From the Research and Development Department, the Canadian Blood Services and the Department of Biochemistry, Microbiology, and Immunology, the University of Ottawa, Ottawa, Ontario K1G 4J5, Canada
Received for publication, May 31, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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The coagulation cofactor Va (FVa) is a
noncovalent heterodimer consisting of a heavy chain (FVaH) and a light
chain (FVaL). Previously, the fibrinolytic effector plasmin (Pn) has
been shown to inhibit FVa function. To understand this mechanism, the
fragmentation profile of human FVa by Pn and the noncovalent
association of the derived fragments were determined in the presence of
Ca2+ using anionic phospholipid (aPL)-coated
microtiter wells and large (1 µm) aPL micelles as affinity matrices.
Following Pn inactivation of aPL-bound FVa, a total of 16 fragments
were observed and their NH2 termini sequenced. These had
apparent molecular weights and starting residues as follows (single
letter abbreviation is used): 50(L1766), 48(L1766), 43(Q1828),
40(Q1828), 30(S1546), 12(T1657), and 7(S1546) kDa from FVaL; and
65(A1), 50(A1), 45(A1), 34(S349), 30(L94), 30(M110), and 3 small
<5(W457, W457, and K365) kDa from FVaH. Of these, 50(L1766), 48(1766),
43(Q1828), and 40(Q1828) spanning the C1/C2 domains, and 30(L94), but
not the similar 30(M110), positioned within the A1 domain remained
associated with aPL. These were detected antigenically during Pn- or
tissue plasminogen activator-mediated lysis of fibrin clot formed in
plasma. Chelation by EDTA dissociated the 30(L94)-kDa fragment, which
was observed to associate with intact FVaL upon recalcification,
indicating that the Leu-94 to Lys-109 region of the A1 domain plays a
critical role in the FVaL and FVaH
Ca2+-dependent association. By using
domain-specific monoclonal antibodies and an assay for thrombin
generation, loss of FVa prothrombinase function was coincident with
proteolysis at sites in the A2 and A3 domains resulting in their
dissociation. Inactivation of FV or FVa by Pn was independent of the
thrombophilic R506Q mutation. These results identify the molecular
composition of Pn-cleaved FVa that remains bound to membrane as largely
A1-C1/C2 in the presence of Ca2+ and suggest that Pn
inhibits FVa by a process involving A2 and A3 domain dissociation.
Factor V (FV)1 is a
central pro-cofactor in the blood coagulation cascade, which must be
proteolytically activated to factor Va (FVa) for clot formation (1-4).
Activated platelets, monocytes, and endothelial cells present at the
site of vascular injury expose anionic phospholipid (aPL;
e.g. phosphatidylserine (PS)) on their membrane surface
providing a site of association for FVa and the serine protease factor
Xa (FXa) in the presence of Ca2+. Once formed, this
macromolecular complex, termed prothrombinase, enhances the
FXa-mediated activation of prothrombin to thrombin by 5 orders of
magnitude (5), which is necessary for physiological clot formation.
Human FV is secreted into plasma as a single chain glycoprotein (2196 amino acids) composed of three homologous A domains and two homologous
C domains and a unique B domain arranged in the order
NH2-A1-A2-B-A3-C1-C2-COOH (6, 7). FVa is a
heterodimer composed of an NH2-terminally derived heavy
chain (FVaH; A1/A2 domains; molecular mass = 104 kDa, amino
acids 1-709) and a COOH-terminally derived light chain (FVaL; A3/C1/C2
domains; molecular mass = 74/71 kDa; amino acids 1546-2196). FVaH
and FVaL are noncovalently associated in the presence of a
Ca2+ (Kd = 6 × 10 Proteolytic activation and inactivation of FV/FVa plays a central role
in regulating coagulation. At the site of vascular injury, FV is
activated through a series of cleavages predominantly by thrombin, at
Arg-709, Arg-1018, and Arg-1545, which excises the B domain to yield
FVa (7). Factor V is initially activated by FXa in an aPL and
Ca2+-dependent manner (2). FVa is also
inactivated by proteolysis. The well studied anticoagulant, activated
protein C (APC), inhibits FVa by cleaving FVaH (14), leading to
dissociation of the A2 domain (15). The known APC-dependent
cleavage of FVaL has no known function. Both FV and FVa can also be
inactivated by an additional protease, the fibrinolytic protease
plasmin (Pn), although inactivation of FV is preceded by a brief phase
of activation (16, 17). Pn further influences coagulation by
inactivating factor VII (18), factor VIII (19), factor IX (20, 21), factor X/Xa (22, 23), and factor XII (24). Work from our laboratory has
suggested a further communication mechanism between coagulation and
fibrinolysis where inhibition of aPL-associated FVa coagulation
activity by Pn results in a species that accelerates Pn generation
(25). These combined observations strongly suggest that Pn participates
in maintaining balance between the opposing hemostatic pathways,
coagulation and fibrinolysis. To understand the anticoagulant function
of Pn, the structural basis by which FVa coagulation cofactor activity
is inhibited by Pn is reported in the current work. The unique
fragmentation profile mediated by Pn has further enabled localization
of the Ca2+-dependent contacts between FVaL and FVaH.
Chemicals and Reagents--
HEPES, EDTA, PS, phosphatidylcholine
(PC), bovine serum albumin, polyethylene glycol (PEG, average molecular
weight 8000) (Sigma), aprotinin (Calbiochem),
H-D-Phe-pipecolyl-Arg-pNA.2HCl (S2238, Chromogenix),
Tween 20 (Fisher), and a chemiluminescent detection system (ECL,
Amersham Pharmacia Biotech) were obtained commercially. Small
unilamellar vesicles (SUV; average diameter 50 nm) consisting of
75:25% mixture of PC/PS were prepared (25) and quantified as described
(26). Large vesicles (LV; 300-600 nm) were made by extrusion using a
Liposofast Basic apparatus (Avestin Inc.). LV consisting of 1-4 mg of
75:25% PC/PS was suspended in 1 ml of methanol in glass tubes and
dried to a thin film under a steady stream of nitrogen at 4 °C. The
phospholipid was resuspended in 0.5-1 ml of 20 mM HEPES,
300 mM sucrose, pH 7.4, vortexed for 1 min, and subjected
to 10 freeze-thaw cycles. The suspension was then extruded through two
1-µm filters (Avestin, Inc.) and collected. The vesicles were then
mixed 1:1 with 20 mM HEPES, 150 mM NaCl, pH 7.4 (HBS/Ca2+), and harvested by centrifugation at 13,000 × g at 22 °C for 5-10 min. The pellet was resuspended
in HBS and quantified using an assay for total phosphorus (27).
Proteins--
Human FVa, FXa, prothrombin, plasminogen, Pn,
monoclonal antibodies (mAb) specific for human FVaH (AHV-5146) and
human FVaL (AHV-5112), and polyclonal sheep anti-human FV (PAHFV-S)
were from Hematologic Technologies, Inc. mAb specific for FVa A3 domain (anti-FVaA3) was produced by Dr. R. Lemieux (Hema-Quebec) for which
in-house recombinant FVaL produced using the baculovirus system and
purified by electroelution was used as antigen. Polymerase chain
reaction-screened R506Q homozygous plasma was a generous gift from Dr.
M. C. Poon, University or Calgary, Canada. Rabbit antiserum to
human plasminogen (Calbiochem), tissue plasminogen activator (tPA,
Genentech), peroxidase-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch Inc.), peroxidase-conjugated goat anti-rabbit IgG
(Promega), assayed reference plasma (Helena Laboratories), and
thromboplastin (Sigma) were commercially obtained.
Factor Va Proteolysis by Pn--
FVa (0.1 µM) was
incubated with Pn (0.1 µM) in the presence of SUV (250 µM) or LV (600 µM) and Ca2+ (2 mM) in HBS/Ca2+ at 22 °C. The digests were
sampled over time and heated at 95 °C for 5 min in sample buffer
(2% SDS, 2% 2-mercaptoethanol, 0.325 M Tris base, pH 6.5, 10% glycerol and 0.001% bromphenol blue). The digests were subjected
to 10% SDS-PAGE (28), stained with Coomassie Blue, or
electrotransferred to polyvinylidene fluoride (PVDF; Immobilon-P,
Millipore) (29). The PVDF membrane was blocked at 22 °C for 1 h
in milk (5%), and cleavage products of FVa were detected by Western
analysis using chemiluminescence (ECL).
FVa was also cleaved by Pn when bound to aPL-coated microtiter wells
prepared as described previously (25) except that the wells were
blocked with gelatin (5 mg/ml) in HBS/Ca2+, and 0.001%
Tween 20 was included in the wash buffer. After equilibration of the
FVa with the aPL-coated well in HBS/Ca2+, the wells were
washed and Pn (0.1 µM) in HBS/Ca2+ was added.
At selected time points, four wells were quickly washed, and bound
protein was eluted with 25 µl of SDS-PAGE sample buffer/well and
pooled, heated to 95 °C for 10 min, and subjected to 10% SDS-PAGE. To visualize cleavage products, gels were either stained with Coomassie
Blue or transferred to PVDF and detected with anti-FVaA3, anti-FVaL,
anti-FVaH, or anti-human FV.
FVa Proteolysis by Pn in a Clot--
To determine whether
Pn-cleaved FVa fragments observed in a purified system could also be
identified in a physiological setting, FVa fragmentation in a lysing
clot was studied. In these experiments, pooled plasma (2.5 µl of
pooled plasma) was clotted by treatment with thromboplastin (80 µg/µl) after recalcification (10 mM CaCl2). Individual reactions were incubated for various times with Pn (50 nM) or tPA (0.5 nM) at 37 °C in a 7.5-µl
final volume of HBS. The reactions were stopped by SDS, run on 10%
SDS-PAGE, transferred to PVDF, and visualized by Western blotting with
anti-FVaL and anti-FVaH antibodies. To resolve better the fragments of
interest a concentration of acrylamide was used that in most cases does not allow the single chain FV to enter the gel (10%).
Amino Acid Sequencing--
NH2-terminal sequencing
of Pn-mediated FVa fragments transferred to PVDF after reducing
SDS-PAGE was conducted as described (30, 31). To sequence exclusively
the FVa-derived fragments remaining bound to aPL in the presence of
Ca2+ or EDTA, FVa digestions were conducted in the presence
of LV, and bound fractions were separated by centrifugation prior to electrophoresis. Individual bands were excised for sequence analysis.
Effect of EDTA on Dissociation of Pn-cleaved FVa
Fragments--
FVa (0.1 µM) was cleaved with Pn (0.1 µM) for selected time points on aPL-coated wells as
described above except that an excess amount of aprotinin (50 kallikrein-inactivation units/ml; KIU/ml) was added to inhibit residual
Pn activity. aPL-bound fragments were incubated at 22 °C for 3 h in HBS/Ca2+ or in HBS with EDTA (10 mM,
HBS/EDTA) to allow for dissociation of
Ca2+-dependent interactions. Protein samples
were subjected to 12% SDS-PAGE, transferred to PVDF, and visualized
with anti-FVaH and anti-FVaL. To determine whether Pn remains
associated with the cleaved fragments, antiserum to human Pgn (30 ng/ml) was used on identical membranes.
FVa Subunit Dissociation Kinetics--
The 30-kDa fragment and
FVaH dissociation with EDTA (10 mM) was monitored
electrophoretically. FVa bound to aPL-coated wells was cleaved with Pn
(0.1 µM) for 1 h, and Pn proteolytic activity was
inhibited with aprotinin (50 KIU/ml). Wells were washed three times,
and 200 µl of HBS/EDTA (10 mM) was added for selected
time points. Dissociation of FVaH due to Ca2+ chelation was
monitored after SDS-PAGE by Western blotting. In a parallel set of
experiments, FVa cofactor activity for thrombin generation was assessed
chromogenically (0.5 µM S2238). Following the last wash,
factor Xa (0.06 µM) and prothrombin (1.0 µM) in 100 µl HBS/Ca2+ were added at
22 °C for 5 min, and the change in absorbance at 405 nm was
monitored using a Vmax spectrophotometer
(Molecular Devices).
Ca2+-dependent Association of the 30-kDa
Fragment with FVaL--
To determine whether the 30-kDa fragment of
FVaH can associate with FVaL, we had to initially obtain and
concentrate sufficient amounts of the 30-kDa fragment. An aPL-coated
plate was coated with FVa (300 ng/well) that was then cleaved with Pn
(0.1 µM). Supernatants from all the wells (19 ml) were
pooled, and the mixture was concentrated by repeated cycles of
centrifugation on a Biomax-10 membrane (Millipore) to ~2 ml. To
determine whether the 30-kDa fragment can associate with intact FVaL,
FVaH was separated from aPL-immobilized FVaL with EDTA for 3 h at
22 °C. The 30-kDa fragment was then incubated in
HBS/Ca2+ or HBS/EDTA with FVaL remaining bound to the
aPL-coated well at 22 °C for 2 h and then at 37 °C for 30 min. The wells were washed, and remaining protein was eluted with
sample buffer. Association of the 30-kDa fragment was evaluated with
anti FVaH by Western blotting.
Inactivation of FV/FVR506Q and
FVa/FVaR506Q by Pn--
Pooled normal plasma or R506Q
homozygous plasma in HBS/EDTA was added to aPL-coated wells and was
incubated for 30 min at 22 °C. The wells were washed three times
with HBS/EDTA and then once with HBS/Ca2+. In one set of
experiments, wells were incubated with 50 µl of HBS/PEG/Ca2+ for 5 min with gentle shaking. In another set
of experiments, human FXa (5 nM) in
HBS/PEG/Ca2+ was added to the wells and incubated for 5 min
to preactivate the FV. The wells were then washed three times, and Pn
(0.15 µM) was added in HBS/PEG/Ca2+ for
various times. Following three more washes in HBS/Ca2+, FXa
(1.0 nM) and prothrombin (1.0 µM) were added
to the wells and incubated for 3 min. Thrombin activity was assessed
chromogenically with S2238 (0.4 mM final).
FVa Cleavage by Pn--
To identify fragments that are generated
during inactivation of FVa by Pn in the presence of SUV and
Ca2+, SDS-PAGE was conducted, which revealed a sequential
cleavage pattern (Fig. 1). Both the
104-kDa FVaH and the 74/71-kDa doublet of FVaL were rapidly cleaved in
a Pn-dependent manner within 1 h into products of
~50, 48, 45, 40, 34, 30, 12, and 7 kDa, along with several smaller
fragments that migrated close to or at the dye front. To determine
whether the digestion profile was aPL-dependent, experiments were performed in the absence of aPL vesicles but in the
presence of Ca2+. It was found that the rate of digestion
of the light chain was accelerated in the presence of aPL, whereas the
rate of digestion of the heavy chain was not altered (not shown).
Aprotinin completely inhibited the cleavage of FVa by Pn. No
distinction in the size of fragments generated was observed when the
digestion was performed in the absence or presence of aPL, although
additional cleavages were observed when the interaction between FVaL
and FVaH was inhibited by chelation (not shown).
Correlation between Pn-mediated FVa Fragments Remaining Bound to
aPL and Loss of Cofactor Activity--
To determine which fragments
remained noncovalently associated with aPL in the presence of
Ca2+, FVa was equilibrated with aPL-coated microtiter
wells, and fragments that remained bound to the plate after digestion
with Pn were washed with buffer containing Ca2+. The
remaining fragments were analyzed by Western blot using an FVaH- and
two FVaL-specific mAb. These data showed that the fragments of 50, 48, 43, 40, and 30 kDa were light chain-derived (Fig.
2, A and B). Since
the mAb in Fig. 2B recognizes the A3 domain, the completely
different fragmentation pattern suggests the commercial mAb used in
Fig. 2A has specificity for the COOH-terminal part of FVaL
containing C1/C2. As shown, the 50- and 48-kDa FVaL fragments were
converted to 43 and 40 kDa, and all remained bound to aPL. Since all
aPL binding of FVa is mediated by FVaL, these fragments contain at
least part of the Ca2+-independent membrane-binding
site(s). Furthermore, we found that fragments of 50, 45, and 30 kDa
were FVaH-derived and remained bound in the presence of
Ca2+ (Fig. 2C). The 30-kDa FVaH-derived fragment
also remained at least partly bound over the time course of the
experiment. Comparable Ca2+-dependent complex
of fragments was observed when beginning with bovine FVa (not shown).
We did not observe a detectable difference in the rate of cleavage or
the cleavage pattern, between experiments performed on aPL-coated
plates, LV, or SUV (not shown). Furthermore, no detectable difference
in the cleavage pattern was observed when the samples were
electrophoresed under nonreducing conditions, indicating no
disulfide-linked fragments were produced (not shown). Whereas all the
aPL-bound bands, visualized by Coomassie Blue and Silver and polyclonal
staining, were detected with the mAbs (not shown), an additional
unbound 34-kDa fragment, released into solution upon cleavage, was
observed with Coomassie Blue (Fig. 1).
In order to correlate the appearance of degradation products with
observed changes in FVa cofactor activity, fragments remaining bound to
aPL were assayed for FVa function. Inactivation of FVa by Pn was rapid
over the time course of the experiment (Fig. 2D). Following
8 min of incubation with Pn, aPL-bound FVa was completely inactive. The
rapid loss of cofactor activity coincided with appearance of the 30-kDa
A3 and the 48- and minor 50-kDa C1/C2-derived fragments and with the
45- and minor 50-kDa heavy chain-derived fragments.
Cleavage of FV and FVa by Pn in a Fibrin Clot--
By having
established the fragment composition of purified Pn-inactivated FVa, we
determined whether these fragments were also generated under more
physiological conditions. Thromboplastin was added to plasma to
generate a fibrin clot, and the FV/FVa fragmentation pattern was
examined following either Pn- or tPA-induced clot dissolution. Western
blots (Fig. 3) showed that the FVaH- and
FVaL-derived fragmentation pattern was identical to those in our
purified system regardless of whether Pn or tPA was used to induce clot
fibrinolysis. These data support the physiological relevance of the
purified system.
Identification of Pn Cleavage Sites in FVa--
To identify the
specific sites of Pn-mediated cleavage, amino acid sequence analyses
were conducted on all Coomassie Blue-stained fragments derived from
purified FVa. Fig. 4A shows
the banding pattern of the fragments subjected to analysis, and the
sequences are summarized in Fig. 4B. As expected, because of
recognition by a single mAb, the 65-, 50-, 45-, and 30-kDa FVaH
fragments overlap, and each contains at least a large portion of the A1 domain (starting at Leu-94). The 30-kDa FVaH band was found to be two
co-migrating species, which differed by 16 amino acids starting at
residues Leu-94 or Met-110 and hence were referred to as 30(L94) and
30(M110). The 34-kDa fragment not detected by the mAbs corresponded to
the COOH-terminal end of FVaH and, unexpectedly, contained a conserved
polymorphism at residue 352 (Leu to Thr) (Fig. 4B). This is
the first report to our knowledge of this substitution, which is
consistent with FVa being purified from pooled plasma. Once generated,
the 34-kDa fragment was quickly proteolyzed into fragments of less than
7 kDa, of which several were sequenced. FVaL is cleaved by Pn to give
50-, 48-, 43-, and 40-kDa fragments that overlap as predicted by
antigenicity and begin toward the COOH-terminal end of A3. By this
method, a 30-kDa FVaL fragment corresponding to the
NH2-terminal end of the light chain was distinguishable. This fragment was short lived and was processed into several smaller fragments of which two were sequenced.
Ca2+-dependent Association of Pn-mediated
FVa Fragments--
Since FVaH does not contain the aPL-binding site
and is anchored to aPL through divalent metal ion-dependent
interactions with FVaL, we investigated the effect of EDTA on the FVa
fragments remaining bound to aPL. Digestion end point fragments that
remained bound to aPL-coated wells after cleavage of FVa with Pn were
subjected to an additional wash containing 10 mM EDTA.
Interestingly, the 30-kDa component of FVaH and the 50- and 40-kDa
FVaL-derived fragments dissociated from the aPL in the presence of EDTA
(Fig. 5). Conversely, the FVaL 48- and
43-kDa fragments remained associated with aPL following EDTA treatment.
Collectively, these data suggest that regions within the 48/43-kDa
fragments of FVaL and the 30-kDa fragment of FVaH contribute to the
Ca2+-dependent intramolecular association
between FVaL and FVaH. It is unknown at present why apparent
Ca2+-dependent differences exist between the
48/43- and 50/40-kDa fragments.
To determine if the rate of EDTA-mediated dissociation of the
FVaH-derived 30-kDa species from FVaL fragments is representative of
dissociation of intact FVaH from intact FVaL, a time course was
conducted (Fig. 6). Release of the 30-kDa
species (Fig. 6A) or FVaH (Fig. 6B) from
aPL-coated microtiter wells by EDTA was followed by Western blot.
Although the general trend was comparable over the duration of the
experiment, intact FVaH has an apparently faster initial phase of
dissociation than the 30-kDa fragment. This may be indicative of a role
for A2 in FVaL binding. As an additional means of following FVaL and
FVaH dissociation, the effect of EDTA on FVa function was followed by
measuring the loss of thrombin generation in a chromogenic assay (Fig.
6C). As expected, EDTA-mediated inhibition of FVa activity
correlated with the dissociation of the 30-kDa fragment or intact FVaH
from FVaL and are in agreement with the slow dissociation rates
reported previously (8, 12). These results demonstrate that the
Ca2+-dependent interactions observed for
FVaPn are representative of intact FVa.
The dependence of Ca2+ on the association of the 30-kDa
FVaH fragment to intact FVaL was studied to investigate further whether the association we observed between Pn-mediated fragments of FVa reflect those within the intact molecule. aPL-coated microtiter wells
were saturated with FVa and then treated with EDTA to dissociate FVaH,
which was confirmed antigenically. The 30-kDa species of FVaH was then
added in the presence of Ca2+, resulting in association
specifically with intact FVaL (Fig. 6C, inset). Binding was
found to be Ca2+-dependent since no detectable
association was observed in the presence of EDTA. Significant
Ca2+-dependent binding of the 30-kDa fragment
to aPL-coated wells depended on the presence of FVaL (not shown). These
data provide additional evidence that the 30-kDa fragment constitutes
the FVaH-derived portion of a Ca2+-dependent
contact region with FVaL.
Identification of aPL-bound Fragments following Pn
Cleavage--
To identify by sequence analysis the species that
remained bound to aPL in the presence or absence of Ca2+,
LV were utilized as a preparative affinity matrix. In this experiment, aPL-bound FVa was pelleted by centrifugation after Pn treatment. As
before, the 65- and 30-kDa FVaH-derived fragments and the 50-, 48-, 43-, and 40-kDa FVaL fragments were observed to remain bound to aPL in
the presence of Ca2+. Of these only the 48- and 43-kDa
fragments remained bound in EDTA (Fig.
7A). Sequence analyses (Fig.
7B) confirmed their identification. An unexpected
observation was made concerning the co-migrating FVaH-derived 30-kDa
fragments we identified in Fig. 4. After affinity fractionation,
sequencing revealed that 30(L94) remained bound to aPL. In contrast,
the similarly sized fragment starting at Met-110 was not observed to
remain bound in the presence of Ca2+, suggesting that the
94-109-kDa region is important for the
Ca2+-sensitive intrachain contact.
Loss of FVa and FVaR506Q Cofactor Activity by
Pn--
To determine whether Pn-dependent inactivation of
FV/FVa was affected by the common "Leiden" thrombophilic mutation,
normal or R506Q FV was trapped from plasma on aPL-coated microtiter
wells and cleaved by Pn. The activity profile of FV, as measured by thrombin generation in the presence of FXa, is shown in Fig.
8A. Pn treatment resulted in
an initial activation and then inactivation. We observed that this
change in FV activity by Pn was indistinguishable from that of
FVR506Q. Incubation of FXa-preactivated FV with Pn resulted
in an immediate loss of thrombin generating activity (Fig.
8B). An indistinguishable inactivation profile was detected
when FVR506Q was used in these experiments. Collectively,
these results clearly indicate that Pn-mediated activation/inactivation
of FV and inactivation of FVa is independent of the R506Q
substitution.
For over a decade, Pn has been known to induce a complicated
cleavage pattern in FVa that causes complete inhibition of coagulation cofactor function. To understand how this functional effect is achieved, we have identified the location of the cleavage sites and
present here a fragmentation map (Fig.
9A). This revealed that the A2
and A3 domains of FVa are proteolyzed at several sites resulting in
their dissociation, which correlates to inactivation of the molecule.
This process is analogous to the A2 dissociation correlated to the
inactivation of FVa by APC (15) and for the spontaneous decay of FVIIIa
activity (32). However, the loss of A3 from aPL-bound FVa as part of an
inhibition mechanism is novel. Since FXa also cleaves FVa at Arg-348
and Arg-1765 without apparent loss of FVa coagulation cofactor activity
(14), our results demonstrate that additional cleavages within these
fragments and at Lys-1827 ultimately result in the observed complete
loss of FVa activity. Cleavage of FVaH by Pn appears to occur by two cleavage pathways (Fig. 9B) that ultimately lead to the loss
of the A2 domain and the generation of a 30-kDa doublet. In the major cleavage pathway, the 30-kDa species is generated from a prominent 45-kDa NH2-terminal fragment. In the minor cleavage
pathway, the 30-kDa fragment is generated from a 50-kDa intermediate
NH2-terminal fragment that is produced from a 65-kDa
fragment. The COOH-terminal fragments from both pathways are quickly
proteolyzed to smaller fragments that migrate close to or at the dye
front (<5 kDa). In contrast, the 30-kDa fragment was observed to
persist throughout the time of the experiment.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9 M) (8), and both chains are
essential for full biological activity (9, 10). Addition of a chelating
agent such as EDTA disrupts this interaction (9-11) which can then be
regained by recalcification (8, 10, 12, 13) resulting in the expression of a single Ca2+-binding site (Kd = 24 × 10
6 M) (13).
Ca2+ binds to neither purified FVaH nor FVaL (13),
suggesting the Ca2+-binding pocket is conformational or
formed by both FVaH and FVaL. Localization of the
Ca2+-binding site has been consequently elusive.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course of Pn cleavage of FVa bound to
SUV. Purified human FVa (0.1 µM) was incubated with
Pn (0.1 µM) in the presence of aPL-containing SUV (250 µM) in HBS/Ca2+ at 22 °C. At selected time
intervals aliquots were withdrawn from the mixture and stopped
immediately with SDS-containing sample buffer. Samples were separated
on 10% SDS-PAGE and stained with Coomassie Blue. Incubation times
(min) with Pn were as follows: 0 (lane 1), 1 (lane
2), 2 (lane 3), 4 (lane 4), 6 (lane
5), 8 (lane 6), 15 (lane 7), 30 (lane
8), 40 (lane 9), and 60 (lane 10).
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Fig. 2.
Time course of Pn cleavage of FVa subunits
bound to aPL-coated wells. 96-Well microtiter wells were coated
with aPL (0.3 µg) and then blocked with gelatin (5 mg/ml). The wells
were preincubated with FVa (0.1 µM) for 30 min and then
with Pn (0.1 µM) in HBS/Ca2+. At the selected
time points, aPL-bound protein was removed from the wells with sample
buffer and separated on 10% SDS-PAGE. The protein was transferred to
PVDF and detected with anti-FVaL (A), anti-FVaA3
(B), or anti-FVaH (C). D, as in
A, except that following incubation with Pn, FXa (0.06 µM), and prothrombin (1.0 µM) were added.
Thrombin generation was monitored with S2238. The average of four
experiments with standard deviation is shown. Incubation times (min)
with Pn were as follows: 0 (lane 1), 1 (lane 2),
2 (lane 3), 4 (lane 4), 6 (lane 5), 8 (lane 6), 15 (lane 7), and 30 (lane
8), 40 (lane 9), and 60 (lane 10).
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Fig. 3.
Pn-cleaved FVa fragments in a lysing
clot. Microcentrifuge tubes containing recalcified plasma were
incubated with thromboplastin (80 µg/µl) in HBS at 37 °C for 3 min. Following clot formation, Pn (50 nM) or tPA (0.5 nM) was added to individual reactions for the indicated
time points. The reactions were stopped with sample buffer, and the
protein was separated on 10% SDS-PAGE, transferred to a PVDF membrane,
and detected with anti-FVaL (top panels) or anti-FVaH
(bottom panels). Pooled plasma (lane 1)
incubation times (min) with Pn or tPA were as follows: 2 (lane
2), 15 (lane 3), 30 (lane 4), 60 (lane
5), 120 (lane 6), and 240 (lane 7).
View larger version (38K):
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Fig. 4.
Identification of proteolytic cleavages in
FVa by Pn. FVa (1.5 µM) was preincubated with aPL
vesicles (300 µM) in HBS/Ca2+ for 15 min
prior to cleavage by Pn (0.2 µM) at 22 °C for 1 h. The reaction was then stopped with SDS-containing sample buffer,
heated at 95 °C for 5 min, and separated on large (12 × 14 cm)
8% SDS-PAGE. A, protein was transferred to PVDF membrane
and stained with Coomassie Blue R-250 stain. B,
NH2-terminal sequence of visible FVa fragments shown in
A.
View larger version (42K):
[in a new window]
Fig. 5.
Effect of EDTA on aPL-associated FVa
fragments. FVa was treated with Pn (as in Fig. 2, 60 min), except
that after incubating with Pn, Pn activity was inhibited with aprotinin
(50 KIU/ml) in HBS/Ca2+ for 15 min. Wells were then
incubated in HBS/Ca2+ (lane 1) or HBS/EDTA
(lane 3) at 22 °C for 2 h. In lane 2, Pn
was not added to the well prior to the addition of HBS/EDTA. Protein
samples were separated on SDS-PAGE and transferred to PVDF. Western
blot analysis was performed with a mixture of anti-FVaL and
anti-FVaH.
View larger version (39K):
[in a new window]
Fig. 6.
EDTA-induced dissociation of
FVaPn and FVa subunits and association of the 30-kDa
species with FVaL. A, FVa was treated
with Pn (as in Fig. 2, 60 min). After a brief wash with
HBS/Ca2+, the wells were incubated with HBS/EDTA for the
indicated times, and the 30-kDa FVaH fragment remaining bound to the
aPL-coated well was detected by Western blotting. B, as in
A except no Pn was included. C, as in
B, except that following incubation with EDTA, factor Xa
(0.06 µM), prothrombin (1.0 µM), and SUV
(20 µM) were added. Thrombin generation was monitored
with S2238. The average of triplicates with standard deviation is
shown. Inset, as in B, except that following
incubation with EDTA, Western blots were conducted to detect
association of the 30-kDa fragment. Lane 1, no 30-kDa
fragment added; lane 2, the 30-kDa fragment was added in the
presence of HBS/Ca2+; lane 3, the 30-kDa
fragment was added in HBS/EDTA.
View larger version (34K):
[in a new window]
Fig. 7.
Identification of the Pn-cleaved FVa
fragments that dissociate in the absence of Ca2+.
A, FVa (1.5 µM) was incubated with LV (600 µM) in HBS/Ca2+ and Pn (0.2 µM)
as above. Proteolysis was stopped with aprotinin (50 KIU/ml). Following
3 h of incubation with EDTA, LV-bound fragments were separated by
centrifugation. The pellet fraction (lane 1) and the
supernatant fraction (lane 2) were then run on large
(12 × 14 cm) 10% SDS-PAGE, transferred to PVDF, and stained with
Coomassie Blue R-250 stain. B, NH2-terminal
sequence of each fragment.
View larger version (18K):
[in a new window]
Fig. 8.
Effect of Pn on plasma FV/FVa and
FVR506Q/FVaR506Q prothrombinase activity.
Pooled plasma or R506Q plasma was added to aPL-coated wells for 30 min.
A, wells were incubated in HBS/PEG/Ca2+.
B, human FXa (5 nM) was added for 5 min and
washed. Pn (0.15 µM) was added at various times,
inhibited with aprotinin, and then followed by FXa (1.0 nM)
and prothrombin (1.0 µM). Thrombin activity was measured
using S2238.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 9.
Cleavage of FVa by Pn in the presence of
Ca2+. A, Pn-mediated fragmentation map of
FVa was constructed to scale based on NH2-terminal sequence
and the apparent molecular weight of fragments which was relativized to
the average molecular weight of an amino acid (110 Da). The estimated
fragment number of amino acids in each is complicated by glycosylation
not included in the estimate of fragment length. Disulfide linkages are
indicated by thin lines. B, probable order of cleavage is
shown. The predicted minor cleavage pathway is depicted by dotted
arrows.
Cleavage of FVaL with Pn resulted in the rapid proteolysis of the 30-kDa (A3 domain) NH2-terminal fragment ultimately leading to dissociation of the A3 domain from the aPL-bound protein and hence the conversion of the 74/71-kDa FVaL into the 40- and 43-kDa COOH-terminal fragments. We suggest that the 50- and 48-kDa fragments are intermediates of the 40- and 43-kDa fragments, respectively, because of similar Ca2+-dependent aPL-binding properties of the 50/40- and 48/43-kDa species.
To our knowledge, this study is the first to describe the Pn cleavage sites within human FVa, although in a preliminary report Kalafatis and Mann (33) presented NH2-terminal amino acid sequence of Pn-cleaved fragments from bovine FVa. The identity of some of these species differed from those presented here for human FVa, with cleavages at Arg-348, Lys-1656, and Arg-1765 being in agreement.
To determine if the Pn-derived FVa fragments we observed in purified systems can occur in a physiological setting, we tested whether these fragments are generated following either Pn or tPA treatment of plasma-derived clot. The data showed that the 30-kDa fragment of FVaH and the four light chain-derived fragments are generated in clots lysed by either Pn or tPA. Together, these results support the hypothesis that at the site of clot formation, where the local concentration of the coagulation cofactor is elevated, Pn processing of FVa may have a physiological function. In further support of physiological relevance, Tracey et al. (34) previously reported the extent of FV proteolysis during thrombolytic therapy with tPA and found that cleavage of FV is primarily Pn-mediated. As an extension of this report, we found no detectable difference between fragments that remain bound to aPL following Pn treatment of FV or FVa (not shown). Independent observations of a FV fragmentation pattern in clots similar to ours have been made (35) where the authors speculated a Pn-dependent origin. We cannot, however, omit the possibility that cell-dependent processes may influence the cleavage profile, such as the requirement of cell-surface thrombomodulin for APC function or cathepsin G elastase from monocytes (36).
The contribution of aPL to the assembly of prothrombinase components, and to the localization of thrombin production to the site of injury, has been well documented (37-42). Furthermore, it has been shown that FVaL confers all of the aPL-binding function of FVa (11, 43-47) and that this interaction is Ca2+-independent (11, 46, 48). Several studies suggest that the C2 domain contains the aPL-binding region of FVaL (49, 50), whereas others have implicated the A3 (45) or both the A3 and C2 domains (47). By having identified the cleavages induced by Pn in FVa, we determined which fragments remained bound to aPL. Upon cleavage of FVa by Pn, four FVaL-derived fragments and one FVaH-derived fragment remained bound to aPL in the presence of Ca2+. Amino acid sequencing and antigenicity revealed that the four FVaL-derived species overlap, with the 50/48- and the 43/40-kDa fragments originating from a common cleavage site at Arg-1765 and Lys-1827, respectively. Each of these excludes a large portion of the A3 domain and includes the entire C2 domain, providing support for C2 function in aPL binding.
Of the four FVaL fragments that remained bound to aPL, only the 48- and 43-kDa fragments remained in the presence of EDTA. Since these are encompassed by the 50-kDa fragment and span the 40-kDa fragment, both of which dissociated from aPL due to chelation, an explanation is difficult to provide without further study. One possibility is related to the two forms of FVa that have been identified, designated FVa1 and FVa2, that differ in the COOH terminus of FVaL (51, 52). The difference has been suggested to be due to alternative glycosylation at Asp-2181 within the C2 domain (49, 53, 54) leading to a weaker association between FVa1 and aPL (51, 52, 55). Since we found that the 48-kDa fragment associated strongly with aPL, it is possible that the difference in the electrophoretic mobility between the 50- and 48-kDa species is due to derivation from FVa1 and FVa2, respectively. At this time we cannot exclude alternate processing at the COOH terminus by Pn resulting in the weaker aPL-binding 40-kDa fragment originating from the heavier 50-kDa species. An additional explanation for the different aPL-binding properties of overlapping fragments may be found in the two forms of the C2 domain recently observed by x-ray crystallography (56). These differ markedly in one of the three protruding hydrophobic loops implicated in aPL binding. Possibly, the Pn-mediated 50- and 40-kDa fragments originate from the FVaL type suggested by crystallography to have weaker aPL-binding. Notwithstanding, our observations provide further support for a role of the C2 domain in Ca2+-independent aPL binding.
It is well established that EDTA-dissociated FVaL and FVaH chains can
reassociate in the presence of additional Ca2+
(Kd = 6 × 109
M) (8). Although Ca2+ does not bind to either
of the individual FVa chains alone, it has been shown that a single
high affinity binding site becomes available when the two chains are
bound together (Kd = 24 × 10
6 M) (13). The first
experimental evidence localizing the Ca2+-sensitive
intrachain contact comes from our identification of two FVaH fragments
with identical electrophoretic mobility. These differ at the
NH2 terminus by only 16 amino acids. Of these, the fragment
beginning at Leu-94 remained associated with FVaL-derived fragments in
a Ca2+-dependent manner, whereas the similar
species beginning at Met-110 did not interact with FVaL even in the
presence of Ca2+. These data suggest that a
Ca2+-sensitive site may be contained between Leu-94 and
Lys-109. Consistent with this hypothesis, x-ray structure and molecular
modeling, using homologous protein templates based on the ceruloplasmin and factor VIII A-type domains, have suggested a putative
Ca2+ ion-binding site within the A1-A3 interface (57, 58).
Interestingly, of the five negatively charged residues (Glu-96,
Asp-102, Glu-108, Asp-111, and Asp-112) predicted to be involved in the
Ca2+-binding pocket in the A1 domain (57), three residues
are located between amino acids Leu-94 and Lys-109, and the remainder
are neighboring. Although molecular modeling supports our finding that
a Ca2+-sensitive region between Leu-94 and Lys-109 may
exist, direct studies must be conducted to substantiate this likelihood.
To determine whether the observed Ca2+-dependent association between the fragments of Pn-cleaved FVa is representative of the intact FVaH/FVaL interaction, we demonstrated that the rates of EDTA-induced dissociation of the 30(L94) species from FVaPn and FVaH from intact FVa were comparable. Furthermore, the dissociation rate closely resembled the timing of FVa inactivation by EDTA. To demonstrate further the contribution of the region within FVaH corresponding to the 30-kDa fragment in the FVaL interaction, we followed the 30-kDa fragment association with aPL-bound intact FVaL. Our results demonstrate that the 30(L94) fragment can indeed associate with FVaL. Binding to the microtiter well was dependent on Ca2+, FVaL, and aPL. This observation is further evidence that the interaction between FVaH and FVaL is mediated at least in part through Leu-94 to Lys-345 within FVaH and Gln-1828 to Tyr-2196 (COOH-terminal half; i.e. C1/C2) in FVaL. Since EDTA is a nonselective chelator, we cannot exclude the role of other metal ions (10, 59) in this process in vivo; however, based on previous observations (10) and our data showing that EDTA-mediated dissociation of the 30(L94) fragment from FVaL is restored by recalcification, Ca2+ is strongly implicated.
In addition to the inhibitory function Pn has on FVa coagulant activity
(16, 17), previous data from our laboratory has also suggested that Pn
may convert FVa into a tPA accelerator (25). To determine whether these
combined regulatory roles could be applied to a prevalent thrombotic
risk factor, the effect of the R506Q "Leiden" mutation on
Pn-mediated inactivation of FV/Va was studied. Our results showed that
the rate of inactivation of FVR506Q/FVaR506Q by
Pn is indistinguishable from normal FV/FVa, although the COOH termini
of several small FVaH fragments we identified were predicted to be
close to Arg-506. The R506Q mutation is known to inhibit the normal
anticoagulant effects of APC on FV/Va (60). Therefore, the data
presented here suggest that the essential physiological role of APC
highlighted in thrombosis associated with FVaR506Q could be
attenuated by administration of thrombolytics.
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ACKNOWLEDGEMENTS |
---|
We thank N. Lavigne for excellent technical assistance; T. Raynor and M. Derry for review of the manuscript; Dr. M. Issa (Canadian Blood Services, University of British Columbia) for sizing the LV; Dr. M. C. Poon (University of Calgary) for providing R506Q plasma; and S. Smith and Dr. A. Kurosky (Protein Chemistry Laboratory, University of Texas, Galveston Medical Branch) for expert amino acid sequence analysis. We thank Dr. J. Grundy for helpful suggestions regarding the manuscript and experimental design and for providing LV. We also thank Dr. R. Lemieux (Hema-Quebec) and Bogna Lasia for mAb production and purification, respectively.
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FOOTNOTES |
---|
* This work was supported by the Canadian Blood Services Research and Development Fund Grant CB50 928 and the Heart and Stroke Foundation of Canada Grant NA 4143.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: Canadian Blood
Services, Research and Development Department, 1800 Alta Vista Dr.,
Ottawa, Ontario K1G 4J5, Canada. Tel.: 613-739-2201; Fax: 613-739-2426;
E-mail: ed.pryzdial@bloodservices.ca.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M004711200
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ABBREVIATIONS |
---|
The abbreviations used are: FV, factor V; FVa, factor Va; Pn, plasmin; tPA, tissue plasminogen activator; APC, activated protein C; PC/PS, phosphatidylcholine/phosphatidylserine; FVaPn, Pn-cleaved FVa; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; LV, large vesicles; PVDF, polyvinylidene fluoride; mAb, monoclonal antibody; SUV, small unilamellar vesicles; KIU, kallikrein-inactivation units.
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