Dept., Canadian Blood Services, 1800 Alta Vista Dr., Ottawa, Ontario K1G 4J5, Canada. Tel.: 613-739-2462; Fax:
613-739-2426; E-mail ed.pryzdial{at}bloodservices.ca.
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ABBREVIATIONS |
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The abbreviations used are: tPA, tissue plasminogen activator; FX, factor X; FXa, activated FX; FVa, factor Va; proPL, procoagulant phospholipid; PAGE, polyacrylamide gel electrophoresis; KIU, kalikrein inhibitory unit(s); RVV, Russell's viper venom FX activator; GEMSA, 2-guanidino-ethylmercaptosuccinic acid; HBS, HEPES plus NaCl; PVDF, polyvinylidene difluoride.
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