(Received for publication, December 16, 1994)
From the
A poor anticoagulant response of plasma to activated protein C
is correlated with a single mutation in the factor V molecule
(Arg
Gln). Factor V was purified to homogeneity
from plasma of two unrelated patients (patient I, factor V
,
and patient II, factor V
), who are homozygous for this
mutation. The factor V molecule from both patients has normal
procoagulant activity when compared with factor V isolated from normal
plasma in both a clotting time-based assay and in an assay measuring
-thrombin formation. The cleavage and subsequent inactivation by
activated protein C (APC) of the
-thrombin-activated
membrane-bound cofactor (factor Va) from both patients were analyzed
and compared with the cleavage and inactivation of normal human factor
Va. In normal factor Va, cleavage at Arg
generates a M
= 75,000 fragment and a M
= 28,000/26,000 doublet and is necessary for the optimum
exposure of the sites for subsequent cleavage at Arg
and
Arg
. Proteolysis at these sites leads to the appearance
of M
= 45,000 and 30,000 fragments and a M
= 22,000/20,000 doublet. Cleavage at
Arg
is membrane-dependent and is required for complete
inactivation. Following 5 min of incubation with APC (5.4 nM)
membrane-bound normal factor Va (280 nM) has virtually no
cofactor activity whereas under similar experimental conditions factor
Va
and factor Va
retain approximately 50% of
their initial activity. After 1 h of incubation with APC, factor
Va
retains 20% of its initial cofactor activity whereas
factor Va
has 10% remaining cofactor activity. The initial
loss in cofactor activity (
70%) of membrane-bound factor Va
and factor Va
during the first 10 min of the
inactivation reaction is correlated with cleavage at Arg
and appearance of a M
= 45,000
fragment and a M
= 62,000/60,000 doublet.
Subsequently, the M
= 62,000/60,000 doublet
is cleaved at Arg
to generate a M
= 56,000/54,000 doublet resulting in complete loss of
cofactor activity. Both procofactors, factor V
and factor
V
, were inactivated following cleavage at Arg
and Arg
, with APC inactivation rates equivalent to
those observed for normal factor V. Our data demonstrate that: 1)
cleavage at Arg
is required for optimum exposure of the
cleavage sites at Arg
and Arg
and rapid
inactivation of membrane-bound factor Va; and 2) cleavage at
Arg
by APC on membrane-bound factor V occurs at the same
rate in both normal and APC-resistant individuals. Thus cleavage at
Arg
and Arg
and subsequent inactivation of
the membrane-bound procofactor, factor V, does not require prior
cleavage at Arg
for optimum exposure.
The generation of -thrombin is a central event of the blood
coagulation process. This enzyme is generated by the catalyst prothrombinase, which is composed of factor Xa and its
cofactor factor Va associated on a membrane surface in the presence of
Ca
. Factor V is a single chain procofactor molecule
of M
= 330,000, which is activated during
blood clotting to factor Va, by factor Xa and/or
-thrombin(1) . The active cofactor, factor Va, purified
from human plasma, is composed of a heavy chain (M
= 105,000) containing the NH
-terminal part of
the procofactor (residues 1-709, A1-A2 domains) and a light chain (M
= 74,000) containing the COOH-terminal
part of the factor V molecule (amino acids 1546-2196, A3-C1-C2
domains)(1, 2, 3) , which are non-covalently
associated. Factor Va is inactivated by proteolysis of the heavy chain
by activated protein C
(APC)(
)(4, 5, 6, 7) . The
mechanism of inactivation is an ordered and sequential event with
cleavage at Arg
necessary for optimum exposure of the
inactivating cleavage sites at Arg
and
Arg
(6) . Cleavage at Arg
occurs
only on the membrane-bound cofactor(6, 7) .
Resistance to APC is defined as a poor anticoagulant response of
plasma to APC in a clotting assay like-activated partial thromboplastin
time(8) . It has been recently reported that APC resistance is
the most common identifiable defect among patients with deep venous
thrombosis (8, 9, 10) . The abnormal response
to APC has been linked to a plasma protein, which was identified as
factor V because when purified human plasma factor V at 4 times the
normal plasma concentration (76 nM) was added to the plasma
from a patient with severe APC resistance, the patient's plasma
recovered its APC sensitivity(11) . It was thus concluded that
factor V acts as a cofactor for the APC anticoagulant
pathway(11) . The molecular defect in APC-resistant patients
was recently identified in several laboratories as a single point
mutation at nucleotide 1691 in the factor V gene (G A
substitution) predicting a single amino acid substitution in the factor
V molecule, Arg
Gln(12, 13, 14, 15, 16) .
Thus, the bulk of data demonstrate that APC resistance is due to an
abnormal factor V molecule rather than to a deficiency of a cofactor
for APC as initially proposed (11) . However, the possibility
that the mutant factor V is defective with respect to the APC cofactor
activity of the protein S/factor V mixture during factor VIIIa
inactivation (17) cannot yet be excluded. The present study was
undertaken in order to examine the APC-mediated inactivation of the
factor V molecule that does not possess a cleavage site for APC at
amino acid residue 506.
All gels
were produced according to the method described by Laemmli (26) and stained with Coomassie Blue. In some experiments the
proteins were transferred to nitrocellulose as described (27) and probed with the monoclonal antibody
HFVa
#6 as recently detailed (28) using the
chemiluminescent substrate Luminol. NH
-terminal sequencing
of the patient's factor V and factor Va fragments from
polyvinylidene difluoride membranes was performed as described (7) in the laboratory of Dr. Alex Kurosky (University of Texas,
Medical Branch at Galveston, Galveston, TX).
Normal plasma factor V, factor V, and factor
V
in the presence of PC
PS vesicles demonstrate
similar cofactor activity (
750 nM IIa/min) following 15
min of incubation with
-thrombin. Following 5 min of incubation
with APC, normal factor Va lost 95% of the heavy chain and virtually
all of its cofactor activity ( Fig. 1and 2A). The pattern of
cofactor activity loss corresponds to cleavage at Arg
,
followed by cleavage at Arg
and
Arg
(6) . These cleavages result in the appearance
of fragments of M
= 45,000 and 30,000 and a
doublet of M
= 22,000/20,000 (Fig. 2A, where the M
= 75,000 fragment intermediate of the inactivation process (lanes5 and 6) and the M
= 30,000 fragment (lanes5-11) are detected). For
the patient's factor Va, under similar experimental conditions,
following 5 min of incubation of APC, the loss of approximately 90% of
the heavy chain corresponds to a 50% loss in cofactor activity (Fig. 1, inset; Fig. 3A, lanes
4-6; and Fig. 2B, lanes 5-7).
The complete disappearance of the heavy chain of the cofactor when
using patient's factor Va corresponds to cleavage at Arg
as determined by amino acid sequencing of the corresponding
fragments. Cleavage at that position results in the generation of a M
= 45,000 fragment and of a M
= 62,000/60,000 doublet (Fig. 3A, lanes 4-6). Subsequently,
complete loss in cofactor activity correlates with cleavage of the M
= 62,000/60,000 doublet at Arg
and generation of a M
= 56,000/54,000
doublet (Fig. 3A, lanes 7-13). The
position of the cleavage site at Arg
is deduced from our
previous findings (6) and from the fact that the fragments of M
= 62,000 and 56,000 have the same
NH
-terminal sequence starting at amino acid residue 307,
suggesting a precursor-product relationship. Thus, the difference
between the two fragments resides at the COOH terminus. These results
were confirmed using a monoclonal antibody directed against the heavy
chain of the cofactor that recognizes an epitope located between
residues 307-506 of the cofactor. In normal factor Va, during
inactivation by APC, this antibody recognizes the M
= 30,000 fragment (Fig. 2A), whereas when
studying factor Va
inactivation, the antibody recognizes
the M
= 62,000/60,000 and 56,000/54,000
doublets (which contain the region 307-709 and 307-679,
respectively, of the factor Va heavy chain) (Fig. 2B).
Thus, it must be concluded that cleavage only at Arg
is
responsible for the loss of approximately 30% in cofactor activity.
Thus, cleavage at Arg
is necessary for optimum exposure
of the cleavage sites at Arg
and Arg
, which
in turn are required for cofactor inactivation (Fig. 1, filled circles). In the absence of the cleavage site at
Arg
the rate of inactivation of factor Va is slower than
that observed for normal factor Va (Fig. 1, filled triangles and filled squares).
Figure 1:
Inactivation of
membrane-bound factor Va by APC. Normal plasma factor V, factor
V, and factor V
(0.28 µM) were
incubated with PC
PS vesicles (100 µM) for 5 min at
37 °C.
-Thrombin was added at 1 unit/ml (12 nM).
Following 15 min of incubation at 37 °C hirudin was added (20
nM). The activity of factor Va was monitored as described (7, 23) in the presence of 1.4 µM prothrombin, 3 µM dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, and 20
µM PC
PS vesicles, using factor Va and factor Xa at 1
and 10 nM, respectively (final concentrations). APC was then
added (5.4 nM). At selected time intervals aliquots were
assayed for cofactor activity. The initial rates of thrombin formation
were calculated and plotted as percent of initial cofactor activity as
a function of time following addition of APC. Filled circles represent membrane-bound normal plasma factor Va treated with APC;
the filled triangles depict membrane-bound factor Va
treated with APC, whereas the filled squares show
membrane-bound factor Va
inactivated by APC. The inset shows the analysis of the inactivation reaction during the first
10 min. At the same time intervals aliquots were withdrawn and analyzed
by SDS-PAGE. The products resulting from the proteolytic inactivation
of membrane-bound factor Va
(filled triangles)
are shown in Fig. 3A, whereas the comparison of the
immunoreactivity of the fragments resulting from the inactivation of
plasma-derived factor Va and factor Va
toward a monoclonal
antibody that recognizes an epitope between amino acids 307-506
is displayed in Fig. 2, A and B.
Figure 2:
Analysis of factor Va inactivation by APC.
The samples assayed for cofactor activity in Fig. 1(normal
plasma factor Va and factor Va) were also analyzed on a
4-15% linear gradient SDS-PAGE. Following transfer to
nitrocellulose fragments were visualized using monoclonal antibody
HFVa
#6. Panel A, normal factor Va control; panel B, factor Va
. Lane1,
factor V in the presence of PC
PS vesicles, no
-thrombin, and
no APC; lanes 2-4, factor V with
-thrombin at 2, 5,
and 15 min; lanes 5-11, membrane-bound factor Va with
APC at 1, 3, 5, 10, 15, 30, and 60 min. The position of the molecular
weight markers is indicated at left of panelA.
Figure 3:
Cleavage of membrane-bound factor
Va and factor V
by APC. Panel A, factor V
was incubated with PC
PS vesicles and
-thrombin as described in the legend to Fig. 1. Following
15 min of incubation, hirudin and APC were added. At selected time
intervals aliquots of the mixture, before and after addition of
-thrombin, were withdrawn and mixed with 2% SDS, 2%
-mercaptoethanol, heated at 90 °C, and analyzed on a
4-15% linear gradient SDS-PAGE gel. The factor Va subunits and
fragments were visualized by Coomassie Blue staining (the same aliquots
were also analyzed for cofactor activity and are shown in Fig. 1, filled triangles): lane1,
factor V
, control; lanes2 and 3, factor V
with
-thrombin at 5 and 10 min; lanes 4-13, factor Va
and APC at 30 s and
2, 4, 7, 10, 15, 30, 45, 60, and 120 min. Lane14,
factor V
in the presence of PC
PS vesicles following
2 h of incubation at 37 °C. Panel B, factor V
was incubated with PC
PS vesicles and APC as described in
the legend to Fig. 4. At selected time intervals aliquots of the
mixture were withdrawn and mixed with 2% SDS, 2%
-mercaptoethanol,
heated at 90 °C, and analyzed on a 4-15% linear gradient
SDS-PAGE gel (at the same time intervals aliquots were also assayed for
activity and are shown in Fig. 4, filled triangles). Lane1, factor V
, control; lanes
2-10, factor V
and APC at 1, 3, 5, 10, 15, 20,
30, 60, and 120 min. Position of the molecular weight markers (
10
) is indicated at the left of panelA.
Figure 4:
Inactivation of membrane-bound factor V by
APC. Normal plasma factor V, factor V, and factor V
(0.22 µM) were incubated with PC
PS vesicles
(200 µM) for 5 min at 37 °C. APC and hirudin were then
added (4.3 and 20 nM, respectively). At various time intervals
aliquots of the mixture were assayed for cofactor activity. The initial
rates of thrombin formation (at the steady state) were calculated and
plotted as percent of initial activity as a function of time following
addition of APC. Filled circles represent membrane-bound
normal plasma factor V treated with APC; the filled triangles depict membrane-bound factor V
treated with APC,
whereas the filled squares show membrane-bound factor V
inactivated by APC. The inset shows the analysis of the
inactivation reaction during the first 30 min. At the same time
intervals aliquots were withdrawn and analyzed by SDS-PAGE. The
products resulting from the proteolytic inactivation of membrane-bound
factor V
(filled triangles) are shown in Fig. 3B, whereas the comparison of the immunoreactivity
of the fragments resulting from the inactivation of plasma-derived
normal factor V and factor V
toward a monoclonal antibody
that recognizes an epitope located between amino acids residues
307-506 is displayed in Fig. 5, A and B.
The dottedline at the bottom of the graph represents the progress of the reaction in the absence
of added factor V (i.e. the generation of
-thrombin by
factor Xa alone).
Figure 5:
Analysis of factor V inactivation by APC.
The samples assayed for activity in Fig. 4(normal plasma factor
V and factor V) were also analyzed on a 4-15% linear
gradient SDS-PAGE. Following transfer to nitrocellulose, fragments were
visualized using monoclonal antibody
HFVa
#6. Panel A, normal factor V control; panel B, factor
V
. Lane1, factor V in the presence of
PC
PS vesicles and no APC; lanes 2-10,
membrane-bound factor V with APC at 1, 3, 5, 10, 15, 20, 30, 60, and
120 min. The position of the molecular weight markers is indicated at left of panelA.
In contrast to the results seen
for the abnormal factor Va preparations, no difference in the
inactivation rates for membrane-bound normal factor V and
membrane-bound factor V and factor V
was
observed (Fig. 4). These data confirm our previous findings that
optimum cleavage at Arg
on the membrane-bound procofactor
does not require prior cleavage at Arg
(6) .
Analysis of the proteolytic fragments deriving from membrane-bound
factor V
following digestion by APC showed the appearance
of a M
= 45,000 fragment, which is produced
slightly faster than the M
= 54,000
fragment (Fig. 3B). NH
-terminal sequence of
the two fragments demonstrated that the M
=
45,000 fragment has the same NH
terminus as factor V,
whereas the M
= 54,000 fragment has an
NH
-terminal sequence matching a portion of factor V
starting at amino acid residue 307. These data were confirmed using the
monoclonal antibody that recognizes the epitope located between amino
acid residues 307-506 of the cofactor. Following cleavage by APC
the antibody recognizes the M
= 30,000
fragment (Fig. 5A) from normal plasma factor V, whereas
for factor V
inactivation, the antibody recognizes the M
= 54,000 fragment (Fig. 5B). Altogether these data lead to the conclusion
that cleavage at Arg
and Arg
occurs almost
simultaneously during inactivation of membrane-bound factor V
and factor V
.
The inactivation of membrane-bound normal factor Va is an
ordered and sequential event with the first cleavage occurring at
Arg of the heavy chain (Fig. 6). This is followed
by cleavage at Arg
and Arg
, which results
in loss of cofactor activity. The M
=
75,000 fragment, which is the consequence of cleavage at
Arg
, is therefore an intermediate in the inactivation
process (Fig. 6). Our data demonstrate that membrane-bound
factor Va from patients with APC resistance lacking the cleavage site
at Arg
is still inactivated following cleavage at
Arg
and Arg
(Fig. 6). However, the
rate of inactivation is slower than in the normal molecule. Thus,
cleavage at Arg
appears to promote, but is not essential
for, cleavage at Arg
and Arg
. In contrast,
no differences in the inactivation rates are observed when studying
inactivation of membrane-bound factor V from normal or APC-resistant
individuals. Direct activity measurements, NH
-terminal
sequencing, and immunoblotting experiments confirmed our previous
findings (6) that, whereas rapid inactivation of membrane-bound
factor Va requires cleavage at Arg
prior to cleavage at
Arg
and Arg
, membrane-bound human factor V
does not require prior cleavage at Arg
for inactivation.
Since inactivation by APC of only the abnormal factor Va, not factor V,
is retarded (hence factor Va lingers at the place of vascular injury)
and since individuals with Arg
Gln mutation have a
poor anticoagulant response to APC, which in turn is associated with an
increase in risk of developing deep vein thrombosis (7-fold increase
for the heterozygous and 80-fold increase for the
homozygous)(29) , it is likely that the primary physiological
substrate for APC in vivo is factor Va, not factor V.
Figure 6:
Schematic representation of the cleavage
of human factor Va heavy chain during inactivation by APC. The heavy
chain of the human cofactor (containing 709 amino acids) is composed of
two A domains (A1-A2) associated through a connecting region (amino
acids 304-316)(2) . Normal plasma factor Va is rapidly
inactivated following three cleavages of the heavy chain; cleavage at
Arg (k
), which is required for
optimum exposure of the cleavage sites at Arg
and
Arg
(k
), produces a M
= 75,000 intermediate. Thus, in the absence of a cleavage
site at Arg
, cleavage at Arg
(k`
) and Arg
(k`
), which are required for cofactor
inactivation, occur much slower than on the precleaved (at
Arg
) heavy chain.
Recent data demonstrate that, in the absence of a membrane surface,
while no cleavage at Arg occurs, factor Va is cleaved at
Arg
followed by cleavage at Arg
resulting
in loss of approximately 30% of cofactor activity(6) . We have
also demonstrated that cleavage at Arg
on the
membrane-bound cofactor is impaired when using recombinant APC with the
Glu
Ala substitution (APC
).
Impaired cleavage at Arg
results in a cofactor that is
cleaved at Arg
and Arg
and still possesses
60% cofactor activity(30) . The present data, obtained using a
factor V molecule that does not possess a cleavage site for APC at
position 506, together with our previous findings (6, 30) suggest that: 1) cleavage at Arg
on the membrane-bound cofactor is responsible for the loss in
approximately 70% of cofactor activity; 2) cleavage at
Arg
, which is lipid-independent, is responsible for loss
in the remaining (30%) cofactor activity. The overall data indicate
that cleavage at Arg
alone in normal factor Va has no
consequence on cofactor activity; however, this cleavage is necessary
for efficient exposure of the cleavage sites at Arg
and
Arg
, which are associated with cofactor inactivation. In
the absence of cleavage at Arg
the inactivation process
proceeds at a significantly reduced rate.
The study of the plasma from patients with APC resistance has been the subject recently of intense investigation worldwide. Surprisingly, the bulk of functional data generated has created much confusion among the scientific community rather than understanding(31, 32, 33, 34, 35, 36) . It has even been proposed that APC resistance may have disappeared with time in certain patients(35) . The possibility of an acquired and transient APC resistance has also been proposed(36) . Our data may explain some of these assay discrepancies.
It is preferable
that individuals that suffered a thrombotic event should first be
screened for the mutation as suggested (32) by DNA analysis and
the results confirmed by the APC resistance assay. Finally, it is
important to recognize that APC resistance (by clotting assay) is
observed in healthy individuals (5% of the population) carrying the
Arg
Gln mutation, who do not present symptoms of
deep vein thrombosis(9, 12, 13) .
Additionally, it is well known that a single hit is in general
insufficient to promote thrombosis. Thus, the existence of other
(perhaps compensatory) mutations in the factor V gene that can
influence APC sensitivity (i.e. alterations in the lipid
binding sites of factor V or in the APC binding sites of the cofactor
or a mutation in the phosphorylation site at Ser
or a
mutation that changes the three-dimensional structure of factor V)
cannot and should not be excluded.