From the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260
Received for publication, August 17, 2002, and in revised form, October 17, 2002
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ABSTRACT |
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Activation of prothrombin, as
catalyzed by the prothrombinase complex (factor Xa,
enzyme; factor Va and phosphatidylserine (PS)-containing
membranes, cofactors), involves production and subsequent proteolysis
of two possible intermediates, meizothrombin (MzIIa) and
prethrombin 2 plus fragment 1.2 (Pre2 & F1.2).
Vmax, Km, or
Vmax/Km for all four
proteolytic steps was determined as a function of membrane-phospholipid
concentration. Proteolysis was monitored using a fluorescent thrombin
inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants
for the conversion of MzIIa and Pre2 & F1.2 to thrombin
were determined directly. Pre2 & F1.2 conversion was linear in
substrate concentration up to 4 µM, whereas
MzIIa proteolysis was saturable. First order rate constants
for formation of MzIIa and Pre2 & F1.2 could not be
determined directly and were determined from global fitting of the data
to a parallel, sequential model, each step of which was treated by the
Michaelis-Menten formalism. The rate of direct conversion to thrombin
without release of intermediates from the membrane-Va-Xa complex (i.e.
"channeling") also was adjusted because both the membranes and
factor Va have been shown to cause channeling. kcat, Km, or
kcat/Km values were
reported for one lipid concentration, for which all Xa was
likely incorporated into a Xa-Va complex on a
PS membrane. Comparing previous results, which were obtained either
with factor Va (Boskovic, D. S., Bajzar, L. S., and
Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C.,
Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002)
Biochemistry 41, 935-949), with results presented here we
conclude that both factor Va and PS-containing membranes
induce similar rate increases and pathway changes. Moreover, we have
determined: 1) factor Va has the greatest effect in
enhancing rates of individual proteolytic events; 2) PS-containing
membranes have the greatest role in increasing the preference for the
MzIIa versus Pre2 pathway; and 3) PS membranes cause ~50% of the substrate to be activated via channeling at 50 µM membrane concentration, but factor Va
extends the range of efficient channeling to much lower or higher
membrane concentrations.
Factor Xa catalyzes the activation of human
prothrombin by proteolytic cleavages at
Arg273-Thr274 and at
Arg322-Ile323. The polypeptide chain composed
of residues 1-273 (fragment 1.2) is released as an activation peptide.
Residues 274-322 comprise the A chain of Both factor Va and phosphatidylserine (PS)-containing
membranes, or a soluble form of PS, affect the rates of each of the five proteolytic events involved in prothrombin activation (Fig. 1)
(1-3). In doing so, these two cofactors 1) increase the overall rate
of prothrombin activation, 2) define the intermediate for activation,
and 3) promote channeling. The rate-enhancing effect of both cofactors
has long been recognized (4, 5), with either cofactor enhancing the
first order rate constant for prothrombin activation by roughly 3 orders of magnitude but both together enhancing it by 200,000-fold (5).
Until recently, it was accepted that factor Va was
responsible for directing activation via the MzIIa pathway
(4, 6). However, more recent results have shown that in the absence of
factor Va, PS-containing membranes alone increase the
preference of human factor Xa for the MzIIa pathway by more than 550-fold relative to the Pre2 pathway (2) and that
this is because of the binding of individual PS molecules to specific
sites on factor Xa (7). Factor Va also enhances the preference of bovine factor Xa for the
MzIIa versus Pre2 pathway, although only by
25-fold (1). The cofactors also control access to pathway E. In the
presence of bovine factor Va, 40% of the substrate was
channeled directly to thrombin (1), whereas 50% processivity occurred
in the presence of an optimal concentration of PS-containing membranes
(2).
Because both cofactors influence similarly the pathway of prothrombin
activation, one might ask why both factor Va and PS membranes should be needed, as apparently they are under physiological conditions, to promote efficient thrombin generation in
vivo. To answer this question, we establish here the pathway of
prothrombin activation by human factor Xa in the presence
of saturating concentrations of factor Va and various
PS-membrane concentrations. Furthermore, for one optimal membrane
concentration, we estimate the second order rate constants for each of
the reactions shown in Fig. 1 and compare these with the rates observed
when either of the cofactors is present individually. We find that
factor Va contributes most to enhancing the rate of
activation and that PS dominates the choice of the MzIIa
pathway. Together the cofactors promote more efficient activation than
either does alone; the overall rate of thrombin formation is at least
two orders of magnitude higher and occurs almost exclusively through
channeling that minimizes the appearance of intermediates in the
solution. In addition, a new role for factor Va has
been found, namely making channeling the stable pathway of activation
over the wide range of membrane concentrations that one might expect to
find in vivo.
Materials
Ecarin from Echis carinatus snake
venom and EGTA were purchased from Sigma. Bovine brain PS and
dioleoyl-phosphatidylcholine (DOPC) were purchased from Avanti Polar
Lipids Inc. (Alabaster, AL). Outdated human plasma was obtained from
the American Red Cross Center, Durham, NC. Human prothrombin, factor X,
and factor Xa were obtained by published procedures (8),
and MzIIa was prepared as described by Pei et
al. (9). Pre2 and F1.2 were obtained as described by Wu et
al. (2). Purified human factors Xa, Va,
and prothrombin also sometimes were purchased from Haematologic Technologies Inc. (Essex Junction, VT). The activity of Factor Xa was assayed with the synthetic substrate S-2765 (2).
Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine (DAPA) was obtained from Haematologic Technologies Inc.
Methods
Preparation of Phospholipid Vesicles--
Extruded large
unilamellar vesicles (LUV) composed of 25/75 bovine brain PS and DOPC
were prepared, and the phospholipid concentration was determined by the
methods described in Wu et al. (2).
Fluorescence Stopped Flow Measurements--
Measurements of
thrombin formation from prothrombin, MzIIa, or Pre2 (with
equimolar fragment 1.2) were performed using DAPA and exploiting the
large change in fluorescence intensity that accompanies the binding of
this fluorophore to the products of the prothrombinase reaction.
Stopped flow measurements were performed using a SLM-Aminco
Milliflow® stopped flow reactor (Spectronic Instruments, Inc.,
Rochester, NY) attached to the SLM 48000® spectrofluorometer (Spectronic Instruments). Broadband fluorescence of DAPA was monitored at 37 °C with a 515-nm cut-off filter in the emission path and with
the excitation monochromator set at 280 nm, slit = 4 nm.
Reactions were initiated by rapidly mixing an equal volume (400 µl)
of the contents of the two driving syringes. Syringe A contained
substrate solution and DAPA in 50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5 (Buffer
R); and syringe B contained pre-assembled prothrombinase
(Xa, Va, and bovine brain PS/DOPC) in the same buffer. The final concentrations of factors Xa and
Va in the reaction chamber were maintained as 2.5 and 30 nM, respectively, for the substrate MzIIa and
prethrombin 2. The substrate/DAPA ratio was always kept at 1/5. The
initial fluorescence intensity (F0) was obtained
from mixing experiments with the substrate and DAPA in syringe A and
the lipid only in syringe B. F0 as well as other fluorescence intensities were corrected for light scattering by subtracting the scattering intensity recorded during a push involving only the lipid and factor Xa with no DAPA (2). Prior to
mixing, both syringes were allowed to incubate at 37 °C for 2-3
min. Fluorescence intensity at the completion of the reaction, after
which no further changes in fluorescence intensity occurred, was
considered to represent quantitative conversion of prothrombin to
thrombin and was used to convert the fluorescence intensity change per
unit time into units describing thrombin active site formation as a function of time. The initial rate of thrombin generation thus was
determined from the initial rate of fluorescence intensity change
normalized to the intensity at complete thrombin formation in
accordance with Wu et al. (2).
Rapid Chemical Quench Measurements--
Quench flow methodology
was used to determine the initial rate of MzIIa and
IIa appearance. Rapid quench experiments were performed
using the Chemical-Quench-Flow Model RQF-3 from Kintek Corp. (State
College, PA). The temperature was maintained at 37 °C by a
circulating water bath. Prothrombin solution in Buffer R with
0.6% polyethylene glycol, which was used to limit thrombin adsorption
to surfaces, was loaded into one sample loop; also in Buffer R,
preassembled prothrombinase complex, at a concentration used in the
stopped flow fluorescence studies, was loaded into another sample loop.
The reaction was initiated by pushing both solutions into the reaction
loops and then was allowed to proceed for various times. The reaction
was stopped by mixing the solutions with a quench solution (100 µl of
100 mM Na2EGTA solution, pH 7.5) from the
quench syringe. The mixture then was expelled into a collection tube.
Aliquots from collected samples were assayed for amidolytic activity
using S-2238 in a VersaMaxTM microplate reader (Molecular
Devices Corp., Sunnyvale, CA), and the concentration of the active site
(IIa plus MzIIa) was determined by comparing
the initial rate of S-2238 hydrolysis to a standard curve, as described
by Wu et al. (2). To measure the amount of
MzIIa formed, the amidolytic activity was measured after
incubating the reaction mixture aliquot for 1 min in the presence of
heparin (10 µg/ml) and anti-thrombin III (300 nM),
thereby blocking the thrombin but not the MzIIa active site
(10). The initial rate of thrombin formation was obtained by
subtracting the rate of MzIIa appearance from the initial
rate of total active site formation.
Analysis of Prothrombin Activation by SDS-PAGE--
These
experiments were performed as described for rapid quench experiments
except that aliquots of 40 µl at 0.5, 1, and 1.5 s were taken
and subjected to gel electrophoresis (1.5 mm of 12% polyacrylamide)
with and without reduction with 5% (v/v) 2-mercaptoethanol (11) for
analysis. Protein bands were visualized with colloidal Coomassie Blue
staining (12).
Kinetic Modeling--
To determine the kinetic constants for the
first two proteolytic events (reactions A and C;
Fig. 1), three experimentally determined time courses (thrombin
formation, MzIIa appearance, and Pre2 appearance) were
modeled according to a steady state kinetic scheme that assumed a
parallel, sequential reaction mechanism, as described in detail by Wu
et al. (2). A fraction of prothrombin consumed was
considered to convert directly to thrombin without the escape of an
intermediate from the membrane-enzyme-cofactor complex (2). All
three sets of data were fit simultaneously by adjusting first order
rate constants Ra (for reaction A;
Fig. 1), Rc
(reaction C), and Re (reaction
E), subject to experimental constraints, namely
Rb and Rd (for reactions
B and D), which were fixed at the values
determined independently in this paper. This global data fitting was
carried out using the SigmaPlot 2000 least-squares regression package
(SPSS Inc., Chicago, IL). The details of these procedures are described
by Wu et al. (2).
Effects of Factor Va and PS/DOPC Membranes
on Prothrombin Activation by Prothrombinase--
To establish
the appropriate factor Va concentration for our studies, we
monitored prothrombin activation using the fluorescent active site
inhibitor DAPA at several concentrations of phospholipid and factor
Va (see "Methods") (Fig.
2). It was not possible to saturate
prothrombinase activity even at 30 nM factor Va
in the presence of only 20 µM PS membranes
(circles in Fig. 2). However, above this lipid
concentration, prothrombinase activity always was saturated by 30 nM factor Va (Fig. 2), reflecting optimal assembly of prothrombinase at that lipid concentration. Based on this
result, we fixed the factor Va concentration at 30 nM in all other experiments. We note that the saturating
activity at 30 nM Va was a function of lipid
concentration, with a maximum at 100-200 µM lipid, as
seen in the inset in Fig. 2. This is not surprising as
several laboratories have reported inhibition of prothrombinase
activity by high phospholipid concentration (13-16), and we have
reported a similar behavior for Xa even in the absence of
factor Va (2).
Activation of Meizothrombin and of Prethrombin 2 plus
Fragment 1.2 to Thrombin--
Fig.
3, A and B, shows
the initial rates of activation to thrombin of MzIIa as
well as Pre2 and F1.2, as obtained from the initial rates of change in
DAPA fluorescence intensity. As noted previously (6, 17), activation of
these intermediates to thrombin reflects the prothrombinase-catalyzed
cleavage of individual peptide bonds,
Arg273-Thr274 in MzIIa and
Arg322-Ile323 in Pre2.
Because these reactions involve a single proteolytic event, it seems
appropriate to apply the Michaelis-Menten model and obtain the apparent
first order rate constants
(Vmax/Km) as a function of
membrane concentration. Plots of initial rates of MzIIa
activation versus substrate concentration thus were fit to
this model. The Km values determined from these fits showed no clear trend with lipid concentrations within experimental and
fitting errors and had an average value of 0.9 ± 0.1 µM. The apparent first order rate constants
(Vmax/Km) for proteolysis of
MzIIa were nearly independent of lipid concentrations above 50 µM but did decrease slightly at 2000 µM
lipid (filled triangles, Fig.
4A). A similar behavior was
seen for Pre2 and F1.2 proteolysis (filled circles, Fig.
4A). For activation of Pre2 and F1.2 as well as
MzIIa, a somewhat lower rate was observed at 20 µM lipid (Fig. 4A), at which only 45% of the
substrate was membrane-bound and for which the rate of prothrombin
activation was not saturated in Va (Fig. 2).
Determination of the Kinetic Constants of the Initial Proteolytic
Steps--
To establish the pathway of activation, we do not need the
rates of reactions B and D but instead those of
reactions A and C. The rates of these reactions
are difficult if not impossible to measure directly because of the
rapid consumption of their products in the second step of prothrombin
activation (Fig. 1). To overcome this problem, we have used an approach
described earlier (2). In Fig. 5, the
time courses for appearance of thrombin plus MzIIa
(circles) and MzIIa alone (squares)
are measured by the S-2238 assay at five lipid concentrations, and the
time courses of thrombin plus MzIIa (open
circles), MzIIa (open squares), and Pre2
(open triangles) appearance are measured by PAGE at 200 µM lipid. The fact that the rate of thrombin formation
was greater than the rate of MzIIa appearance at all lipid
concentrations means either that significant thrombin formation
occurred through the Pre2 pathway or that a large fraction of
MzIIa and/or Pre2 rapidly was processed directly to
thrombin and not detected as a released intermediate. An extensive
SDS-PAGE analysis was performed at one lipid concentration (200 µM) with the results matching the quench flow results and
showing that no Pre2 formation was detectable (Fig. 5). Single time
points confirmed that Pre2 could not be detected by SDS-PAGE at any
other lipid concentration, implying that a fraction of intermediate(s)
was processed directly to thrombin (reaction E in Fig.
1).
To test quantitatively for this possibility, we fit our data as
described elsewhere (2) with the measured first order rate constants
(Vmax/Km)
Rb and Rd (Fig. 4) fixed
and the values of Ra, Rc,
and Re, adjusted to obtain the best possible fit
to these experimental time courses. The parameter values
Ra (open triangles) and
Rc (open circles), resulting in the
fits shown by the solid lines through the data in the
left panels of Fig. 5, are plotted as a function of
phospholipid concentration in Fig. 4B. The rates of Pre2
formation were set at a constant upper limit (Ra = 0.001 s
Fig. 4B also shows, as a function of lipid concentration,
the rate of direct conversion of prothrombin
(Re, channeling; open squares) that
was necessary to obtain the fits shown in Fig. 5. Shown in Fig. 4 are
the relative rates of prothrombin activation via the following
pathways: the channeling pathway (Re
relative = Re/(Ra + Rc + Re); open
squares, panel E); the MzIIa pathway (open circles, panel D), or the Pre2 pathway
(open triangles, panel C).
The purpose of this work was to ask why factor Va and
PS membranes both seem to be required for efficient prothrombin
activation. The results obtained here, combined with other results
obtained in the presence of either of the two cofactors alone (1-3,
6), suggest the following answers: (i) the main role of the cofactor Va and the membrane is rate enhancement; (ii) the two
co-factors direct prothrombin activation through the meizothrombin
pathway, with PS membranes having the major role in this function;
(iii) both cofactors contribute to channeling, i.e. to the
creation of a fast pathway of direct conversion of prothrombin to
thrombin; and (iv) factor Va allows factor Xa
to function efficiently as a robust channeling enzyme over a wide range
of membrane concentration. We will discuss each conclusion in order.
Rate Enhancement--
To show how PS membranes and factor
Va together enhance the rate of prothrombin activation
compared with the individual cofactors alone, we must obtain from our
results estimates of second order rate constants. Second order rate
constants are difficult to obtain because of a lack of precise
knowledge about the total amount of factor Xaa
in a reaction mix actually complexed with factor Va on a
membrane to form the active prothrombinase complex. The difficulty in
defining Xa-Va-membrane complex concentration
derives from the fact that studies of prothrombin activation normally
are carried out on discrete membrane vesicles so that the efficiency of
association between factors Va and Xa is a
function of lipid concentration (14, 15). This has been suggested to
account, at least in part, for the phenomenon of lipid inhibition that
we note in the inset in Fig. 2. There is no quantitative
treatment of this effect that would allow prediction of the
concentration of Xa-Va complex at different
lipid concentrations based on first principles. Thus, we must be
content to make simplifying assumptions, as in earlier studies, so that
we might compare rate constants with them. Nesheim et al.
(4) reported rate constants for bovine prothrombinase under conditions
for which activation was maximal with respect to both lipid and
Va concentrations, whereas Krishnaswamy et al.
(6) based human prothrombinase concentration on an apparent assembly
dissociation constant estimated from the dependence of prothrombin
activation rate on factor Va concentration. Based on the
results summarized in Fig. 4A, cleavage of individual bonds
was optimal at 50 µM lipid. This lipid concentration is
also low enough to avoid complications because of incomplete distribution of Va and Xa between discrete
vesicles (7 Xa and 80 Va/vesicle). The apparent
dissociation constant (1.7 ± 1 nM) obtained from the
data of Fig. 2 using the approach of Krishnaswamy et al. (6)
also supports the assumption that all factor Xa (at 2.5 nM) is incorporated into the Va-Xa
complex at 30 nM Va and 50 µM
lipid. Based on this assumption, the second order rate constants
(kcat/Km) are
calculated and presented in Table I for 50 µM lipid.
These constants are in close agreement with the only results in
the literature to which we can make a direct comparison, 2.9 × 107 and 2.3 × 107
M
There is no simple means by which to judge how PS membranes and
factor Va increase the rate of prothrombin activation
because they can affect any of five different reactions shown in Fig. 1. To begin, we focus on the cleavages of two bonds in prothrombin that
lead to intermediate formation. The rates of these bond cleavages in
prothrombin (reactions A and C) were very
different (Table I), as both PS membranes and factor Va
plus membranes inhibited Arg273-Thr274
cleavage in reaction A and promoted
Arg322-Ile323 cleavage in reaction
C dramatically. This shifts activation to the
MzIIa intermediate almost exclusively, as we will discuss in the next section. Based on the data recorded in Table I, in the
presence of PS membranes, the rate-enhancing effect of factor Va on the reaction producing MzIIa
(1800) is roughly twice that of PS membranes (750). Based on
results obtained with bovine proteins (1, 18, 19), in the absence of PS
membranes, the rate of enhancement of this reaction due to factor
Va alone can be estimated to be 14,000. From either
comparison, we conclude that factor Va plays the dominant
role in enhancing the rate of MzIIa formation.
We turn attention next to the activation of intermediates to thrombin.
Our results show that the rate constants for proteolysis of the
Arg273-Thr274 and
Arg322-Ile323 bonds in MzIIa and
Pre2, respectively, were quite similar in the presence of
Va and PS membranes, in agreement with the conclusion reported by Krishnaswamy et al. (6) for human prothrombinase assembled in the presence of 20 µM PS/phosphatidylcholine
(25/75) small unilamellar vesicles. Because the formation of Pre2 is so slow in the presence of PS membranes, we can focus attention for all
practical purposes solely on MzIIa activation. PS
membranes alone result in a 100-fold enhancement of the rate of human
MzIIa activation (2), whereas factor Va alone
results in a 940-fold enhancement of bovine MzIIa
activation (18); therefore, factor Va accounts for roughly
a 9-fold greater rate of MzIIa activation than do PS
membranes. Comparing, at optimal membrane concentrations, the second
order rate constant for reaction D for the full
Xa-Va-PS membrane complex (Table I) to that
reported for factor Xa bound to PS membranes (2), we see a
20-fold increase in rate because of adding factor Va to the
PS membrane. Based on this comparison (9-fold versus
20- fold ± membranes), it would seem that binding to PS membranes
does not alter significantly the ability of factor Va to
enhance the rate of MzIIa activation, and, as with
MzIIa formation, factor Va seems to be the
major contributor to the cofactor-mediated rate enhancement of
MzIIa consumption.
Shifting Prothrombin Activation to the Meizothrombin
Pathway--
Aside from rate enhancement, the principle role of factor
Va and PS-containing membranes in prothrombin activation is
to direct the pathway of activation. To discuss this effect of the
cofactors, we do not need absolute rates; the relative rates recorded
in Fig. 4, C-E, will suffice. We note first that 96% of
the initial proteolysis of human prothrombin by factor Xa
leads to Pre2 formation (2). From the relative rates recorded in Fig.
4, it is clear that bovine factor Va alone reduces this
fraction to roughly 31% (X in panel C) (1),
whereas PS membranes reduce this fraction to roughly 4% at optimal
membrane concentration (cross-filled triangles in
panel C) (2). It is notable that neither cofactor alone is sufficient to completely eliminate formation of Pre2 intermediate. However, in the presence of both PS membranes and factor
Va, no Pre2 could be detected (Fig. 5), and the fraction of
prothrombin initially converted to Pre2 was reduced to zero at all
membrane concentrations (open triangles in panel
C). The fact that the fraction of prothrombin activated to
MzIIa was lower for the full prothrombinase studied here
(open circles in panel D) than for Xa
bound to PS membranes alone (cross-filled circles in
panel D) (2) simply reflects a large amount of activation via channeling (panel E), as will be discussed next. These
relative rates establish that factor Va and PS membranes
together direct activation exclusively via the MzIIa
intermediate and/or via channeling to a greater extent than either
cofactor can accomplish alone.
Effects of PS and Va Cofactors on Channeling--
Both
factor Va and PS membranes have been shown to promote
conversion of prothrombin to thrombin without release of the
intermediate from the enzyme complex (1, 2), i.e. to promote
intermediate channeling (reaction E in Fig. 1). Fig.
4E summarizes our results as well as results presented in
the recent literature with respect to channeling. Here we see that
bovine factor Va alone causes factor Xa
initially to process roughly 39% of prothrombin via channeling
(X in Fig. 4E) (1). At optimal PS membrane
concentration (50 µM), 55% of prothrombin initially is
processed via channeling (cross-filled squares) (2),
although channeling was much less efficient at lower and higher
membrane concentrations. The addition of factor Va to PS
membranes had little effect on the fraction of prothrombin initially
activated via the channeling pathway at 50 µM PS
membranes but had a significant influence at higher and lower membrane
concentrations (open squares, Fig. 4E), as will
be discussed below.
Factor Va in the Presence of PS Membranes Creates a
Robust Channeling Enzyme--
Even though the relative rates
presented in Fig. 4 show that only about 55-68% of prothrombin
initially is processed via channeling, the calculated initial time
courses of thrombin generation through different pathways show that, at
all lipid concentrations, the presence of factor Va and
PS-containing membranes causes thrombin formation to proceed
exclusively through channeling, at least at the early stage of the
reaction. This finding is depicted graphically in the right-hand
panels of Fig. 5 (channeling, small filled circles; Pre2 intermediate, dashed lines; and MZIIa
intermediate, small filled triangles). Although these two
observations at first might seem to be inconsistent, they are not
because in the presence of factor Va and PS membranes,
channeling occurs at a rate similar to those of meizothrombin formation
and activation to thrombin (Table I). Under these circumstances, a
two-step, sequential mechanism will be roughly 100 times slower than a
channeling mechanism. By contrast, in the presence of PS membranes but
not factor Va, MzIIa formation and channeling
had comparable rate constants, but MzIIa activation
occurred roughly 100 times faster (2). Thus, the presence of factor
Va on a PS membrane enhances the rates of channeling and
MzIIa formation substantially (~1700-fold at 50 µM lipid; Table I) but enhances the rate of
MzIIa activation by only 20-fold (Table I). This converts
prothrombin activation from a process that proceeds via both
MzIIa and channeling in the presence of PS membranes (2) to
one that proceeds exclusively via channeling when factor Va
is bound to the membranes (Fig. 5).
The most remarkable effect of factor Va is that it makes
factor Xa an efficient channeling enzyme over the two
orders of magnitude range of lipid concentrations we considered
(open squares in Fig. 4E). This could be a
great advantage in vivo where the concentrations of
platelet membrane vesicles (20, 21) promoting prothrombin activation
could vary greatly within a wound area, even though it would be
important to have stable production of thrombin under all conditions.
Channeling occurs in the absence of factor Va, but it is
limited to a narrow range of membrane concentrations (cross-filled squares, Fig. 4E) (2). The ability
of factor Va to support channeling at higher membrane
concentrations seems to be responsible for the maximal overall
activation rate seen at 100-200 µM lipid in the
inset in Fig. 2 compared with the minimal lipid dependence
seen for individual proteolytic steps of activation (Fig. 4,
A and B). It is not yet clear whether the ability
of PS membranes and factor Va to support channeling at high
membrane concentrations is a result of the cooperative effect of the
two cofactors or the binding of PS to factor Va to enhance
its channeling activity. Clarification of this issue must await
a full analysis, analogous to the ones presented here and elsewhere (1,
2) of channeling in the newly described solution-assembled
prothrombinase (22).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin, which is linked
by a disulfide bond to the B chain of thrombin (residues 323-581). As
two cleavages are required in the prothrombin molecule to produce
thrombin, two kinetic pathways are possible in the activation of
prothrombin (Fig. 1). A single cleavage at
Arg273-Thr274 (reaction A) yields
fragment 1.2 (F1.2)1 and an
intermediate species known as prethrombin 2 (Pre2). Pre2 is composed of
the A and B chains of thrombin with the crucial bond at
Arg322-Ile323 intact, making Pre2
catalytically inactive. Further proteolysis at this bond (reaction
B) converts prethrombin 2 to
-thrombin. Alternatively, a
single cleavage in the prothrombin molecule at Arg322-Ile323 (reaction C) produces
a species known as meizothrombin (MzIIa), a catalytically
active intermediate. Further proteolysis of meizothrombin by factor
Xa at the Arg273-Ile274 bond
(reaction D) yields as products F1.2 and catalytically
active
-thrombin. In two recent studies, a third possible pathway of activation, processive proteolysis of the intermediate without release
from the enzyme, was demonstrated (reaction E in Fig. 1) (1,
2).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of human prothrombin
activation. Prothrombin consists of a membrane-binding
N-terminal domain (Fragment 1.2) and a C-terminal
catalytic domain (Prethrombin 2). Two peptide bonds
(Arg273-Thr274 (site
(2)) and
Arg322-Ile323 (site
(1))) must be hydrolyzed by factor Xa to
activate prothrombin. This creates two possible pathways of activation,
A (cut (2)) and B (cut (1))
as well as C (cut (1)) and D (cut
(2)), and also creates two possible released intermediates,
Pre2 and MzIIa. A third possible pathway of activation is
envisioned (E, fast consecutive cuts (1) and
(2)) in which no intermediate is released, i.e.
extensive channeling occurs.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Initial rate of prothrombin activation is
shown as a function of added factor Va in the presence of
25/75 PS/DOPC vesicles (LUV). Activation was initiated by
stopped flow mixing of equal volumes of prothrombin (final
concentration of 0.5 µM) and DAPA (final concentration of
2.5 µM) in one syringe with prothrombinase complex in
another syringe. The prothrombinase was assembled using 2.5 nM factor Xa, 0-30 nM factor
Va, and 20 µM (circles), 50 µM (squares), 100 µM
(diamonds), 200 µM (triangles), and
2000 µM (inverted triangles) 25/75 PS/DOPC
vesicles (LUV). A plot of the initial rates of prothrombin activation
(Vini) obtained in the presence of 30 nM factor Va versus lipid
concentration ([PL]) is shown in the
inset.
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Fig. 3.
Initial rates of IIa formation
from intermediates as a function of substrate concentrations.
Activation was initiated by stopped flow mixing of equal volumes of
substrate and DAPA (in a ratio of 1/5) in one syringe with
prothrombinase complex in another syringe. The final concentrations in
the reaction chamber were 2.5 nM factor Xa and
30 nM factor Va. The substrates were
MzIIa (A) and an equimolar mixture of Pre2 and
F1.2 (B). The concentration of lipid was 20 µM
(circles), 50 µM (squares), 200 µM (triangles), 1200 µM
(open inverted triangles), and 2000 µM
(open hexagons).
View larger version (18K):
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Fig. 4.
First order rate constants (Ra,
Rb, Rc, Rd, and Re) for
the five proteolytic events of prothrombin activation as a function of
phospholipid concentrations. Panel A contains
experimentally determined rates (filled symbols) of second
steps, whereas Panel B shows rates of initial steps obtained
from modeling (open symbols). Rates of
Arg273-Thr274 cuts (reactions A and D) are
recorded as triangles, whereas
Arg322-Ile323 cuts (reactions B and C) are
shown as circles. The channeling rate,
Re, is shown in squares.
Panels C-E show relative rates of the three
possible initial prothrombin activation steps: Pre2 formation
(R Ra/(Ra + Rc + Re);
triangles, panel C); MzIIa formation
(R
Rc/(Ra + Rc + Re); circles, panel D);
and channeling
(R
Re/(Ra + Rc + Re); squares, panel E).
The relative rates of channeling without factor Va also are
reproduced (cross-filled symbols) from the findings of Wu
et al. (2). The X symbols on the ordinates mark
the relative rates in the presence of factor Va and in the
absence of membrane, as reported by Boskovic et al.
(1).
View larger version (30K):
[in a new window]
Fig. 5.
Best fits of simulations to observed
prothrombin proteolysis data at 20, 50, 200, 1200, and 2000 µM PS/DOPC LUV concentrations. Left
panel, prothrombin (0.5 µM) proteolysis
catalyzed by Factor Xa (2.5 nM) in the presence
of 5 mM CaCl2, factor Va (30 nM), and different concentrations of PS/DOPC (25/75)
([PL]) at 37 °C. The formation of thrombin plus
MzIIa (circles) and the appearance of
MzIIa (squares) during prothrombin proteolysis
was followed by generation of amidolytic activity toward the synthetic
substrate S-2238 at all lipid concentrations and by SDS-PAGE
(open symbols) at 200 µM (see "Methods").
Pre2 could not be detected by SDS-PAGE (open triangles). All
experimental curves were fitted simultaneously using the
SigmaPlot simulation program to obtain estimates for the rate constants
Ra, Rc, and Re (channeling).
Simulated curves were calculated using best fit kinetic
parameters and are shown as solid lines. Right
panel, thrombin formation through different pathways: total
IIa (solid line), IIa formed through
Pre2 pathway (dashed line), IIa formed through
MzIIa pathway (small triangles), and
IIa formed through channeling (small
circles).
1) consistent with the fact that Pre2 was never detected.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1s
1 for reactions B and D,
respectively (6).
Kinetic parameters (kcat/Km) for activation of
prothrombin and its intermediates
1 s
1. The changes are given as the
ratio of the absolute rate of the current row to the rate of the
previous row rounded to two significant figures. PC,
phosphatidylcholine.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant HL45916 (to B. R. L.).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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, CB # 7260, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260. Tel.: 919-966-5384; E-mail: uncbrl@med.unc.edu.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M208423200
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ABBREVIATIONS |
---|
The abbreviations used are:
F1.2, fragment 1.2;
DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine;
LUV, large unilamellar vesicles;
S-2238, H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline
dihydrochloride (synthetic substrate for thrombin);
S-2765, N--benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline
dihydrochloride (synthetic substrate for factor Xa);
DOPC, dioleoyl-phosphatidylcholine;
Pre1, prethrombin 1;
Pre2, prethrombin 2;
IIa, thrombin;
MzIIa, meizothrombin;
PS, phosphatidylserine.
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REFERENCES |
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