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Membrane Binding by Prothrombin Mediates its Constrained Presentation to
Prothrombinase For Cleavage.
Harlan N. Bradford†, Steven J. Orcutt
†¶ and Sriram Krishnaswamy
†‡§
†Research Institute, Children’s Hospital of Philadelphia &
‡Department of Pediatrics, University of
Pennsylvania, Philadelphia, PA 19104
Running Head: Membrane Binding and Zymogen Activation
§Correspondence: Sriram Krishnaswamy, Children’s Hospital of Philadelphia, 310 Abramson, 3615 Civic
Center Boulevard, Philadelphia, PA 19104 Voice: (215) 590-3346 Email:[email protected]
Background: Prothrombin variants lacking
membrane binding have probed the contribution
of the substrate-membrane interaction in
thrombin formation by prothrombinase.
Results: Loss of membrane binding yields
modest changes in rate but affects the pathway
for substrate cleavage.
Conclusions: Membrane binding by the
substrate constrains the presentation of
prothrombin for cleavage by prothrombinase.
Significance: New insights into how the action
of prothrombinase on prothrombin is regulated.
SUMMARY
Long-standing dogma proposes a profound
contribution of membrane binding by
prothrombin in determining the rate at which
it is converted to thrombin by
prothrombinase. We have examined the
action of prothrombinase on full-length
prothrombin variants lacking -
carboxyglutamate modifications (desGla)
with impaired membrane binding. We show
an unexpectedly modest decrease in the rate
of thrombin formation for desGla
prothrombin but with a major effect on the
pathway for substrate cleavage. Using desGla
prothrombin variants in which the individual
cleavage sites have been singly rendered
uncleavable, we find that loss of membrane
binding and other Gla-dependent functions in
the substrate leads to a decrease in the rate of
cleavage at Arg320
and a surprising increase
in the rate of cleavage at Arg271
. These
compensating effects arise from a loss in the
membrane component of exosite-dependent
tethering of substrate to prothrombinase and
a relaxation in the constrained presentation
of the individual cleavage sites for active site
docking and catalysis. Loss of constraint is
evident as a switch in the pathway for
prothrombin cleavage and the intermemdiate
produced but without the expected profound
decrease in rate. Extension of these findings
to the action of prothrombinase assembled on
platelets and endothelial cells on fully
carboxylated prothrombin reveal new
mechanistic insights into function on
physiological membranes. Cell-dependent
enzyme function is likely governed by a
differential ability to support prothrombin
binding and the variable accumulation of
intermediates from the two possible pathways
of prothrombin activation.
Thrombin, the pivotal proteinase of blood
coagulation, is produced by the proteolytic
activation of prothrombin (1). Prothrombinase,
the physiological catalyst for this reaction, is an
enzyme complex assembled by reversible
protein-protein and protein-membrane
interactions between the proteinase, factor Xa,
its cofactor, factor Va and membranes
containing phosphatidylserine (2). Membrane-
binding by the cofactor and proteinase, mediated
by specific domains, greatly enhances both the
kinetics and thermodynamics of prothrombinase
assembly (1,2). Reactions between membrane-
bound Xa and Va proceed with very high rate
constants because of approximation arising from
dimensional and orientational restrictions (3).
Tight binding interactions, further enhanced by
linkage effects allow the membrane-bound
proteins to bind each other at sub-nanomolar
concentrations (4). Prothrombin also binds
reversibly to these membranes in a Ca2+
-
http://www.jbc.org/cgi/doi/10.1074/jbc.M113.502005The latest version is at JBC Papers in Press. Published on August 12, 2013 as Manuscript M113.502005
Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
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dependent fashion by virtue of -
carboxyglutamate residues at its N-terminus (5).
Consequently, the reversible interaction between
substrate and membranes is also expected to
result in its accelerated and oriented delivery to
membrane-bound prothrombinase (1,6). These
features are considered necessary for normal
hemostasis, allowing accelerated thrombin
formation on activated platelets or other cells
expressing phosphatidylserine on their outer
leaflet and localized at the site of vascular
damage (7).
The essential role for membrane binding in
blood coagulation is obvious from bleeding
associated with deficiencies in the vitamin K-
dependent reactions required for the post-
translational carboxylation of specific
glutamates to form -carboxyglutamate (8).
Warfarin and its derivatives, which interfere
with -carboxylation, are widely used for the
therapeutic control of thrombosis and need
careful dosing to avoid bleeding (9). However,
interference with -carboxylation impacts Ca2+
-
and membrane binding of all the vitamin K-
dependent coagulation proteins (9). In the case
of prothrombinase, this would impair membrane
binding by Xa as well as prothrombin, thereby
impacting the assembly of prothrombinase as
well as membrane-dependent substrate delivery.
Thus, warfarin effects do not permit incisive
inferences regarding the importance of the
substrate-membrane interaction in function.
The contribution of membrane-binding by
prothrombin towards thrombin formation has
been extensively investigated (6). Although
some controversies linger, it is widely accepted
that membrane binding by prothrombin plays an
essential role in affecting the rate of thrombin
formation. Ideas in the field are dominated by an
influential model proposed almost 30 years ago
based on co-concentration effects of
prothrombinase and prothrombin in a
microscopic sub-space bordering the membrane
surface (10,11). The model predicts a
catastrophic decrease in rate, at least by a factor
of 3,500, associated with a loss in membrane
binding by the substrate (10). The proposed
magnitude of functional loss is not fully
consistent with kinetic studies done in solution
or with various prothrombin fragments lacking
the membrane binding domain (12,13).
However, some of those interpretations could be
compromised by unanticipated effects associated
with proteolytic elimination of approximately
30% of the polypeptide structure of
prothrombin.
A complexity, insufficiently considered in the
foregoing, arises from the fact that the
conversion of prothrombin to thrombin requires
the action of prothrombinase on two sites in two
sequential enzyme catalyzed reactions (14). For
prothrombinase assembled on phospholipid
vesicles containing an optimal fraction of
phosphatidylserine, the reaction exclusively
proceeds by initial cleavage at Arg320
followed
by cleavage at Arg271
yielding meizothrombin
(mIIa)1 as an intermediate (for clarification, see
Scheme II below) (15,16). The basis for such
ordered cleavage, despite the fact that both sites
are accessible for proteolysis, lies in the
constrained way the substrate is tethered to the
enzyme through exosite interactions (17,18).
These interactions, between enzymic sites
removed from the active site and sites on the
substrate distant from the cleavage site, facilitate
the constrained presentation of the cleavage sites
for docking at the active site of Xa within
prothrombinase (14,18,19). While such ideas
have centered on protein-protein contacts, the
substrate-membrane interaction in the vicinity of
prothrombinase would also constitute a
component of exosite binding that might further
contribute to the constrained presentation of the
substrate to prothrombinase. This idea is
supported by previous studies hinting at an
altered order of bond cleavage of prothrombin
species isolated from the blood of warfarin-
treated cows and by studies done in the absence
of membranes (13,20).
We now use newer developments in the
understanding of substrate recognition by
prothrombinase as a framework to investigate
this problem. We employ a series of full length
recombinant prothrombin variants lacking -
carboxylation (desGla, dG) to permit an
assessment of the contribution of membrane
binding by the substrate on all four possible
cleavage reactions. Our findings yield surprising
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insights into how membranes regulate function
with bearing on prothrombinase function on
physiologic membranes that likely express
insufficient amounts of phosphatidylserine for
robust prothrombin binding.
EXPERIMENTAL PROCEDURES
Reagents—Human plasma used for protein
isolation was a generous gift of the
Plasmapheresis Unit of the Hospital of the
University of Pennsylvania. D-phenylalanyl-L-
proline-L-arginine chloromethyl ketone (FPRck,
Calbiochem), Alexa488-maleimide, (Invitrogen),
dansylarginine-N-(3-ethyl-1,5-pentanediyl)-
amide (DAPA, Haematologic Technologies) and H-D-phenylalanyl-L-pipecolyl-L-arginine-p-
nitroanilide (S2238, DiaPharma) were from the
indicated suppliers. Concentrations of stock
solutions prepared in water were determined
using = 8,270 M
-1.cm
-1 (S2238) and =
4,010 M-1
.cm-1 (DAPA). The acetothioacetyl
adduct of FPRck (ATA-FPRck) was prepared by
reacting FPRck to completion with an excess of
succinimidyl acetothioacetate (Invitrogen) and
purification as previously described (21). Small
unilamellar phospholipid vesicles (PCPS)
composed of 75% (w/w) hen egg L-α-
phosphatidylcholine and 25% (w/w) porcine
brain L-α-phosphatidylserine (Avanti) were
prepared and quality controlled as described
(12). Large unilamellar vesicles containing
97.5% (w/w) L-α-phosphatidylcholine and 2.5%
(w/w) L-α-phosphatidylserine were prepared by
extrusion and quality controlled as before (15).
Concentrations of PCPS were determined by
hydrolysis and colorimetric determination of
inorganic phosphate (22). Kinetic measurements
were conducted in 20 mM Hepes, 0.15 M NaCl,
0.1% (w/v) polyethyleneglycol (Mr=8K), 5 mM
CaCl2 pH 7.5 (Assay Buffer) at 25o
C. Protein
substrates were exchanged into Assay Buffer by
centrifugal gel-filtration before use.
Proteins—Prothrombin, factor X and factor V
were isolated from human plasma by established
procedures (23,24). Factor Xa and factor Va
were purified and quality controlled following
preparative activation of factor X by the purified
activator from Russell’s viper venom or of factor
V by thrombin as before (15,25). Thrombin and
prethrombin 2 (P2) were purified following
preparative proteolysis of prothrombin as
described (26). Recombinant tick anticoagulant
peptide (rTAP) was prepared as before (27).
Fully carboxylated wild type recombinant
prothrombin (IIWT) and its variants containing
Gln replacing Arg271
(IIQ271), Gln replacing
Arg320
(IIQ320) and Gln replacing Arg271
and
Arg320
(IIQQ) were produced in stably
transformed HEK293 cell lines and purified as
before (15). Uncarboxylated prothrombin
variants (dG-IIWT, dG-IIQ271, dG-IIQ320 and dG-
IIQQ) were produced by culturing the same stable
cell lines in serum free media without vitamin K.
Purification of these forms employed the scheme
utilized for the carboxylated forms except that
the pool from the first chromatography step was
precipitated with 80% saturated (NH4)2SO4
instead of barium citrate and their elution
positions in the subsequent chromatography
steps were clearly different from the
carboxylated counterparts. All recombinant
prothrombin variants were quality controlled by
N-terminal sequencing, time-of-flight mass
spectrometry and quantitative analysis of -
carboxyglutamate following base hydrolysis
(28). Uncarboxylated F12 (dG-F12) was
obtained by preparative activation of dG-IIS195A
by prothrombinase and purified using
procedures similar to those described for
carboxylated F12. Uncarboxylated prothrombin
(dG-IIWT, 32 µM) in 20 mM Hepes, 0.15 M
NaCl , 5 mM CaCl2 and 5 µM CoCl2 was
preparatively activated with 0.42 µM ecarin in
the presence of ATA-FPRck (0.16 mM) and the
resulting inactivated meizothrombin (dG-mIIai)
was isolated as before (15). A fraction of dG-
mIIai was reacted with an excess of Alexa488
maleimide in the presence of 0.1 M NH2OH and
the resulting singly labeled fluorescent adduct
(dG-mIIaAlexa488) was purified as described
(15,29).
The cDNA encoding Ecarin with Ser170 replaced
with Pro, a His6 extension at the COOH
terminus and flanked by HindIII and EcoR1 sites
was synthesized based on the published
sequence (30). Digestion with these enzymes
allowed for cloning into pcDNA 3.1+
(Invitrogen) and subsequent transfection of AV-
12 cells to obtain stable clones. Highest
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producing clones were identified by
measurements of prothrombin activation and
verified by western blotting using a mouse anti-
His-4 antibody (Qiagen). Ecarin was expressed
on a large scale in serum free medium
essentially following procedures described for
the prothrombin variants (15). Purification was
done by an initial capture step with Q-Sepharose
Fast Flow (Pharmacia) followed by
chromatography using Poros HQ-150 (Applied
Biosystems) using buffers and gradient
conditions described for prothrombin
purification (15). The pool containing Ecarin
activity was dialyzed into 20 mM Hepes, 10 mM
imidazole, 0.15 M NaCl, pH 7.4 and applied to a
1 ml HisTrap FF column (GE Healthcare)
charged with Ni2+
. Bound protein was eluted
with the same buffer containing 500 mM
imidazole, dialyzed into 20 mM Hepes, 0.15 M
NaCl, 5 µM CoCl2, pH 7.4 and stored frozen in
aliquots.
Protein concentrations were determined using
the following molecular weights and extinction
coefficients ( ): Xa, 45,300, 1.16 (31); all
prothrombin variants, 72,000, 1.47 (32);
thrombin or P2, 37,500, 1.89 (26); Va, 175,000,
1.78 (33); F12, 34,800, 1.2 (32); rTAP, 6,980,
2.56 (27); ecarin, 88,000, 1.0.
Cleavage of Prothrombin Variants—Reaction
mixtures (800 µl) containing either 5 µM or 1.4
µM prothrombin variant, 30 µM DAPA, 30 µM
PCPS, 30 nM Va in Assay Buffer at 25o
C were
initiated by the addition of either 0.4 nM or 0.15
nM Xa. Aliquots (40 µl) withdrawn at the
indicated times were quenched by mixing with
16 µl of 0.2 M Tris, 6.4% (w/v) SDS, 32% (v/v)
glycerol, 0.04% (w/v) bromphenol blue, 50 mM
EDTA, 50 mM dithiothreitol, pH 6.8
supplemented with 36 µM FPRck. Following
heating at 85 °C for 5 min., samples were
subject to electrophoresis using 10% Tris-
Glycine gels (Invitrogen), and protein bands
were visualized by staining with Coomassie
Brilliant Blue R250 for experiments done at the
high substrate concentration or by the Colloidal
Blue stain and infra-red imaging for the low
substrate concentration (34). Quenched samples
from the low concentration data set were also
visualized following quantitative western
blotting as previously described (34). For
measurements of proteolytic activity, aliquots
(10 µl) were quenched by mixing with 40 µl of
Assay Buffer containing 1 µM rTAP and 50 mM
EDTA in place of CaCl2. The concentration of
proteinase formed was determined from initial
velocities of S2238 hydrolysis as described (35).
Kinetic Studies— Initial velocity measurements
of proteinase formation from dG-IIQ271 were
determined discontinuously as previously
described (15). Reaction mixtures (200 µl)
containing the indicated concentrations of dG-
IIQ271, 30 µM PCPS and 30 nM Va at 25 °C,
were initiated with 0.5 nM Xa. Aliquots (10 µl)
were removed at 0, 0.5, 1.0, 1.5, 2 and 3 min
following initiation and quenched by mixing
with 40 µl Assay Buffer containing 1 µM rTAP
and 50 mM EDTA in place of CaCl2. The
concentration of proteinase formed in the
quenched samples was inferred from the rate of
S2238 hydrolysis and its linear dependence on
known concentrations of thrombin established
with each experiment. Initial velocities of
proteinase formation were then determined from
the linear appearance of proteinase with time
established with at least 4 of the 6 quenched
samples. For experiments with alternate
substrates or inhibitors, initial velocities with
increasing concentrations of dG-IIQ271 were
determined in the presence of the indicated fixed
concentrations of dG-IIQQ or dG-IIQ320.
Initial velocities for the conversion of the dG-
F12/P2 mixture to thrombin (cleavage at Arg320’
)
were determined in the same way with varying
concentrations of P2 and the dG-F12
concentration maintained at either 1.2 or 1.5
molar equivalents of P2.
The kinetics of action of prothrombinase on dG-
mIIai (cleavage at Arg271’
) was inferred from the
fluorescence increase seen upon conversion of
dG-mIIaAlexa488 to thrombin analogous to the
change seen in fluorescein modified but fully
carboxylated mIIa (15). Reaction mixtures (200
µl), prepared in black 96-well plates (Corning),
contained 0.1 µM dG-mIIaAlexa488 and increasing
concentrations of dG-mIIai to achieve the
indicated total concentration of substrate, 30 µM
PCPS and 30 nM Va in Assay Buffer. Reactions
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were initiated by the addition of 1 nM Xa and
product formation was monitored at 25o
C in a
Spectramax Gemini (Molecular Devices) using
EX=488 nm and monitoring broadband
fluorescence with a 510 nm long pass filter in
the emission beam. Initial, steady state velocities
of the fluorescence increase were converted to
concentration terms using the limits of the
progress curves as described (15,29).
Platelets— Platelets were isolated from blood
freshly drawn by venipuncture from aspirin-free,
volunteer donors following written consent
using a protocol approved by the institutional
review board. Blood (45 ml) was drawn into 5
ml of anticoagulant composed of 65 mM citric
acid, 85 mM Na3citrate, 2% (w/w) dextrose.
Platelet rich plasma was obtained by
conservative aspiration of part of the upper layer
after centrifugation (1000xg, 18 min, 25 o
C) in a
swinging bucket rotor. Aliquots of platelet rich
plasma (5 ml) were applied in parallel to
columns of sepharose 2B-CL300 (2.5x10.5 cm,
50 ml) equilibrated in 3.8 mM HEPES, 0.38 mM
Na2HPO4, 137 mM NaCl, 2.68 mM KCl, 0.98
mM MgCl2, 5.55 mM dextrose, 0.2% (w/v) BSA
pH 7.3 (Hepes/Tyrodes/BSA). Platelets eluting
in the void volume, well separated from the
plasma fraction, were pooled and counted using
a Hemavet 1500FS (CDC Technologies). Yields
were typically ~300,000 platelets/µl blood and
were essentially free of other cell types. Platelets
were maintained at room temperature and used
in experiments within ~2 hr of isolation.
Endothelial Cells — Human umbilical vein
endothelial cells (HUVECs, 1-7 passages) were
a generous gift of Dr. Long Zheng, Children’s
Hospital of Philadelphia. The cells were grown
to near-confluence in 6 well plates using EBM-2
medium (Lonza). Prior to the experiment,
HUVECs were washed 3 times with 3 ml of
HEPES/Tyrodes/BSA supplemented with 5 mM
CaCl2. Following washing, 0.5 ml of the same
buffer was added to the wells in preparation for
thrombin activation.
Prothrombin Cleavage on Cell Membranes—
Freshly purified platelets, adjusted to 2x108/ml,
were supplemented with 1 M stocks of Tris pH
7.5 and CaCl2 to achieve final concentrations of
20 mM and 5 mM respectively. Platelets were
activated by thrombin (10 nM, 3 min) followed
by the addition of 12 nM hirudin. Reaction
mixtures (800 µl, 25 °C) were prepared by
mixing equal volumes of the activated platelet
preparation and a solution prepared in Assay
Buffer containing 2.8 µM prothrombin variant,
60 µM DAPA and 60 nM Va. Cleavage
reactions were initiated by the addition of 0.5
nM Xa, sub-sampled at the indicated times for
SDS-PAGE analysis as above and analyzed by
quantitative western blotting (34). Washed
HUVECs were activated with thrombin (20 nM,
3 min) followed by the addition of 25 nM
hirudin. Reaction mixtures (1 ml) were prepared
in the wells of the 6-well plate by the addition of
2.8 µM prothrombin variant, 60 µM DAPA and
60 nM Va in Assay Buffer (0.5 ml). The
activation reaction was initiated by the addition
of 0.2 nM Xa and allowed to proceed with rotary
shaking (400 rpm, Thermomixer R, Eppendorf )
at 25 °C. Reaction mixtures were sampled at the
indicated times for SDS-PAGE analysis as
above and analyzed by quantitative western
blotting (34).
Data Analysis— Concentrations of PCPS and
Va were chosen to saturate Xa based on the
measured equilibrium constants for
prothrombinase assembly (4). The concentration
of enzyme was considered equal to the limiting
concentration of Xa in each experiment and used
to normalize initial velocities.
Quantitative densitometry of stained gels or
from quantitative western blotting was
performed as previously validated and described
in detail (12,15,16,34). Estimates of initial
velocity from progress curves constructed in this
way were obtained by analysis according to the
logarithmic approximation (36). Observed
steady state kinetic constants were determined
by non-linear least squares analysis according to
the Henri-Michaelis-Menten equation. Global
analysis according to Scheme I to obtain
intrinsic constants was done with the rapid
equilibrium assumption using Dynafit (Biokin)
(37). Provided this assumption holds, the
kinetics for cleavage at the individual sites in
dG-IIQ271 and dG-IIQ320 can be described by the
Henri-Michaelis-Menten equation (18,35).
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Intrinsic constants for either of the two cleavage
events are related to the observed kinetic
constant by:
(1)
( ⁄ )
(2)
where KEXO is the cleavage site independent
equilibrium dissociation constant for exosite
binding. Kmobs, (V/E)obs, Ks* and kcat would be
subscripted with either 271 or 320 depending on
the single cleavage site substrate variant used.
These relationships were used to compute terms
otherwise inaccessible from global analysis
according to Scheme I. All fitted constants are
listed ± 95% confidence limits. Errors from
fitted constants were propagated for the
calculated terms (38). With the exception of the
data set obtained for the cleavage of dG-IIQ271 or
dG-IIQ320 by Xa partially saturated by µM
concentrations of Va in solution, the reported
data are representative of two or more
experiments performed at a comparable level of
detail, frequently with different protein
preparations.
RESULTS
Prothrombin Variants— As in previous work,
we have employed recombinant variants of
prothrombin in which the individual cleavage
sites at Arg271
and Arg320
have been rendered
uncleavable, either singly or in combination, by
substitution with Gln (15). Culture of the stable
cell lines expressing these variants in the
absence of vitamin K yielded fully
uncarboxylated protein. Quantiative analysis of
-carboxyglutamic acid (Gla) content following
base hydrolysis yielded the expected 10 moles
Gla/mole protein for the proteins expressed in
the presence of vitamin K (15). In contrast, a Gla
peak was undetectable for the dG variants
providing an upper limit estimate of 0.3
Gla/mole protein. N-terminal sequencing
verified correct processing of the propeptide
during secretion and mass spectrometry yielded
the expected molecular weight for full-length
prothrombin (not shown). A desGla variant
(dG-IIWT) produced minimal changes in light
scattering when titrated with increasing
concentrations of PCPS. In a parallel
experiment, the fully carboxylated protein (IIWT)
produced a large and saturable change in light
scattering intensity signifying binding (not
shown). In accordance with the literature (20),
we estimate that the desGla variants of
prothrombin bind to PCPS membranes with at
least 500-fold weaker affinity than the fully
carboxylated protein.
Activation of Prothrombin— The contribution
of prothrombin -carboxylation to its function as
a substrate for prothrombinase was first assessed
by progress curves of thrombin formation (Fig.
1). As expected, prothrombinase yielded
thrombin at a lower rate from dG-IIWT in
comparison to IIWT (Fig. 1). However, in
contrast to the profound reduction in rate
expected based on the literature, we were
surprised to find that the initial rate of thrombin
formation from dG-IIWT was only modestly
lower by a factor of ~5 (10).
Progress curves were further analyzed by SDS-
PAGE and protein staining (Figs. 2A & 2B).
The cleavage patterns observed for IIWT (Fig.
2A) were consistent with sequential cleavage at
Arg320
to yield mIIa followed by cleavage at
Arg271
to produce thrombin as previously
reported (15). The signature features of this
cleavage pathway are the transient accumulation
of the F12-A fragment, the delayed appearance
of F12 following the conversion of mIIa to
thrombin and the lack of any detectable
prethrombin 2 (P2). In contrast, consumption of
dG-IIWT, only modestly slower than that of IIWT,
yielded no obvious evidence for F12-A
formation but instead produced P2 in a transient
way (Fig. 2B). Thus, the modestly decreased
rate of thrombin production from dG-IIWT (Fig.
1) obscures major differences in the way the
substrate is cleaved by prothrombinase.
Nevertheless, proteinase formation as judged by
peptidyl substrate cleavage from either
carboxylated or uncarboxylated prothrombin
matches well with the appearance of the
thrombin B chain determined by quantitative
densitometry (Fig. 1).
Cleavage of the Individual Bonds within
Prothrombin— Initial evidence implying altered
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selectivity for bond cleavage within dG-IIWT by
prothrombinase was further pursued by studies
with carboxylated and uncarboxylated versions
of the cleavage site mutants (Fig. 2 C-F). IIQ271
was rapidly consumed by prothrombinase from
cleavage at Arg320
to produce mIIa (Fig. 2C).
The use of dG-IIQ271 yielded much slower
cleavage at Arg320
(Fig. 2D). Cleavage at Arg271
in IIQ320 proceeded very slowly (Fig. 2E)
reflecting the preference of prothrombinase for
cleavage at Arg320
in intact prothrombin (15).
We were surprised to find that dG-IIQ320 was
cleaved at a greater rate than IIQ320 (Fig. 2F).
While this observation has bearing on the altered
cleavage patterns seen with dG-IIWT, it also
illustrates that loss of membrane binding by the
substrate is not uniformly deleterious and
unexpectedly produces a gain in a selected
aspect of function.
Because F12 from the desGla variants migrates
as an indistinct smear, initial studies were
conducted at 5 µM substrate to facilitate reliable
interpretation of SDS-PAGE analyses (Fig. 2).
To rule out the possibility that our unexpected
findings may reflect a peculiarity of this choice
in concentration, we pursued further work at the
physiological concentration of prothrombin (1.4
µM) using infra-red detection of stained gels or
by quantitative western blotting with near-IR
fluorescence detection (34). The findings were
equivalent, borne out by the quantitative analysis
of prothrombin consumption from the two types
of measurements (Fig. 3). Loss of membrane
binding in IIWT only yielded a modest ~2.5 fold
decrease in initial rate of prothrombin
consumption (Fig. 3A). This modest decrease
arose from opposing effects producing a larger
decrease in rate (~15-fold) for cleavage at Arg320
in dG-IIQ271 (Fig. 3B) but an increase in rate (~4-
fold) for cleavage at Arg271
in dG-IIQ320 (Fig.
3C).
Initial rates of prothrombin consumption (Table
1) illustrate that prothrombinase consumes
carboxylated prothrombin by preferential
cleavage at Arg320
with a minor contribution
from cleavage at the alternate site. In contrast,
prothrombinase acts on dG-IIWT at a slightly
reduced rate but by preferential cleavage at
Arg271
while cleavage at Arg320
also proceeds at
a significant rate. This carboxylation-dependent
switch in selectivity, thus far interpreted to
reflect the contribution of membrane binding by
prothrombin, accounts for the change in
cleavage patterns seen in the action of
prothrombinase on IIWT and dG-IIWT (Fig. 2 A &
B).
Altered Cleavage Site Selectivity in desGla
Prothrombin— Further kinetic analyses using
the initial velocity and rapid equilibrium
assumptions were pursued using a model
developed in previous studies with carboxylated
prothrombin (Scheme I). This model is rooted in
kinetic and equilibrium binding measurements
illustrating that equivalent exosite binding
interactions are responsible for tethering the
substrate to the enzyme regardless of cleavage
site (16,39). Active site docking by Arg271
in
exosite-bound IIQ320 or Arg320
in enzyme-bound
IIQ271 then occurs in a mutually exclusive way
for catalysis at the individual sites (Scheme I).
IIQQ is uncleavable because it cannot engage the
active site of prothrombinase (39). Alternate
substrate studies measured the rate of dG-mIIa
formation from varying concentrations of dG-
IIQ271 in the presence of different fixed
concentrations of dG-IIQ320 or dG-IIQQ (Fig. 4).
Global analysis of the data according to Scheme
I yielded adequate fits and permitted meaningful
assessment of KEXO, Ks*320, Ks*271 and kcat320
(Fig. 4). Because the product of dG-IIQ320
cleavage is not measured here, kcat271 was
estimated in combination with initial rates
determined from densitometry measurements
(Table 1). These parameters reflect the intrinsic
constants governing the ability of
prothrombinase to discriminate between the two
sites within intact but desGla prothrombin
(Table 2).
Comparisons with values previously determined
for fully carboxylated prothrombin (Table 2),
reveal how impaired membrane binding by
desGla prothrombin affects its recognition by
prothrombinase. For the desGla variants, exosite
binding is ~70-fold weaker than for the
carboxylated substrate. Membrane binding and
possibly other Gla-related functions play a major
role in the exosite-dependent tethering of
prothrombin to prothrombinase. In fully
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carboxylated prothrombin, the unimolecular
binding constant for active site docking by
Arg320
(Ks*320) is ~200-fold more favorable than
for Arg271
(Ks*271) (Table 2). Such
discrimination is lost in desGla prothrombin
wherein active site docking by Arg320
and Arg271
occurs with approximately equal and low
affinity (Ks*320 Ks*271 2) (Table 2). It would
appear that in desGla prothrombin, the
constrained presentation of the substrate for
active site docking has been altered, resulting in
the scrambling of preferential active site docking
of one cleavage site over the other. The intrinsic
kcat for cleavage at Arg320
remained unaffected
by the carboxylation state of the substrate.
Surprisingly, kcat271 was increased ~30-fold for
the desGla variant in comparison to
carboxylated prothrombin (Table 2). This
implies that loss of membrane binding and/or
other Gla-related functions mediated by the N-
terminus of prothrombin detectably affect distant
structures surrounding the Arg271
site.
Kinetics of the Four Possible Cleavage
Reactions— The observed steady state kinetic
constants for each of the four possible half-
reactions allows prediction of flux towards
thrombin formation. Steady state kinetic
constants were measured and/or calculated for
the action of prothrombinase at Arg271
and
Arg320
in otherwise uncleaved desGla
prothrombin (Table 2). Initial velocity studies
were also performed to examine the action of
prothrombinase at the remaining site in singly
cleaved desGla variants (Fig. 5). Steady state
kinetic constants for the cleavage at Arg271’
following initial cleavage at Arg320
were
obtained using dG-mIIa as substrate (Fig. 5A).
Cleavage at Arg320’
following initial cleavage at
Arg271
was assessed using P2 reconstituted with
dG-F12 (Fig. 5B). Equivalent initial velocities
obtained at two ratios of dG-F12:P2 support the
contention that P2 was essentially saturated with
dG-F12 at all concentrations used (Fig. 5B).
Observed steady state kinetic constants (Table
2) with representative values listed in Scheme II
provide the basis for the further consideration of
the action of prothrombinase on desGla
prothrombin.
Initial rates calculated at the physiological
concentration of prothrombin reveal that desGla
substrate forms are cleaved more slowly (by at
least a factor of 10) than the equivalent
carboxylated species for three of the four
possible half-reactions (Scheme II). The
exception is cleavage at Arg271
in intact
prothrombin that is increased for desGla
substrate in accordance with the rates detailed
for dG-IIQ320 (Table 1). Thus, asymmetry in
action of prothrombinase at Arg271
in intact
prothrombin versus mIIa in the carboxylated
forms is altered for the desGla substrate (15).
Consequently, ~80% of the rate of consumption
of desGla prothrombin is expected to result in
the formation of dG-F12/P2 and ~20% from the
formation of dG-mIIa. In accordance with
observations (Fig. 2), minor amounts of dG-
mIIa are predicted to accumulate as an
intermediate while abundant amounts of P2 are
expected as a long lasting intermediate given its
slower conversion to thrombin. Simulations with
these steady state kinetic constants (not shown)
indicate that the initial rate of proteinase
formation (mIIa+IIa) through the initial cleavage
of desGla prothrombin at Arg320
would be ~6-
fold greater than proteinase formation via the P2
pathway. This point highlights the dangers
inherent in inferring the predominant pathway
for product formation in such systems solely on
the basis of amounts of intermediate observed.
Correlation with Membrane Binding— A
diagnostic difference in the action of
prothrombinase on desGla versus fully
carboxylated substrate lies in the relative rates
for cleavage at the two sites within intact
prothrombin (Scheme II). In the case of
carboxylated substrate cleavage at Arg320
is ~30-
fold greater than cleavage at Arg271
. For desGla
prothrombin, cleavage at Arg271
is ~4-fold
greater than cleavage at Arg320
. At issue is
whether such effects can be wholly ascribed to a
loss in membrane binding by the desGla variants
rather than other -carboxyglutamate-dependent
effects on the substrate.
To address this uncertainty, we determined the
relative rates of cleavage at the two sites within
fully carboxylated prothrombin but in the
absence of membranes. Progress curves for the
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cleavage of IIQ320 and IIQ271 in the absence of
added membranes were constructed by
quantitative blotting following the addition of 1
nM Xa partially saturated (~50%) with 2 µM Va
(Fig. 6A). Comparable initial rates were
obtained for cleavage at the individual sites in
otherwise fully carboxylated intact prothrombin
(Fig. 6A). While the ~30-fold discrimination
seen for cleavage at Arg320
relative to Arg271
seen for the action of membrane bound
prothrombinase on carboxylated substrate was
abolished in the absence of membranes, this
reaction system only partly replicated the
findings seen with the desGla prothrombin
variants (Table 1). Cleavage at Arg271
in IIQ320
yielded v/E equivalent to that observed for the
action of membrane-bound prothrombinase on
dG-IIQ320. Therefore, the gain in function seen
for cleavage at Arg271
in intact desGla
prothrombin and only a fraction of the loss in
function seen for cleavage at Arg320
can be
replicated with fully carboxylated prothrombin
but in the absence of membranes. Since
essentially equivalent results were obtained
using dG-IIQ320 and dG-II271 (Fig. 6A) the data
implicate subtle differences between membrane
assembled prothrombinase and Xa saturated
with Va in solution as the reason for the
discrepancy. The findings are consistent with the
conclusion that membrane binding by the
substrate contributes in a major way to its
constrained presentation to prothrombinase and
the subsequent ability of the enzyme to
discriminate between the two cleavage sites.
Cleavage Site Selectivity on Physiological
Membranes— Relative rates of cleavage of
fully carboxylated IIQ271 and IIQ320 were
employed to investigate how the substrate-
membrane interaction may control
prothrombinase function on physiological
surfaces. This comparative approach allows
secure conclusions without knowledge of the
concentration of productively-bound
prothrombinase. Studies with activated platelets
revealed that cleavage at either Arg320
or Arg271
in intact prothrombin proceeded at equivalent
rates (Fig. 6B). This indicates that
prothrombinase assembled on the platelet
surface does not discriminate between the two
cleavage sites in prothrombin much as Xa bound
to Va in the absence of membranes (Fig. 6A).
Thus on the platelet surface, prothrombin
consumption is expected to proceed
approximately equally through the P2 and mIIa
pathways. In contrast, when assembled on
activated HUVECs, prothrombinase acted on
Arg320
in IIQ271 ~2-fold faster than it cleaved
Arg271
in IIQ320 (Fig. 6C). For these cells,
cleavage of prothrombin would partition
between the formation of mIIa and P2 in a ratio
of 2:1. The findings are consistent with a greater
contribution from membrane-binding by
prothrombin on HUVECs in comparison to
platelets. The exact contribution of the two
possible pathways to thrombin formation would
require knowledge of the kinetic constants for all
four cleavage reactions. However, the
implications are that variable contributions of
prothrombin cleavage via both possible
pathways will determine the amount of
intermediates observed in a cell type-dependent
fashion. Such observations might be expected to
correlate with the variable ability of the
membrane surfaces to support prothrombin
binding.
These predictions were tested by quantitative
western blotting to analyze IIWT cleavage by
prothrombinase assembled on platelets or
HUVECs previously activated with thrombin.
With platelets, bands corresponding to both P2
and mIIa (F12-A) accumulated transiently (Fig.
7A). In agreement with prior work, only a trace
band arising from mIIa (F12-A) was evident
(Fig. 7A) (40). In contrast, bands corresponding
to mIIa and P2 were more prominent with
HUVECs yielding approximately equal peak
concentrations of F12-A and P2 (Fig. 7B). In
order to relate these findings to the limited
ability of these activated cells to support
prothrombin binding, we pursued studies with
IIWT and synthetic vesicles containing 2.5%
(w/w) phosphatidylserine. The intent was to
replicate the low amounts of phosphatidylserine
expected in the outer leaflet of these cells which
would facilitate the high affinity interactions
required for prothrombinase assembly but
significantly impact prothrombin binding and
density of bound substrate (7). Accordingly,
bands corresponding to the transient formation
of both P2 and mIIa were observed with these
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synthetic membranes approximating the pattern
of cleavage seen with the activated cells (Fig.
7C). Taken together with predominant cleavage
of IIWT via the formation of mIIa seen with
vesicles containing 25% (w/w)
phosphatidylserine (Fig. 2A), the data present a
consistent picture implicating a limited but
variable ability of physiological membranes to
support substrate binding as a major determinant
of the pathway for prothrombin activation.
DISCUSSION Our strategy of using appropriate recombinant
prothrombin derivatives to probe the details of
the newly expanded model for the action of
prothrombinase on prothrombin now sheds new
light on a long-standing problem in coagulation
enzymology. Prevailing dogma predicts a
catastrophic decrease in the rate of action of
prothrombinase on prothrombin with impaired
membrane binding. Instead, by the use of full-
length prothrombin variants lacking Gla
modifications, we find only modest changes in
rate attributable to a loss in membrane binding
by the substrate at its physiological
concentration. These modest decreases belie
major mechanistic changes in the way
prothrombin is recognized by prothrombinase.
The findings reveal unexpected insights into
how the interaction between the substrate and
membrane in the vicinity of prothrombinase is a
major determinant of the constrained
presentation of prothrombin to the membrane-
assembled enzyme complex.
Membrane binding by the substrate, lost in the
desGla variants, contributes in a prominent way
to the initial exosite-driven tethering of the
substrate to prothrombinase (Scheme I). This
point is evident from a ~70-fold increase in KEXO
for the desGla variants. High selectivity (~200-
fold) for subsequent active site docking by
Arg320
over that of Arg271
seen with carboxylated
prothrombin is lost in the desGla variants. For
these species with impaired membrane binding,
the unimolecular binding constants for active
site docking indicate that two cleavage sites can
engage the active site of Xa within
prothrombinase with approximately equal
probability. Scrambling of selectivity for the two
cleavage sites is consistent with the loss of a
subset of exosite binding interactions that
otherwise position bound substrate in a
constrained way for preferential active site
docking by Arg320
. Coupled with the increased
intrinsic kcat271 these altered constraints in
exosite tethering of the substrate provide the
mechanistic basis for the qualitative change in
cleavage pattern observed in the action of
prothrombinase on IIWT versus dG-IIWT.
We intentionally qualify inferences of the
contribution of the substrate-membrane
interaction to function from studies with the
desGla variants. Altered function of these
variants could arise from a loss of additional
Gla-dependent functions beyond simply a loss in
membrane binding. In support of this possibility
are the findings with fully carboxylated
prothrombin variants and Xa partially saturated
with Va in solution that do not fully replicate the
observations made with desGla variants and
membrane assembled prothrombinase. However,
the fact that equivalent rates of consumption
were observed with IIQ271, IIQ320, dG-IIQ271 and
dG-IIQ320 in the absence of membranes suggests
that this small discrepancy most likely lies in
differences in the properties of Xa saturated with
Va in solution relative to the membrane
assembled enzyme. A second concern is
reflected by the previously documented
interaction between a peptide derived from the
prothrombin Gla domain and factor Va (41).
Again, compromised interactions between
desGla prothrombin variants and Va are unlikely
to be a major source of the present findings
based on the equivalent rates of consumption of
the fully carboxylated and desGla variants in the
absence of membranes (Fig 7A). However, the
need for cautious interpretation is suggested by
the obvious effects of the loss of Gla on the
intrinsic kcat for cleavage at Arg271
. It remains
uncertain whether this solely arises from
alterations in the constrained way that the
exosite-tethered substrate is presented to
prothrombinase.
Qualifications notwithstanding, our findings
ranging from studies with desGla prothrombin
variants, to studies with fully carboxylated forms
in the absence of membranes, and with
prothrombinase assembled on physiological
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surfaces or on synthetic membranes with
limiting amounts of phosphatidylserine are
internally consistent. They portray a unifying
picture wherein membrane binding plays an
essential role in dictating the presentation of
substrate to prothrombinase. Altered interactions
that affect these constraints are evident as
changes in the intermediates observed and an
apparent change in the pathway for prothrombin
cleavage. These ideas highlight, both empirically
and conceptually, the dangers in interpreting the
major pathway for thrombin formation from the
relative abundance, or lack thereof, of the two
intermediate species (40,42). They also likely lie
at the heart of some of the variable results and
recent controversies in the field (43-45).
Recent studies have proposed a fundamental
difference in the molecular architecture of
prothrombinase assembled on platelets relative
to synthetic membranes based on the formation
of prethrombin 2 as an intermediate and the lack
of observable intermediates in the fluid phase
(42). An adequate kinetic explanation for these
findings as well as justification for the major
pathway for thrombin formation will need to
await the determination of steady state kinetic
constants of each of the four possible cleavage
reactions. However, our results, point to the
inefficient binding of prothrombin to activated
platelet membranes as a parsimonious
mechanistic explanation. Parallels in the
selectivity of prothrombinase for prothrombin in
the absence of membranes and the ability of low
phosphatidylserine containing vesicles to
replicate the findings of prothrombin cleavage
seen on physiological membranes lend support
to this contention. Mechanistic issues aside, the
findings with HUVECs and platelets illustrate
that different cell types may variably support the
formation of mIIa during thrombin formation
perhaps reflecting a differential ability to
support prothrombin binding (46-48). Given its
zymogen-like character and skewed substrate
specificity of mIIa for the anticoagulant
activities of thrombin, this may have bearing on
the spatial regulation of coagulation (34).
Our findings contrast with prevailing dogma
associating impaired membrane binding by
prothrombin with a profound loss in the rate of
thrombin formation (10,11). Indeed, when
considered in the context of the four possible
reactions of prothrombin activation, the desGla
substrate variants exhibit a very modest decrease
in rate for three of the four cleavage reactions.
The increased rate observed for one of the steps
even more surprisingly illustrates that loss of
Gla-dependent functions is not uniformly
deleterious to the function of prothrombin as a
substrate for prothrombinase. Clearly, bleeding
associated with incorrect dosing with warfarin
cannot be ascribed to the faulty function of
desGla prothrombin as a substrate. However, the
reduced amounts of anticoagulant-specific mIIa
produced as an intermediate during the
activation of desGla prothrombin could have
bearing on warfarin-induced thrombosis (9).
It could also be argued that prothrombin
completely devoid of Gla is not a good facsimile
of partially carboxylated zymogen forms
expected in the blood of patients being
therapeutically treated with warfarin. This
argument is weak because an average Gla
content of 3 moles/mole protein seen in the non-
membrane binding fraction of prothrombin from
treated individuals reveals nothing of the
fractional distribution of the various
prothrombin isoforms with a Gla content
varying between 0 and 10 (49). It should be
noted that prothrombin devoid of Gla has been
isolated from the blood of warfarin-treated cows
(20). In keeping with the cooperative nature of
Ca2+
binding by the Gla residues, mixtures of
bovine prothrombin isoforms lacking 3-4 of the
full Gla complement of 10 were seen to exhibit
greatly impaired membrane binding (20).
A recent structure of a variant of prothrombin
lacking residues 1-44 encompassing the Gla
domain from the Di Cera laboratory has
provided evidence for flexibility in the linker
between the fragment 1 and fragment 2 domains
as well as the disorder in the region surrounding
the Arg271
cleavage site (50). Such flexibility
may provide an explanation for how the desGla
substrate tethered to prothrombinase, in the
absence of additional constraints imposed by
membrane binding, may allow active site
docking of two distant sites with approximately
equal probability. However, the associated claim
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that Arg271
is solvent accessible while Arg320
is
buried has yielded the major conclusion that
prothrombinase must cleave first at Arg271
before the Arg320
site becomes exposed (50).
This conclusion regarding the molecular
mechanism of prothrombin activation cannot be
reconciled with a large body of evidence
regarding the cleavage of IIWT by
prothrombinase on synthetic PCPS membranes
(15,17,43,51,52). The present work further rules
out the relevance of such a conclusion for
prothrombinase function on natural membranes
(platelets or HUVECs), in solution or even on its
action on desGla-IIWT. We are nonplussed by the
obvious disparity between the structure-based
proposal and the existing literature particularly
as the authors cite a paper from 1872 yet fail to
acknowledge a broad swathe of contemporary
work in the field that runs counter to their claim
(15,17,43,51-54).
In summary, our studies with desGla
prothrombin variants provide surprising insights
into a long-standing problem in coagulation
enzymology. The findings point to a prominent
role for the substrate-membrane interaction in
mediating exosite-dependent binding to
prothrombinase and the constrained presentation
of the substrate for cleavage. Our findings bear
on how the varied ability of physiological
membranes to affect such constrained
presentation of prothrombin might underlie the
regulation of the pathway for prothrombin
cleavage and the intermediate produced.
Whether such differential proportioning via the
formation of mIIa relative to the zymogen P2
has a significant regulatory role remains to be
established.
ACKNOWLEDGEMENTS
This work was supported by grants HL-074124 and HL-108933 (to SK) from the NIH. We acknowledge
the assistance of Long Zheng in providing HUVECs and expertise with their culture. We are also grateful
to Rodney Camire and William Church for critical review of the manuscript.
FOOTNOTES ¶ Present address: Janssen Pharmaceuticals Inc., Spring House, PA
1Abbreviations:
DAPA: dansylarginine-N-(3-ethyl-1,5-pentanediyl)-amide
desGla, dG: lacking -carboxyglutamic acid
F12: fragment 1.2 (prothrombin residues 1-271)
F12-A: fragment 1.2-A chain (prothrombin residues 1-320)
FPRck: D-phenylalanyl-L-proline-L-arginine chloromethyl ketone
ATA-FPRck: acetothioacetyl-FPRck
Gla: containing -carboxyglutamic acid
HUVECs: human umbilical vein endothelial cells
IIWT: wild type and fully -carboxylated human prothrombin
IIQ271: fully -carboxylated human prothrombin containing Gln in place of Arg271
IIQ320: fully -carboxylated human prothrombin containing Gln in place of Arg320
IIQQ: fully -carboxylated human prothrombin containing Gln in place of Arg271
and Arg320
mIIa: meizothrombin (prothrombin cleaved at Arg320
with residues 1-320 and 321-579 in
disulfide linkage)
mIIai: mIIa covalently inactivated with ATA-FPRck
P2: prethrombin 2 (prothrombin residues 272-579)
PCPS: small unilamellar vesicles composed of 75% (w/w) L-α-phosphatidylcholine and 25%
(w/w) L-α-phosphatidylserine
PS: L-α-phosphatidylserine
rTAP: recombinant tick anticoagulant peptide
S2238: H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide
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FIGURE LEGENDS
Figure 1. Proteinase Formation from Carboxylated and Uncarboxylated Prothrombin. Progress curves
for proteinase formation by prothrombinase were measured with 0.4 nM Xa, 30 nM Va, 50 µM PCPS and
5.0 µM IIWT () or dG-IIWT (). Product formation was measured by initial velocities of S2238
hydrolysis (closed symbols) or by the amount of B-chain determined by quantitative densitometry
following SDS-PAGE (open symbols). Product concentration was normalized to the initial concentration
of substrate and the lines are arbitrarily drawn.
Figure 2. Cleavage of Carboxylated and Uncarboxylated Prothrombin Variants. Samples from reaction
mixtures containing 0.4 nM Xa, 30 nM Va, 30 µM DAPA, 50 µM PCPS and 5.0 µM indicated
prothrombin variant were analyzed by SDS-PAGE following disulfide bond reduction and visualized by
staining with Coomasie Blue R250. Reaction times (in minutes) following initiation with Xa are listed on
the bottom margin. Relevant prothrombin fragments are identified on the left margin and molecular
weights of standards (x103) are shown on the right margin. Images of two gels have been aligned in each
panel.
Figure 3. Progress Curves for Prothrombin Consumption. Samples obtained from reaction mixtures
containing 0.15 nM Xa, 30 µM PCPS, 30 nM Va, 30 µM DAPA and 1.4 µM prothrombin variant were
analyzed by SDS-PAGE. The fate of the indicated prothrombin variant was inferred from densitometry
analysis of gels with colloidal blue (open symbols) or western blotting (closed symbols). Each panel
illustrates the fate of the indicated fully carboxylated () or desGla () variant normalized to the
starting concentration. The lines were arbitrarily drawn. Initial rates of prothrombin consumption are
listed in Table 1.
Scheme I. The Action of Prothrombinase on Intact Prothrombin. The substrate is initially tethered to
prothrombinase (E) by exosite binding (KEXO) independent of cleavage site availability. Arg320
in IIQ271
then engages the active site in a unimolecular binding step (Ks*320=E.IIQ271/E*.IIQ271) followed by
catalysis (kcat320) to yield mIIa. Equivalent steps determine active site docking (Ks*271= E.IIQ320/E*.IIQ320)
and catalysis (kcat271) by Arg271
in IIQ320. The double mutant (IIQQ) participates in exosite binding but does
not engage the active site of prothrombinase.
Figure 4. Kinetics of the Action of Prothrombinase on dG-IIQ271. Initial velocities of proteinase
formation were determined in reaction mixtures containing increasing concentrations of dG-IIQ271, 30 µM
PCPS, 30 nM Va, 0.5 nM Xa and different fixed concentrations of dG-IIQ320 (Panel A) or dG-IIQQ (Panel
B). Fixed concentrations of dG-IIQ320 correspond to 0, 5, 15, 20 and 30 µM (top to bottom, Panel A) and
of dG-IIQQ correspond to 0, 10, 20 and 40 µM (top to bottom, Panel B). The lines are drawn following
global analysis according to Scheme I with fitted constants listed in Table 2.
Figure 5. The Action of Prothrombinase on Singly Cleaved desGla Intermediates. Panel A: Initial
velocities for cleavage at Arg271’
in dG-mIIa were determined by fluorescence measurements using
reaction mixtures containing 0.1 µM dG-mIIaAlexa488, increasing concentrations of dG-mIIai, 30 µM PCPS,
30 nM Va and 1 nM Xa. Panel B: Initial velocities of thrombin formation measured using increasing
concentrations of P2 premixed with either 1.2 () or 1.5 () equivalents of dG-F12, 30 µM PCPS, 30
nM Va and 0.5 nM Xa. The lines are drawn following analysis using the observed steady state kinetic
constants reported in Table 2.
Scheme II. Kinetic Constants for the Four Half-Reactions of Prothrombin Activation. Observed steady
state kinetic constants for the action of prothrombinase (E) on desGla substrate species are listed for each
half-reaction. Relative rates for the carboxylated (Gla) versus uncarboxylated (desGla) substrate forms are
those calculated at 1.4 µM substrate.
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Figure 6. Membrane-Dependent Discrimination Between Arg320
and Arg271
by Prothrombinase. Progress curves for the consumption of 1.4 µM IIQ271 by cleavage at Arg
320 () or of 1.4 µM IIQ320 by
cleavage at Arg271
() were determined by quantitative western blotting using reaction mixtures
containing 1 nM Xa and 2 µM Va in the absence of membranes (Panel A), 0.5 nM Xa, 30 nM Va and 108
thrombin activated platelets/ml (Panel B) or 0.2 nM Xa, 30 nM Va and confluent HUVECs activated by
thrombin (Panel C). Panel A also illustrates data obtained with 1.4 µM dG-IIQ271 () or dG-IIQ320 ()
under the same conditions as used for the fully carboxylated variants. The lines are arbitrarily drawn.
Figure 7. Cleavage of Prothrombin by Prothrombinase assembled on Physiological Membranes. Western blots imaged with near-IR fluorescence illustrate the fate of 1.4 µM IIWT following cleavage
using Panel A: 0.5 nM Xa, 30 nM Va and 108 thrombin activated platelets/ml; Panel B: 0.2 nM Xa, 30
nM Va and confluent HUVECs activated by thrombin and Panel C: 0.5 nM Xa, 30 nM Va and 30 µM
extruded membranes containing 2.5% phosphatidylserine and 97.5% phosphatidylcholine. Reaction times
(in minutes) are listed in the lower portion of each panel. Two images have been aligned in each panel.
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Table 1: Normalized Rates of Prothrombin Consumption by Prothrombinasea
Prothrombin Derivative Bond(s) Cleaved v/E (1.4 µM substrate) v/E (5 µM substrate)
IIWT Arg320, Arg271 0.99 1.00 IIQ271 Arg320 1.14 1.09 IIQ320 Arg271 0.07 0.04 dG-IIWT Arg320, Arg271 0.39 0.63 dG-IIQ271 Arg320 0.08 0.15 dG-IIQ320 Arg271 0.29 0.57
a Initial rates of prothrombin consumption were measured from progress curves determined by quantitative densitometry following SDS-PAGE at either 1.4 µM or 5 µM substrate. Initial rates were normalized for differences in the concentration of enzyme (v/E) and are expressed in multiples of the rate observed at 5 µM IIWT.
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Table 2: Kinetic Constants for the Cleavage of Carboxylated and Uncarboxylated Substrate
Substrate Constant Units desGla Substrate
Source Gla Substrate
Source
Intrinsic Constants
KEXO µM 17.2 ± 0.5 1 0.25 ± 0.01 7 IIQ271 Ks*320 Dimensionless 1.9 ± 0.2 1 0.02 ± 0.002 8 IIQ271 kcat320 s-1 109 ± 7 1 94 ± 2 8 IIQ320 Ks*271 Dimensionless 1.7 ± 0.2 1 4.3 ± 0.11 8 IIQ320 kcat271 s-1 375 ± 78 2 ~13 9
Observed Constants
IIQ271 Kmobs,320 µM 11.2 ± 1.2 3 0.18 ± 0.01 10
11.0 ± 0.8 4 (V/E)320 s-1 38.3 ± 0.6 3 94 ± 2 10
37.4 ± 0.2 4 IIQ320 Kmobs,271 µM 10.8 ± 1.6 3 0.35 ± 0.01 10 (V/E)271 s-1 139 ± 13 5 ~ 3 10 F12/P2 Kmobs,320’ µM 20.1 ± 0.7 6 0.18 ± 0.01 10 (V/E)320’ s-1 13.5 ± 0.3 6 76 ± 1 10 mIIai Kmobs,271’ µM 11.3 ± 0.9 6 0.28 ± 0.02 10 (V/E)271’ s-1 67.0 ± 2.9 6 114 ± 3 10
1. Globally fitted according to Scheme 1 and Figure 4. 2. Calculated from (V/E)271 and Equation 2. 3. Calculated using globally fitted parameters and Equations 1 and 2. 4. Steady State constants measured in the absence of inhibitor (Fig. 4A). 5. Calculated from rates in Table I, Kmobs,271, Kmobs,320 and (V/E)320. 6. Measured in Figs 5A and 5B. 7. Representative Ki for inhibition by IIQQ, taken from reference 15. 8. Derived from direct binding measurements taken from reference 16. 9. Estimated from (V/E)271 (taken from reference 15) and Equation 2. 10. Taken from reference 15.
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Figure 1
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Figure 2
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Figure 3
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Scheme I
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Figure 4
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Figure 5
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Scheme II
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Figure 6
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Figure 7
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Harlan N. Bradford, Steven J. Orcutt and Sriram KrishnaswamyProthrombinase For Cleavage.
Membrane Binding by Prothrombin Mediates its Constrained Presentation to
published online August 12, 2013J. Biol. Chem.
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