(Received for publication, December 14, 1995)
From the
When a recombinant variant of prothrombin with the cleavage site
mutations R155A, R271A, and R284A (rMZ) is exposed to either
prothrombinase or ecarin, a form of meizothrombin (rMZa) is generated
that is stable for weeks in the presence of Ca (Côté, H. C. F.,
Stevens, W. K., Bajzar, L., Banfield, D. K., Nesheim, M. E., and
MacGillivray, R. T. A.(1994) J. Biol. Chem. 269,
11374-11380). In the absence of Ca
however,
rMZa is rapidly cleaved within a disulfide bonded loop in the F1 domain
at Arg
in the sequence RTPR
DKL, yielding a molecule
with 3 chains joined by two disulfide bonds (rMZa*). Cleavage kinetics
are first order regardless of the rMZa concentration, indicating an
intramolecular cleavage. This cleavage does not occur at Ca
concentrations in excess of 1.0 mM. To assess the role
of the F1 domain in rMZa activity, another variant lacking the R155A
mutation (rMZdesF1) was expressed, which when activated yields
meizothrombin lacking the F1 domain (rMZdesF1a). Rates of hydrolysis of
the tripeptide substrate S2238 by rMZa or rMZa* increase from 60% to
90% that of recombinant thrombin as Ca
,
Mg
, or Mn
concentrations are varied
from 0 to 10 mM. K
and k
values for rMZa in the absence and presence of
5 mM Ca
are 1.9 and 2.2 µM and
65 and 105 s
. TAME esterase activity of rMZa also
increases with 5 mM Ca
. No such metal
ion-dependent effects are obtained with either thrombin or rMZdesF1a.
Fibrinogen clotting activities, relative to that of thrombin, increase
in a manner analogous to those obtained with small substrates, for rMZa
and rMZa* but not rMZdesF1a. Complexes of the active site probe
dansylarginine N-(3-ethyl-1,5-pentanediyl)amide with rMZa and
rMZa*, but not thrombin or rMZdesF1a exhibit large cation-dependent
decreases in fluorescence intensity, suggesting that metal ion binding
in the F1 domain alters the environment of the probe at the active
site. These results indicate that in the absence of divalent cations,
the activity of rMZa is inhibited, perhaps by obstruction of the active
site by the F1 domain, and that Ca
binding to the F1
domain modulates the properties of not only the F1 domain but also the
protease domain.
Prothrombin is one of a group of plasma proteins involved in
blood coagulation that require vitamin K-dependent carboxylation of
several amino-terminal glutamic acid residues for full biological
function(1) . In prothrombin, 10 of the first 33 residues are
-carboxyglutamic acid (Gla) (
)residues and define the
Gla domain. This domain mediates the metal ion-dependent interaction of
prothrombin and the other vitamin K-dependent coagulation factors with
negatively charged membrane surfaces(2) . Unlike other
Gla-containing coagulation factors such as factor IX, factor X, and
protein C, prothrombin loses its Gla domain following activation to the
serine protease thrombin.
Prothrombin activation is catalyzed by the
prothrombinase complex(3) , composed of factor Xa (a serine
protease), factor Va (an essential protein cofactor), negatively
charged phospholipids, and calcium ion. Prothrombinase-catalyzed
activation of prothrombin to thrombin results in peptide bond cleavage
at two sites in the molecule (Fig. 1). Cleavage occurs initially
at Arg, within a disulfide loop, forming the active
intermediate meizothrombin (4) . Further cleavage at
Arg
produces thrombin and the amino-terminal activation
peptide referred to as fragment 1.2. Thrombin itself can cleave
prothrombin. Two major sites are recognized, one at Arg
,
between the kringle domains of prothrombin, and a second at
Arg
, within the A chain of the protease domain. Thrombin
has also been reported to cleave prothrombin at a third site, at
Arg
within a disulfide loop between the Gla and first
kringle domains(5) . Two of the intermediates of prothrombin
activation, meizothrombin and meizothrombin des-fragment 1, may also
catalyze the cleavage of prothrombin at these sites.
Figure 1:
Schematic representation of the
activation of recombinant prothrombin and the mutants rMZ and rMZdesF1.
Activation of recombinant prothrombin (rII) to thrombin (rIIa) proceeds
via the intermediate meizothrombin (rmIIa), formed by cleavage within
the protease domain at Arg. Further cleavage can occur at
Arg
, Arg
, and Arg
, to yield
thrombin (rIIa) and the activation peptides, fragments 1 and 2. The
variant rMZdesF1 has the cleavage site mutations R271A and R284A and,
upon activation, proceeds through meizothrombin to meizothrombin
des-fragment 1 (rMZdesF1a). A stable recombinant form of meizothrombin
(rMZ) was also produced with the additional mutation R155A. This
species, when activated and stored in the presence of calcium ion,
remains as meizothrombin (rMZa), but rapidly autocatalyzes cleavage at
Arg
in the absence of calcium ion to form a three-chain
form of meizothrombin (rMZa*).
In contrast to
thrombin, the prothrombin activation intermediate meizothrombin retains
an intact Gla domain. Although naturally unstable, this intermediate
was recently obtained as a stable product by introducing three
mutations (R155A, R271A, R284A) which remove susceptibility to cleavage
by prothrombinase at Arg and by thrombin at the two
feedback cleavage sites(6) . If Arg
is retained,
cleavage by either thrombin or meizothrombin produces a species without
the Gla domain and the first kringle, known as meizothrombin
des-fragment 1 (Fig. 1).
Recent studies of the epidermal
growth factor-like and Gla domains of coagulation factors IXa and X
suggest that calcium ion binding in the Gla domain may affect
properties of the other domains of these proteases. For example,
calcium ion binding in the Gla domain of factor IXa alters the
conformation of the serine protease domain(7) , and a study of
amino-terminal fragments of factor X (8) revealed that
Ca binding in the Gla domain of factor X influences
Ca
binding in the amino-terminal of the epidermal
growth factor-like domain. Previous studies of meizothrombin activity (6, 9, 10) have not examined activities at
varying levels of Ca
.
To understand the
consequences of retaining the amino-terminal portion of the prothrombin
molecule on meizothrombin activity, recombinant prothrombin and two
site-directed mutants were prepared. One mutant was prothrombin
(R155A,R271A,R284A) designated rMZ, which upon activation with either
the prothrombinase complex or ecarin yields full-length
meizothrombin(6) . A second mutant prothrombin (R271A,R284A)
designated rMZdesF1 yields meizothrombin des-fragment 1 upon
activation. These and recombinant prothrombin (rII) were expressed,
isolated, and activated, and their activities against small substrates
and fibrinogen were compared in the presence of varying amounts of
Ca.
Figure 2:
Calcium ion protection of rMZa from
autocatalysis to rMZa*. A sample of rMZ (100 µg/ml) in 0.02 M HEPES, 0.15 M NaCl, 5 mM Ca,
pH 7.4, was incubated with ecarin (3 µg/ml), and samples were
withdrawn and quenched with acetic acid (0.2 M, 2 volumes) at
1, 5, 20, and 60 min as indicated on the gel. After 60 min, the sample
was split in two, and to one aliquot EDTA was added (10 mM final concentration). Subsequent samples from the incubations with
(+) and without(-) EDTA were quenched 1, 5, 20, and 60 min
later. The samples were lyophilized, dissolved in gel loading buffer,
and analyzed by NaDodSO
-polyacrylamide gel electrophoresis
under reducing conditions. Bands corresponding to intact rMZ, the two
chains of rMZa (F1.2A and B chains), and the cleaved F1.2A chain from
rMZa* (F1.2A*) are indicated.
Further
analysis revealed that the cleavage giving rise to rMZa* occurred only
in the absence of Ca and was prevented by storage of
rMZa in 5 mM CaCl
. Analysis of the time course of
cleavage indicated that rMZa has a half-life in excess of 28 days in
the presence of Ca
, but only 8 min in the absence of
Ca
. It is highly unlikely that this cleavage is a
result of a trace contaminant in the ecarin preparation, as identical
results were obtained when the prothrombinase complex was used as the
activator. The cleavage follows first order kinetics, with the percent
conversion following Ca
removal identical at three
concentrations of the enzyme between 25 and 100 µg/ml (Fig. 3). The rate of cleavage is not affected by equimolar
concentrations of bovine F1 or a recombinant human prothrombin variant
lacking factor Xa and thrombin cleavage sites (data not shown) nor are
these species cleaved during the time course of the experiment.
Although the concentration range studied was limited for technical
reasons, modelling the meizothrombin proteolysis as an intermolecular
reaction occurring with either high (
10 µM),
intermediate (1 µM), or low (
0.1 µM)
affinity using Kinsim(17) , failed to produce a reasonable fit
to the data, whereas modelling the reaction as an intramolecular event
(first order) produced an excellent global fit to the data (Fig. 3). These results indicate that the cleavage at Arg
is an intramolecular rather than intermolecular event.
Figure 3:
Time course of conversion of rMZa to rMZa*
at varying concentrations in the absence of Ca.
Samples of rMZa were diluted to 100, 50, and 25 µg/ml (
,
, and
respectively) in buffer containing 2 mM CaCl
. At time zero, EDTA (5 mM final
concentration) was added to chelate the Ca
, and, at
the indicated times, samples were quenched with 0.2 M acetic
acid. Samples were reduced to dryness, redissolved in gel loading
buffer, and run on 5-15% polyacrylamide gradient gels under
reducing conditions in the presence of NaDodSO
. The extent
of cleavage at each time point was ascertained by laser densitometry of
the Coomassie Blue-stained gels. The line represents a global fit
determined by regression to a first order rate
equation.
Figure 4:
Calcium ion requirement for maintenance of
intact rMZa. Aliquots of rMZa (1 µM) in 0.02 M HEPES, 0.15 M NaCl, 5 mM CaCl were
added to 0.02 M HEPES, 0.15 M NaCl, pH 7.4,
containing various concentrations of calcium ion. After a 60-min
incubation at 22 °C, the samples were quenched and analyzed as
described in the legend for Fig. 3. From the determinations of
the amount of rMZa remaining intact, the rate constants for cleavage at
the varying Ca
concentrations were determined.
Calculated half-lives are inset.
Figure 5:
Activity of rIIa, rMZa, rMZa*, and
rMZdesF1a against the amidolytic substrate S2238 in the presence of
varying concentrations of Ca. Aliquots of rIIa, rMZa,
rMZa*, and rMZdesF1 (50 µl) (
,
,
, and
,
respectively) to yield final concentrations of 0.25 nM were
incubated for 5 min at 37 °C in microtiter wells with 150 µl of
0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80 with
varying concentrations (0-10 mM) of CaCl
.
The substrate S2238 (0.5 mM final concentration in the
incubation buffer) was added, and initial rates of hydrolysis were
determined by following the absorbance at 405 nm at 1-min
intervals.
Figure 6:
The effect of calcium ion concentration on
fibrinogen clotting by rMZa, rMZa*, and rMZdesF1a relative to rIIa.
Fixed concentrations of rIIa (1 nM), rMZa (15 nM), or
rMZdesF1a (3.6 nM) to give approximately equal clotting times
in the presence of 5 mM CaCl were added to the
wells of a microtiter plate containing varying concentrations of EDTA
to give a range of Ca
concentrations (0-5
mM). Fibrinogen cleavage was initiated with the addition of 2
mg/ml fibrinogen in 0.02 M HEPES, 0.15 M NaCl, 0.01%
Tween 80, pH 7.4, and the time to onset of clotting was determined by
monitoring turbidity at 320 nm. Rates of fibrin formation relative to
rIIa are plotted for rMZA (
) and rMZa* (
). The clot times
for all species (rIIa, rMZa, rMZa*, and rMZdesF1 are
,
,
, and
, respectively) are inset.
Figure 7:
Titration of rIIa, rMZa, rMZa*, and
rMZdesF1a with calcium ion in the presence of DAPA. Aliquots of rIIa,
rMZa, rMZa*, and rMZdesF1a (100 nM) (,
,
,
and
, respectively), in the presence of 200 nM DAPA in
0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80 were
titrated with a solution of CaCl
, while DAPA fluorescence
was continuously monitored (
280 nm,
545 nm). The results are expressed as a percentage of the
original fluorescence signal.
Previous studies of the binding of prothrombin and its
activation intermediates to hirudin tail peptides(19) ,
thrombomodulin, and exosite antibodies(20) , have suggested
that the fragment 1 domain may in some way block access to the primary
anion binding exosite. Through measurements of the Ca dependence of meizothrombin autoproteolysis at Arg
,
activity toward a variety of substrates, and energy transfer to the
active site probe DAPA, this study provides further evidence for
interactions between the fragment 1 and protease domains of
meizothrombin.
Cleavage of prothrombin at Arg has
previously been reported following lengthy incubation of prothrombin
with thrombin in the absence of Ca
(5) . In
the present studies, however, the cleavage of rMZa at Arg
follows first order kinetics, with the time course of the
reaction being identical at three different concentrations of rMZA,
indicative of an intramolecular cleavage event. These results imply a
direct interaction between the amino-terminal portion of the fragment 1
domain with the active site of meizothrombin. Cleavage of rMZa at
Arg
occurs rapidly in the absence of Ca
with a half-life under 10 min, but is profoundly attenuated in
the presence of Ca
.
Structural changes in the
fragment 1 domain of prothrombin have been shown to accompany divalent
metal ion binding by a number of methods including sedimentation
velocity(21) , intrinsic fluorescence(21) , circular
dichroism(22) , and immunologic techniques(23) . Due to
the sensitivity of cleavage at Arg to levels of
Ca
, and the location of the cleavage site within the
fragment 1 domain, this event is likely modulated by metal ion binding
in the Gla domain. Indeed, cleavage of prothrombin by thrombin at
Arg
is also inhibited in the presence of
Ca
, and the protective effect has been attributed to
conformational changes mediated by Ca
binding to the
Gla domain(24) .
Although the three-dimensional structures
for the apo- and Ca-bound forms of human fragment 1
are not available, those of bovine fragment 1 in the absence and
presence of Ca
have been
determined(25, 26) . The structure of the Gla domain
is disordered in the absence of Ca
, but the disulfide
loop containing the cleavage site at Arg
is defined in
both structures. Interestingly the region around Arg
changes significantly upon Ca
binding, with a trans to cis conformational change at Pro
and a 90° rotation of Arg
relative to its
neighboring residues, enabling the formation of new salt links between
Arg
and Gla
, Leu
, and
Gla
. These changes at the Arg
cleavage site
likely explain the insensitivity of Ca
-bound
meizothrombin to autoproteolysis. Another contributing factor to the
stability of rMZa in the presence of Ca
may be the
disruption of interactions between the negatively charged residues of
the Gla domain with positively charged portions of the protease domain
(possibly the primary anion binding exosite) upon Ca
binding. In any case, autocatalytic cleavage of rMZa at
Arg
necessitates direct interaction between the fragment 1
and protease domains.
In addition to preventing autoproteolysis of
rMZa, Ca increases the activity of rMZa toward small
ester (TAME) and amide (S2238) substrates as well as increasing its
fibrinogen clotting activity. Equivalent increases in activity are
obtained with rMZa*, a species cleaved at Arg
, so the
increase in activity cannot be simply attributed to a
Ca
-mediated protection from autoproteolysis. Neither
rMZdesF1a, lacking the fragment 1 domain, nor thrombin demonstrate this
Ca
-dependent increase in activity, suggesting that
interactions of Ca
with the fragment 1 domain are
responsible for the altered activity of rMZa.
Similar effects on the
activity of rMZa are observed with both small substrates (amide and
ester) and fibrinogen, although fibrinogen binding involves
considerably more complex interactions with the enzyme than do the
small substrates. Thus, obstruction of the active site or reduced
turnover for some other reason, rather than a change in substrate
binding, is responsible for the reduced activity of rMZa in the absence
of Ca. This interpretation is supported by the
observation that values obtained for the K
for
S2238 hydrolysis remain constant (1.9 versus 2.2
µM) in the absence or presence of 5 mM Ca
, while k
values change
(65 versus 105).
Titration of rMZa with Ca in the presence of the reversible fluorescent inhibitor DAPA
results in a 30% decrease in fluorescence energy transfer, with a
half-maximal effect occurring at 0.5 mM Ca
.
This result is similar to that obtained with meizothrombin prepared by
activation of prothrombin with ecarin in the presence of
dansyl-glutamylglycylarginyl chloromethyl ketone, yielding an active
site blocked species with a covalently attached dansyl reporter
group(27) . In both cases, a change in the active site
environment is revealed by a substantial decrease in probe fluorescence
upon the addition of Ca
. It is unlikely that
Ca
has a direct effect on the fluorescence of DAPA,
as direct excitation of DAPA in the absence of protein yields less than
2% of the signal obtained in the presence of rMZa, and the fluorescence
decrement observed with rMZa or rMZa* does not occur with either
rMZdesF1a or rIIa. In addition, both rMZa and rMZa* display much
greater fluorescence than either rIIa or rMZdesF1a in the absence of
Ca
(2.5-3-fold). The decreases in DAPA
fluorescence of rMZa and rMZa*, but not rMZdesF1a, upon the addition of
Ca
are consistent with a Ca
binding
event in the fragment 1 domain affecting the probe environment at the
active site.
The Ca concentrations required for
half-maximal increase in rMZa activity, protection from
autoproteolysis, and decreases in DAPA fluorescence (0.5-1
mM) are significantly higher than those reported necessary for
conformational change in the isolated fragment 1 domain (0.2-0.4
mM)(21, 22) . These levels of Ca
are, however, consistent with those reported necessary to prevent
the proteolytic cleavage of prothrombin at Arg
by
thrombin (0.6 mM)(24, 28) .
Divalent metal
ions have been widely reported to cause self-association of prothrombin
fragment 1 (21, 29) and, under some conditions,
prothrombin(29, 30) . Metal ion-dependent rMZa dimer
formation is unlikely to be responsible for any of the effects observed
in these studies, due to the high protein and metal ion concentrations
necessary for dimerization to occur. In addition, Mg at the concentrations used in this study does not seem to be
capable of inducing fragment 1 or prothrombin
dimerization(21, 29, 30) , but will support
the increase in S2238 hydrolysis observed with rMZa.
Cleavage of
rMZa at Arg has little effect on the rate of S2238
hydrolysis at varying Ca
concentrations. There is,
however, a marked effect on the concentration necessary to produce
half-maximal fibrinogen clotting activity, and decrease in DAPA
fluorescence, with higher (1-1.5 mMversus 0.5
mM) levels necessary to produce these changes in the cleaved
species. These differences may be indicative of a reduced ability of
the cleaved form to undergo the necessary conformational changes to
relieve the enzyme from inhibition at a given Ca
concentration, with both fibrinogen clotting and DAPA
fluorescence being more sensitive to interdomain interactions distant
from the active site than S2238 hydrolysis.
These results together
provide evidence for interaction between the fragment 1 and protease
domains of rMZa at Ca levels below 1 mM. The
consequences of this interaction include meizothrombin autoproteolysis
at Arg
, as well as decreases in catalytic activity toward
small ester (TAME) and small and large amide substrates (S2238,
fibrinogen). Interestingly, the transition for these effects borders on
the range of free Ca
in the plasma (1-1.3
mM)(31) . Finally, these results are indicative of
flexibility in the structure of prothrombin, allowing for association
between the amino-terminal fragment 1 and carboxyl-terminal serine
protease domains.