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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2169-2178
"Normal" Thrombin Generation
By
Saulius Butenas,
Cornelis van't Veer, and
Kenneth G. Mann
From the Department of Biochemistry, University of Vermont,
Burlington, VT.
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ABSTRACT |
We have investigated the influence of alterations in plasma
coagulation factor levels between 50% and 150% of their mean values for prothrombin, factor X, factor XI, factor IX, factor VII, factor VIII, factor V, protein C, protein S, antithrombin III (AT-III), and
tissue factor pathway inhibitor (TFPI) as well as combinations of
extremes, eg, 50% anticoagulants and 150% procoagulants or 50%
procoagulants and 150% anticoagulants in a synthetic "plasma" system. The reaction systems were constructed in vitro using purified, natural, and recombinant proteins and synthetic phospholipid vesicles or platelets with the reactions initiated by recombinant tissue factor
(TF)-factor VIIa complex (5 pmol/L). To investigate the influence of
the protein C system, soluble thrombomodulin (Tm) was also added to the
reaction mixture. For the most extreme situations in which the
essential plasma procoagulants (prothrombin, and factors X, IX, V, and
VIII) and the stoichiometric anticoagulants (AT-III and TFPI) were
collectively and inversely altered by 50%, a 28-fold difference in the
total available thrombin generated was observed. Variations of most of
these proteins 50% above and below the "normal" range, with the
remainder at 100%, had only modest influences on the peak and total
levels of thrombin generated. The dominant factors influencing thrombin
generation were prothrombin and AT-III. When these 2 components were
held at 100% and all other plasma procoagulants were reduced to 50%,
there was a 60% reduction in the available thrombin generated. No
increase in the thrombin generated was observed when the 150% level of
all plasma procoagulants other than prothrombin was evaluated. When only prothrombin was raised to 150%, and all other factors were maintained at 100%, the thrombin generated increased by 71% to 121%.
When AT-III was at 50% and all other constituents were at 100%,
thrombin production was increased by 104% to 196%. The additions of
protein C and protein S over the 50% to 150% ranges with Tm at 0.1 nmol/L concentration had limited influence on thrombin generation.
Individual variations in factors VII, XI, and X concentrations had
little effect on the duration of the initiation phase, the peak
thrombin level achieved, or the available thrombin generated. Paradoxically, increases in factor IX concentration to 150% led to
lowered thrombin generation, while decreases to 50% led to enhanced
thrombin generation, most likely a consequence of factor IX as a
competitive substrate with factor X for factor VIIa-TF. Reductions in
factor V or factor VIII concentration led to prolongations of the
initiation phase, while the reduction of TFPI to 50% led to shortening
of this phase. However, none of these alterations led to significant
changes in the available thrombin generated. Based on these data, one
might surmise that increases in prothrombin and reductions in AT-III,
within the normal range, would be potential risk factors for thrombosis
and that algorithms that combine normal factor levels may be required
to develop predictive tests for thrombosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBIN IS the essential enzyme product
of the blood coagulation enzymatic cascade.1 In humans and
a variety of natural and transgenic animal models, the regulation of
the production of this enzyme is vital to the maintenance of the
hemostatic balance.2-9 Genetic and acquired deficiencies
that cause reductions in, or the absence of, thrombin generation lead
to hemorrhagic syndromes.10-16 Defects in the regulatory
stoichiometric and dynamic processes that downregulate thrombin
generation are associated with thrombotic risk.17-24
At a minimum, the procoagulant process is mediated by an array of at
least 6 plasma proteins (prothrombin, factor VII, factor IX, factor X,
factor V, and factor VIII) and 1 tissue protein, tissue factor (TF).
The anticoagulant process is governed by a minimum of 4 plasma
proteins: antithrombin III (AT-III), protein C, protein S, and the
tissue factor pathway inhibitor (TFPI), and by 1 membrane-bound protein
contributed by vascular tissue, thrombomodulin (Tm).1 Thus,
a minimum of 12 proteins are explicitly and centrally associated with
the maintenance of blood fluidity and protection from vascular injury.
In addition to these proteins, membrane-binding sites essential to both
procoagulant and anticoagulant processes are provided by vascular cell
injury either by trauma and inflammatory mediators and by
platelets.25,26 The latter are essential contributors to
the initiation and propagation of the process.27
Other proteins contributed by the so-called "contact pathway of
coagulation," while apparently not essential for the procoagulant response, may perform important supplemental roles in the coagulation process. These include high-molecular-weight kininogen, factor XII,
prekallikrein, and factor XI. Potential additional anticoagulant roles
are played by heparin cofactor II,  2-macroglobulin, and 1-antitrypsin.28,29
Following mechanical or inflammatory damage to the vascular wall, the
procoagulant reaction is thought to begin by the binding of small
amounts of preexistent 2-chain plasma factor VIIa to TF. The resulting
membrane bound factor VIIa-TF enzyme complex activates the plasma
zymogens factor X and factor IX by limited proteolysis. Factor Xa
generation is further accelerated by the formation of the
membrane-bound intrinsic factor Xase complex composed of factor IXa and
factor VIIIa. Ultimately, the factor Xa formed by both enzyme complexes
binds to membrane-bound factor Va to produce the prothrombinase
complex, which converts prothrombin to thrombin.1 The
composite of these procoagulant reactions leads to the biphasic
generation of thrombin. During an initiation phase, picomolar amounts
of enzymes are produced while the procofactors, factor V and factor
VIII, are nearly quantitatively activated. Subsequently, during a
propagation phase, the bulk of the thrombin is generated.30
The initiation and propagation phases of the coagulation system are
differentially regulated by the stoichiometric inhibitors AT-III and
TFPI and by activated protein C produced dynamically by thrombin-Tm.
TFPI, which has as its principle targets factor Xa, and the factor
VIIa-TF-factor Xa product complex,31 serves principally to
regulate the initiation phase of the reaction.32 AT-III,
which reacts with all of the serine proteases produced in the
coagulation system, is in significant molar excess to its target
enzymes and serves principally to quench enzyme activity once
formed.33 As a consequence, AT-III makes a larger
contribution to ablating the propagation phase than in altering the
initiation phase of the reaction.32 The combinations of
AT-III and TFPI, and TFPI and the protein C system, act in synergy to
produce threshold limits of thrombin generation, which are related to
initiator concentration.32,34
From studies of "normal" human physiology, it is generally
inferred that the range of procoagulant and anticoagulant species in
the average blood sample ranges from 50% to 150% of some mean value,
and the assessment of potential pathology based on clinical laboratory
blood studies, in general, attributes values between 50% and 150% of
any the coagulation factors as within the "normal" range.35,36
In previous studies, empirical and theoretical models of the
coagulation have been provided.30,37-40 We have assessed
the influence of alterations in the concentrations of the vascular components TF and Tm and the stoichiometric inhibitors TFPI and AT-III
in regulating the procoagulant system when the levels of all other
proteins are set at normal, 100% values.32,34 From those
studies, it is clear that the process of the blood coagulation system,
although made up of fairly conventionally functioning complex enzymes,
behaves in such a manner that synergy between procoagulants and
inhibitors produces threshholded responses with respect to
stimuli, and the process of blood clotting functions effectively in a
"yes/no" configuration in which the procoagulant initiating
stimulus must be at a certain level to bring about the uncompromised
generation of thrombin.32,34
A number of epidemiologic studies have shown that concentration
variations of blood coagulation proteins within the 50% to 150%
range, including prothrombin, AT-III, proteins C and S, factors VII,
VIII, and IX, and fibrinogen, are associated with thrombotic risk.19,24,41-44 We therefore elected to evaluate the
influence of alterations in the concentration of each procoagulant and
anticoagulant (zymogens, procofactors, and inhibitors) species of the
extrinsic pathway with respect to its influence on the generation of
thrombin, at a given stimulus.
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MATERIALS AND METHODS |
Materials.
Phospholipid vesicles (PCPS) composed of 25% phosphatidylserine and
75% phosphatidylcholine were prepared as described.45 Phospatidylserine, phosphatidylcholine, and EDTA were purchased from
Sigma (St Louis, MO). Spectrozyme TH was purchased from
American Diagnostica (Greenwich, CT). FPRck was obtained
as a gift from Haematologic Technologies (Essex Junction, VT). The
enzyme-linked immunosorbent assay (ELISA) thrombin-AT-III (TAT) kit
(Enzygnost TAT) was purchased from Behring (Marborg, Germany).
Proteins.
Human coagulation factors VII, X, and IX, and prothrombin and
protein C were isolated from fresh-frozen plasma using the general methods of Bajaj et al,46 and were purged of trace
contaminants and traces of active enzymes as described.32
Human factor V and AT-III were isolated from freshly frozen
plasma.47,48 Human protein S was purified using
Blue-Sepharose chromatography.49 Recombinant factor VIII
and recombinant TF (residues 1-242) were provided as gifts from Drs Shu
Len Liu and Roger Lundblad (Hyland Division, Baxter
Healthcare, Duarte, CA). Recombinant human factor VIIa was purchased
from NOVO Pharmaceuticals (Denmark). Recombinant full-length TFPI
produced in Escherichia coli was provided as a gift from Dr K. Johnson (Chiron, Emeryville, CA). Recombinant soluble Tm (Solulin) was
provided as a gift from Dr J. Morser (Berlex, Richmond, CA) and human
plasma factor XI was a gift from Dr R. Jenny (Haematologic
Technologies). Corn trypsin inhibitor was isolated from popcorn
as described elsewhere.50 Washed platelets were prepared by
the procedure of Mustard et al.51
Coagulation factor activation experiments.
Thrombin generation initiated by factor VIIa-TF in a reconstituted
procoagulant model using mean plasma protein concentrations was studied
as described previously.32,34,37 TF (0.5 nmol/L) was
relipidated into 400 µmol/L PCPS by incubation in 20 mmol/L HEPES,
150 mmol/L NaCl, and 2 mmol/L CaCl2 pH 7.4 (HBS/Ca2+) for 30 minutes at 37°C. The
relipidated TF was incubated with 10 pmol/L factor VIIa for 20 minutes
to allow factor VIIa-TF complex formation. Factor V, factor VIII, and
Tm (when desired) were added to the relipidated factor VIIa-TF complex,
and thrombin generation was started by addition of an equal volume of a
zymogen-inhibitor mixture containing prothrombin, factors X and IX,
TFPI, AT-III, proteins C and S, and factors VII and XI (the last 4 when
desired) prepared in HBS/Ca2+ and preheated at 37°C for
3 minutes. The final concentrations of the proteins in the reaction,
chosen to represent mean plasma values (100%), are indicated in Table
1. The Tm concentration was 0.1 nmol/L or 1 nmol/L, factor VIIa and TF concentrations were 5 pmol/L and 0.25 nmol/L, respectively. In experiments in which PCPS was substituted by
washed platelets at 2 × 108/mL concentration, final
concentrations of factor VIIa and TF were 100 pmol/L and 12.5 pmol/L,
respectively. The concentrations of selected proteins in mixtures were
varied from 50% to 150% of their mean plasma values. Following
initiation of the reaction, at selected time points, 10-µL aliquots
were withdrawn from the reaction mixture and quenched in 20 mmol/L EDTA
in HBS (pH 7.4) containing 0.2 mmol/L Spectrozyme TH and assayed
immediately for thrombin activity. The hydrolysis of the substrate was
monitored by the change in absorbance at 405 nm using a
Molecular Devices Vmax spectrophotometer
(Molecular Devices, Menlo Park, CA). Thrombin generation was calculated
from a standard curve prepared by serial dilutions of -thrombin. The
total active thrombin generated was evaluated by integrating the area
under thrombin versus time curve.
Coagulation in whole blood.
The protocol used is a modification of the protocol of Rand et
al.52
Clotting in freshly drawn, nonanticoagulated whole blood was performed
in 32 tubes (2 series per experiment, 16 tubes per series). All tubes
were loaded with corn trypsin inhibitor (100 µg/mL blood), 12.5 pmol/L relipidated TF/mL blood (PCPS:TF = 2,000) in HBS with 2 mmol/L
CaCl2 (all tubes in each series except phlebotomy control
tube), 50 µg/mL blood of prothrombin or 100 µg/mL blood of AT-III
(all tubes, experiment series only), and equivalent volume
prothrombin/AT-III dilution buffer (HBS, pH 7.4, all tubes control
series only). No more than 35 µL of reagents was loaded in each tube.
The zero tube of each series was pretreated using 1 mL of 50 mmol/L
EDTA and 10 µL of 10 mmol/L FPRck (diluted in 0.01 mol/L HCl). Normal
donor blood was drawn by venipuncture, delivered into the
reagent-loaded tubes, and the tubes periodically quenched with EDTA and
FPRck. The clotting time was monitored visually by two observers. Tubes
were centrifuged and the supernatants were aliquoted for further
analyses. Commercial ELISAs for TAT were performed according to the
manufacturer's protocol. Thrombin generation analyses were
accomplished using 10 µg/mL of a polyclonal burro -human
prethrombin-1 antibody as described elsewhere.52
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RESULTS |
Thrombin generation induced by the factor VIIa-TF complex in a mixture
containing the essential coagulation proteins (factors X, IX, V, and
VIII, and prothrombin) and the inhibitors TFPI and AT-III evolves after
an initiation phase as a peak of thrombin activity, the formation of
which is downregulated and quenched by the action of AT-III. Under
these conditions, explosive thrombin generation becomes a
threshold-limited event with respect to the initiating factor VIIa-TF
concentration.32 Based on this observation, the factor
VIIa-TF enzymatic complex was used at 5 pmol/L, a concentration that
permits observation of thrombin generation even under the least
favorable conditions of this study, ie, the extreme cases when
procoagulants are present at 50% and anticoagulants at 150% of their
mean plasma values. On the other hand, this concentration of initiator
does not eliminate the responsiveness of the system to the variations
of reactant concentrations at the most favorable thrombin generation conditions.
Procoagulants and stoichiometric inhibitors.
Figure 1 displays thrombin generation
profiles, which result when the reaction is initiated by 5 pmol/L
TF-factor VIIa complex on 200 µmol/L PCPS vesicles. Under conditions
in which all procoagulant factors and stoichiometric inhibitors are at
their mean plasma concentrations, thrombin generation occurs after an
initiation phase and reaches a maximum concentration of approximately
300 nmol/L. The formation and inhibition rates are equivalent by 2.5 minutes; subsequently declining until the thrombin is largely inhibited
by 10 minutes into the reaction. When AT-III and TFPI are present at
150% and the procoagulants are reduced to 50% of mean values, a
severely depressed thrombin generation profile is observed. The area
under the curve (total thrombin) under these conditions is reduced to
approximately 25% of the normal profile. Conversely, a decrease in the
concentration of the anticoagulants by 50% in combination with an
increase of the concentration of all procoagulants to 150% results in
an approximately 700% increase (from control) in total thrombin
generation, which reaches a maximum concentration of 1 µmol/L. The
observed stable level of thrombin at the latter time points of this
profile is the result of the consumption of AT-III in this situation
(1.7 µmol/L) and the quantitative conversion of prothrombin (2.1 µmol/L) to thrombin. The initial rate of thrombin generation is
altered approximately 16-fold between extremes (8.9 nmol/L/s and 0.57 nmol/L/s, respectively), which results from accumulated 50% variations
from the mean concentrations of coagulation factors and inhibitors.
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| Fig 1.
Thrombin generatin on 200 µmol/L PCPS at varying
procoagulant and anticoagulant concentrations in the absence of protein
C pathway. Thrombin generation was initiated by 5 pmol/L factor VIIa-TF
in the presence of all proteins at mean plasma concentrations ( ), in
the presence of procoagulants (prothrombin, factors V, VIII, IX, and X)
at 150% and anticoagulants (AT-III and TFPI) at 50% of their mean
plasma concentrations ( ), and in the presence of procoagulants at
50% and anticoagulants at 150% of their mean plasma concentrations
( ).
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Figure 2 illustrates data for experiments
in which only prothrombin and AT-III are varied over the range from
50% to 150% with factors X, V, VIII, and IX, and with TFPI maintained
at 100% of mean plasma value. The control is 100% of all species. The contrast between 150% prothrombin and 50% AT-III, and 50%
prothrombin and 150% AT-III, is most similar to the extremes
represented in Fig 1 when all constituents were varied over this range.
This suggests that prothrombin and AT-III are the principle agents responsible for the major excursions observed in Fig 1. This
observation is supported by the data of Fig
3, which demonstrates that variations of
prothrombin concentration over the range from 50% to 150% with other
proteins present (including AT-III) at 100% cause an approximately 4-fold increase in maximum thrombin concentration (from 200 nmol/L to
800 nmol/L) and in total thrombin (Fig 3; Table
2). The variation of AT-III concentration
with other proteins present at 100% leads to an approximately 50%
decrease in the maximum thrombin concentration and in total thrombin
generated (Table 2).
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| Fig 2.
Thrombin generation on 200 µmol/L PCPS at varying
prothrombin and AT-III concentrations in the absence of protein C
pathway. Thrombin generation was initiated by 5 pmol/L factor VIIa-TF
in the presence of all proteins at their mean plasma concentrations
(), in the presence of prothrombin at 150% and AT-III at 50% of
mean plasma concentrations (), in the presence of prothrombin at
50% and AT-III at 150% of mean plasma concentrations (), in the
presence of prothrombin at 125% and AT-III at 75% of mean plasma
concentrations ( ), and in the presence of prothrombin at 75% and
AT-III at 125% of mean plasma concentrations ( ). Factors V, VIII,
IX, and X and TFPI were present at mean plasma concentrations in all
experiments.
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| Fig 3.
Prothrombin titration on 200 µmol/L PCPS in a
coagulation protein activation experiment in the absence of protein C
pathway. Thrombin generation was initiated by 5 pmol/L factor VIIa-TF
in the presence of prothrombin at 150% (), 125% (), 100%
(), 75% (), and 50% () of its mean plasma concentration. All
other proteins were present at mean plasma concentrations.
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Table 2.
Total Thrombin, Maximum Thrombin Concentration, and
Initiation Phase Duration (%) Dependence on Protein Concentration
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In a protein activation experiment in which washed platelets were
substituted for PCPS vesicles, variations of prothrombin and AT-III
concentrations over the 50% to 150% range led to results similar to
experiments conducted on PCPS (Fig 4;
Table 2).
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| Fig 4.
Thrombin generation on 2 × 108/mL platelets
at varying prothrombin concentrations in the absence of protein C
pathway. Thrombin generation was initiated by 5 pmol/L factor VIIa-TF
in the presence of prothrombin at 100% (), 150% (), and 50%
() of its mean plasma concentration. All other proteins were present
at mean plasma concentrations.
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Adding the dynamic anticoagulant system.
Tm is an essential thrombin cofactor required for protein C activation.
Since the local concentration of Tm on endothelial cells may vary
widely, an explicit knowledge of effective Tm concentration in vivo is
not available. We chose to use soluble Tm at 0.1 nmol/L and 1 nmol/L
concentrations based on previously published data that indicated these
concentrations of Tm are able to support protein C activation in the
reconstituted model.34 The inclusion of soluble Tm at these
concentrations and protein C and protein S at their mean plasma
concentrations into the reaction mixture does not significantly change
the response of the system to variations of prothrombin and AT-III. The
total thrombin and maximum levels of thrombin generated are similar to
those observed in the absence of the protein C pathway. However, the
duration of the initiation phase is affected by the variations of
prothrombin and AT-III concentrations when protein C, protein S, and Tm
are present (Table 2). Thus, a decrease in prothrombin concentration by
50% extends the initiation phase by 25% (Fig
5A), whereas a 50% decrease in AT-III
concentration leads to the shortening of this phase by 20% (Fig 5B).
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| Fig 5.
Thrombin generation on 200 µmol/L PCPS at varying
prothrombin (A) or AT-III (B) concentrations in the presence of protein
C pathway. Thrombin generation was initiated by 5 pmol/L factor VIIa-TF
in the presence of prothrombin or AT-III at 100% (), 150% (),
and 50% () of their mean plasma concentration. All other proteins
were present at mean plasma concentrations.
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Individual variations in the concentration of factors V, VIII, VII,
IX, X, and XI, TFPI, and proteins C and S.
Individual variations in the concentration of these proteins over the
range from 50% to 150% do not alter significantly the levels of total
available thrombin generated or maximum thrombin concentration (Table
2). However, the influence of these variations on the initiation phase
is not identical for all proteins that were tested. This group of
proteins can be divided into 2 subgroups: (1) those for which varying
concentrations have an observable influence on this phase (factors V,
VIII, and IX, and TFPI); and (2) those that do not alter the initiation
phase (factors X, VII, and XI and proteins C and S).
Individual variations in the concentrations of factors V, VIII, and
IX, and TFPI.
When factor V concentrations are varied over the 50% to 150% range,
the initiation phase of the reaction is prolonged by more than 1 minute
(from 4 minutes to 5.5 minutes) when factor V levels are reduced to
50% of the nominal average value (Fig 6A).
The initiation phase is only slightly shortened when factor V is
present at 150%. A similar observation is made when factor VIII levels are varied (Table 2); however, the initiation phase is influenced by
factor VIII concentration to a lesser extent than for factor V. Prolongation of the initiation phase at 50% of factor VIII is similar
to the shortening of this phase obtained at 150% of this protein
( 0.5 minutes). The reduction of TFPI from the mean plasma value to
50% results in a significant shortening (by 1 minute) of the
initiation phase of the reaction (Table 2). However, the peak
concentrations of thrombin generated and the total thrombin generated
over the time course of the experiment are only moderately influenced
by TFPI concentration changes within the "normal" range. Raising
the TFPI concentration to 150% produced little change over 2.5 nmol/L
TFPI.
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| Fig 6.
Thrombin generation on 200 µmol/L PCPS at varying
factor V (A), factor IX (B), protein C (C), and factors V, VIII, IX,
and X simultaneously (D) concentrations. Thrombin generation was
initiated by 5 pmol/L factor VIIa-TF in the presence of either factor V
or factor IX, or protein C, or factors V, VIII, IX, and X
simultaneously at 100% (), 150% (), and 50% () of their
mean plasma concentrations. All other proteins were present at mean
plasma concentrations.
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Paradoxically, variations in the procoagulant factor IX concentration
led to a similar pattern of thrombin generation as variations in TFPI
concentration. Thus, a shortening of the initiation phase is observed
at 50% levels of factor IX (by 1 minute), while a prolongation (by
0.5 minute) of the initiation phase is observed when factor IX
levels are increased to 150% of the nominal average value (Fig 6B;
Table 2). The total thrombin generated is also inversely related to the
factor IX concentration. At the lowest level of factor IX, the highest
peak levels of thrombin are produced (310 nmol/L), while the highest
level of factor IX gives the lowest peak thrombin level (175 nmol/L).
Individual variations in the concentrations of factors X, VII, and
XI, and proteins C and S.
Variations in these protein levels from 50% to 150% produces only a
slight (if any) effect on the duration of the initiation phase, the
peak thrombin levels achieved, and the total available thrombin (Table
2). A representative illustration is shown for protein C in Fig 6C.
As stated earlier, thrombin generation is not influenced by changes in
factor VII or factor XI concentrations over the range from 50% to
150% (Table 2). The experimental conditions used did not permit a
direct comparison of alterations in factor VII levels because an excess
of TF was used to insure complete complexation of the factor VIIa
added. Factor VIIa generated from factor VII forms a complex with
excess TF, which increases the concentration of the initiator until all
TF is bound to factor VIIa. The increased concentrations of the factor
VIIa-TF complex caused by the addition of factor VII shorten the
initiation phase of the reaction at all concentrations of factor VII
tested (5 to 15 nmol/L) compared with experiments performed in the
absence of this zymogen; however, variation in factor VII concentration
from 50% to 150% does not influence this phase (data not shown).
The data presented thus far suggest that while individual variations in
factors IX, X, V, and VIII have some influence on thrombin generation,
the contributions of these components are relatively small when
compared with the contributions of prothrombin and AT-III to the
overall process. Their combined influence was tested by analysis of
thrombin generation on 200 µmol/L PCPS in the absence of protein C
pathway proteins with prothrombin and AT-III at 100% by varying all
other procoagulants in the reaction system from 50% to 150%. The data
presented in Fig 6D demonstrate that an increase in all the
procoagulants, save prothrombin, had little influence on the thrombin
generation profile. In contrast, a reduction by 50% of all these
constituents delays the onset of the propagation phase by approximately
3 minutes, reduces peak thrombin levels by 66% and total thrombin by
60%.
The collected data, in terms of total thrombin produced, for the
variations of prothrombin, AT-III, factors X, IX, VIII, V, VII, and XI,
as well as TFPI and proteins C and S in the reaction system are
illustrated in Fig 7. The total thrombin
data are extracted from the experimental curves of Figs 3 and 6, as
well as from similar data not shown. The relative magnitudes of
thrombin production change with variations of the 7 procoagulants and
the 4 anticoagulants illustrate the dominance of prothrombin and AT-III
to the overall generation of thrombin. Halving the level of AT-III
leads to more than a doubling of the total thrombin generated, while a
50% increase in prothrombin concentration leads to a 75% increase in
thrombin relative to that generated at nominal average prothrombin
concentrations. In contrast, a reduction in prothrombin concentration
to 50% of the mean level leads to a halving of the total thrombin
available in the reaction system. Similar alterations of all other
procoagulants lead to a 60% reduction relative to that obtained at the
nominal plasma concentrations. In contrast to prothrombin, a 50%
increase in all other procoagulants has virtually no effect on the
total available thrombin. Individually, factor X and factor V at 50% levels each produces only a 20% reduction in total thrombin.
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| Fig 7.
Total thrombin generated at varying protein
concentrations. Data were taken from Figs 3 and 6 and from Table 2. The
following proteins were varied in the range from 50% to 150% of their
mean plasma value: prothrombin (), AT-III (), factor V (),
factor VIII (), factor VII (), factor IX ( ), factor X ( ),
factor XI ( ), factors V, VIII, IX, and X simultaneously
( ), TFPI (⊠), protein C
( ), and protein S ( ).
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Additions of prothrombin or AT-III to whole blood.
An experimental model to study the TF pathway of coagulation in
minimally altered whole blood was developed previously in our
laboratory.52 In this model, clotting of fresh blood is initiated by TF under conditions in which contact activation is suppressed. This system allows for the testing of theories of blood
coagulation under the conditions in which the native state of blood
components is preserved. This model has been validated in analyses of
blood from healthy persons,52 as well as from patients with
hemophilia A and factor XI deficiency.50 Direct comparison
of the synthetic plasma system and the whole blood models indicates
that activation initiated by the same concentration of TF leads to
similar thrombin generation profiles in both experiments (data not
shown). This suggests that conclusions drawn from the reconstituted
model data can be applied to whole blood as well. To test this
hypothesis, we evaluated how thrombin generation is affected by
additions of either prothrombin or AT-III to whole blood. The indicator
of thrombin generation in this experiment is thrombin-AT-III complex
(TAT) formation. The concentration of free thrombin was also evaluated
by immunoblotting to quantitate thrombin B chain.
When a 50% supplement of prothrombin (50 µg/mL) is added to contact
pathway suppressed whole blood and coagulation is initiated by 12.5 pmol/L relipidated TF, the clotting time is shortened by almost 1.5 minutes relative to the control (4:40 minutes v 6:06 minutes)
and the maximum rate of TAT formation in this case is approximately
50% greater than that in the control (4.0 nmol/L/s v 2.7 nmol/L/s, respectively) (Fig 8). The
maximum levels of TAT observed are also elevated when excess
prothrombin is added to whole blood (1,500 nmol/L v 1,100 nmol/L in control after 20 minutes of activation). Detectable amounts
of free thrombin are observed by approximately 1 minute earlier in the
excess prothrombin experiment (Fig 9) than
in the control and remain higher during all 20 minutes of the
experiment.
View larger version (13K):
[in this window]
[in a new window]
| Fig 8.
TAT generation in whole blood when 50 µg/mL of
prothrombin is added. Whole blood clotting was initiated by 12.5 pmol/L
relipidated TF in unaltered blood () or after the addition of 50 µg/mL of prothrombin (). TAT concentrations were evaluated by
ELISA. Clot times are indicated by arrows.
|
|
View larger version (13K):
[in this window]
[in a new window]
| Fig 9.
Thrombin generation in whole blood when 50 µg/mL of
prothrombin is added. Concentration of free thrombin evaluated from
Western blots by densitometry in control () and in excess
prothrombin () experiments.
|
|
The addition of 0.1 mg/mL AT-III (50% of mean plasma value) to whole
blood increases the clotting time by 1.3 minutes and the initiation
phase of TAT formation is slightly prolonged (data not shown). However,
the maximum rate of this complex generation is only marginally altered
by the addition of AT-III.
| |
DISCUSSION |
Numerous epidemiologic studies indicate that deficiencies in blood
coagulation proteins cause serious bleeding problems, whereas the
deficiencies in coagulation inhibitors and certain mutations in
coagulation factors may lead to thrombotic disorders. From clinical
studies it is assumed that the normal concentration range of proteins
involved in blood coagulation and regulation of this process may vary
in the average blood sample from 50% to 150% of their mean plasma
values.35,36 However, a number of epidemiologic studies
have shown that concentration variations of some of these proteins
within the normal range are associated with thrombotic risk.19,24,41-44
Data presented in this study indicate that of all the procoagulants and
stoichiometric inhibitors that influence thrombin generation,
prothrombin and AT-III are the standout contributors to significant
alterations. Varying these two proteins (in combination or
individually) within their felt to be "normal" concentration range causes significant alterations in the amount of total thrombin available, as well as in its maximum levels and rates of generation. Any prothrombin concentration used in this study (0.7 to 2.1 µmol/L) is below saturation for the prothrombinase complex.53 The
term "KM" for membrane-bound reactions is complex,
involving not only the intrinsic association of enzyme and substrate,
but also accumulation of substrate by the membrane.54 The
value reported for this constant for prothrombin activation by the
prothrombinase complex in the presence of 20 µmol/L PCPS is 1.1 µmol/L.53 Our data indicate that the KM for
this process in the presence of 200 µmol/L PCPS is 1.0 µmol/L
(manuscript in preparation). Thus, further increases in
the substrate (prothrombin) concentration would be associated with an
increased rate of thrombin generation. A reduced rate of AT-III
inhibition55 would allow elevated levels of thrombin during
the initiation phase, thus accelerating factor V and factor VIII
activation and shortening this phase. Therefore, in the whole blood
experiments, the clotting time, which occurs at the inception of the
propagation phase, is significantly influenced by the addition of
either prothrombin or AT-III. In both synthetic plasma and whole blood,
a similar response of the initiation phase duration to the
concentration of prothrombin or AT-III is observed. Increased levels of
prothrombin produce significant increases in the thrombin generation.
Recently, the in vivo influence of elevated prothrombin levels
associated with prothrombin gene mutation G20210A has been studied
intensively. Population studies suggest that increased prothrombin
levels are associated with multiple thrombotic
disorders,5,41,56-59 particularly venous thrombosis,
although conclusions on this subject are still
controversial.60 The importance of prothrombin levels for
TF initiated coagulation-related processes in plasma of
warfarin-treated rabbits was also well established.61
No substantial differences in total thrombin were observed when the
concentrations of factors V, VIII, IX, and X were individually varied
in the range used in this study. The duration of the initiation phase
was also not very sensitive to increased levels (150%) of these
proteins. Only when factor V was reduced to 50% of its mean plasma
value, was the initiation phase prolonged by 40%, whereas in the
presence of 50% factor IX, this phase was reduced by 25%. However,
the combined reduction of these 4 proteins to 50% of their mean plasma
values caused a substantial decrease in total thrombin and
significantly prolonged the initiation phase. These conclusions are
consistent with the observations that individuals with any of these
proteins at concentrations exceeding 25% to 50% of their mean plasma
values do not exhibit any bleeding problems.14,62 On the
other hand, acquired or hereditary multiple coagulation factor
deficiency may cause bleeding problems, sometimes severe or even
lethal.63-66
The lack of response in thrombin generation to increased concentrations
of all four coagulation factors (V, VIII, IX, and X) is most likely
ascribed to a paradoxical effect of factor IX. This effect on the
initiation phase and the total thrombin and peak thrombin concentration
is probably a consequence of the competition of factor IX and factor X
for the factor VIIa-TF catalyst. At a limited catalyst concentration
and at a higher affinity of factor IX for the factor VIIa-TF
complex,67 the presence of a competing substrate (factor
IX) decreases the rate of factor X activation and, as a consequence,
the initial prothrombinase concentration because factor Xa is a
limiting component of this enzymatic complex during the initiation and
propagation phases.30 A similar effect on thrombin
generation was observed when another competitive substrate of factor X,
factor VII at its mean plasma concentration, was introduced into the
reconstituted model.68
Factor XI appears to perform an accessory function in the TF
coagulation system by its activation to factor XIa by thrombin, which
leads to enhancement of the formation of thrombin by the activation of
factor IX to factor IXa. From whole blood studies, this feedback was
shown to be important only in thrombin generation initiated at very low
levels of TF.50 Therefore, it was predictable that under
the present experimental conditions variations in factor XI
concentration would have only a marginal effect on the reaction system.
Similarly, no bleeding complications are observed in individuals who
display factor XI concentrations greater than 10% of the mean plasma
value.62
Plasma TFPI concentrations are nominally 2.5 nmol/L. However, the true
physiologically effective concentration of TFPI is uncertain. This
inhibitor is sequestered by platelets, bound by the endothelium and
plasma lipoproteins, released by a variety of effectors, and major
portions circulate in plasma as variably truncated
forms.69,70 Thus, the biologically relevant concentration of TFPI may not be reflected by the mean plasma concentration. In
addition, proteolytic processing of TFPI significantly alters it
effectiveness as an inhibitor. These limitations are inescapable in an
interpretation of in vitro concentration changes in TFPI. The results
of a study, which evaluated TFPI levels in normal individuals and
patients with a variety of diseases, indicate that variations of this
inhibitor in a wide range are not associated with thrombotic or
bleeding tendencies.69 Similarly, no significant impact on
thrombin generation was observed in the reconstituted model of this
study with normal factor V when TFPI concentration was varied from 1.25 nmol/L to 3.75 nmol/L. In the presence of mutant factor V, factor
VLEIDEN, a 50% decrease in TFPI concentration led to a
dramatic increase in thrombin levels.71
The absence of a significant response to varying concentrations of
protein S and protein C was predictable on the basis of previous
studies that concluded protein S is not able to inhibit thrombin
generation in a reconstituted model when phospholipids are present at
relatively high concentrations.72 It has also been
established that variations of only protein C concentration in the
"normal" range do not cause coagulation
disorders.24,73,74
The data presented here clearly indicate than only prothrombin and
AT-III when varied within a "normal" range are able to significantly influence the coagulation process. Other proteins involved in blood coagulation and regulation of coagulation are able
only marginally (if at all) to alter this process.
| |
ACKNOWLEDGMENT |
We thank Neal J. Golden and Jason J. Penucci for their technical
assistance with a few of the experiments. We thank Dr Shu Len Liu and
Dr Roger Lundblad for providing us with recombinant factor VIII and
recombinant TF; Dr Kirk Johnson for providing recombinant TFPI; Dr John
Morser for providing recombinant soluble Tm; and Dr Richard Jenny for
providing factor XI.
| |
FOOTNOTES |
Submitted December 23, 1998; accepted June 3, 1999.
Supported by Program Project Grant No. HL 46703 from the National
Institutes of Health (K.G.M.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Presented in part at the XVIth Congress of the International Society on
Thrombosis and Haemostasis, June 6-12, 1997, Florence, Italy (abstr
PS-1653), at the 15th International Congress on Thrombosis, October
16-21, 1998, Antalya, Turkey (abstr 234), and at the 40th Annual
Meeting of the American Society of Hematology, December 4-8, 1998, Miami Beach, FL (abstr 151).
Address reprint requests to Kenneth G. Mann, PhD,
Department of Biochemistry, C401 Given Bldg, University of Vermont,
Burlington, VT 05405-0068.
| |
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G. Lippi, G. L. Salvagno, M. Montagnana, and G. C. Guidi
Reliability of the Thrombin-Generation Assay in Frozen-Thawed Platelet-Rich Plasma
Clin. Chem.,
September 1, 2006;
52(9):
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M. Pinotti, C. Bertolucci, F. Portaluppi, I. Colognesi, E. Frigato, A. Foa, and F. Bernardi
Daily and Circadian Rhythms of Tissue Factor Pathway Inhibitor and Factor VII Activity
Arterioscler Thromb Vasc Biol,
March 1, 2005;
25(3):
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[Abstract]
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K. Vanschoonbeek, M. A.H. Feijge, M. Paquay, J. Rosing, W. Saris, C. Kluft, P. L.A. Giesen, M. P.M. de Maat, and J. W.M. Heemskerk
Variable Hypocoagulant Effect of Fish Oil Intake in Humans: Modulation of Fibrinogen Level and Thrombin Generation
Arterioscler Thromb Vasc Biol,
September 1, 2004;
24(9):
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[Abstract]
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E. Castoldi, J. W. P. Govers-Riemslag, M. Pinotti, D. Bindini, G. Tans, M. Berrettini, M. G. Mazzucconi, F. Bernardi, and J. Rosing
Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation
Blood,
December 1, 2003;
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[Abstract]
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H. Philippou, J. Rance, T. Myles, S. W. Hall, R. A. Ariens, P. J. Grant, L. Leung, and D. A. Lane
Roles of Low Specificity and Cofactor Interaction Sites on Thrombin during Factor XIII Activation: COMPETITION FOR COFACTOR SITES ON THROMBIN DETERMINES ITS FATE
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[Abstract]
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W. L. Chandler and T. Velan
Estimating the rate of thrombin and fibrin generation in vivo during cardiopulmonary bypass
Blood,
June 1, 2003;
101(11):
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[Abstract]
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A. S. Wolberg, D. M. Monroe, H. R. Roberts, and M. Hoffman
Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk
Blood,
April 15, 2003;
101(8):
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[Abstract]
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L. R. Berry, P. Klement, M. Andrew, and A. K. C. Chan
Effect of Covalent Serpin-Heparinoid Complexes on Plasma Thrombin Generation on Fetal Distal Lung Epithelium
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[Abstract]
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D. M. Monroe and H. R. Roberts
Mechanism of Action of High-Dose Factor VIIa: Points of Agreement and Disagreement
Arterioscler Thromb Vasc Biol,
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K. G. Mann, S. Butenas, and K. Brummel
The Dynamics of Thrombin Formation
Arterioscler Thromb Vasc Biol,
January 1, 2003;
23(1):
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[Abstract]
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V. Schroeder, H. P. Kohler, K. E. Brummel, and K. G. Mann
Factor XIII activation by thrombin depends on FXIIIVal34Leu genotype
Blood,
January 1, 2003;
101(1):
371 - 371.
[Full Text]
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K. G. Mann and M. Kalafatis
Factor V: a combination of Dr Jekyll and Mr Hyde
Blood,
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H. Nagashima
Studies on the Different Modes of Action of the Anticoagulant Protease Inhibitors DX-9065a and Argatroban. I. EFFECTS ON THROMBIN GENERATION
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December 20, 2002;
277(52):
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[Abstract]
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D. M. Monroe, M. Hoffman, and H. R. Roberts
Platelets and Thrombin Generation
Arterioscler Thromb Vasc Biol,
September 1, 2002;
22(9):
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[Abstract]
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M. F. Hockin, K. C. Jones, S. J. Everse, and K. G. Mann
A Model for the Stoichiometric Regulation of Blood Coagulation
J. Biol. Chem.,
May 17, 2002;
277(21):
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[Abstract]
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V. V. Kakkar, D. A. Hoppenstead, J. Fareed, Z. Kadziola, M. Scully, R. Nakov, and H. K. Breddin
Randomized trial of different regimens of heparins and in vivo thrombin generation in acute deep vein thrombosis
Blood,
March 15, 2002;
99(6):
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[Abstract]
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K. C. Odegard, F. X. McGowan Jr, J. A. DiNardo, R. A. Castro, D. Zurakowski, C. M. Connor, D. D. Hansen, E. J. Neufeld, P. J. del Nido, and P. C. Laussen
Coagulation abnormalities in patients with single-ventricle physiology precede the Fontan procedure
J. Thorac. Cardiovasc. Surg.,
March 1, 2002;
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[Abstract]
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A. Undas, K. Brummel, J. Musial, K. G. Mann, and A. Szczeklik
Blood coagulation at the site of microvascular injury: effects of low-dose aspirin
Blood,
October 15, 2001;
98(8):
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[Abstract]
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A. Undas, K. E. Brummel, J. Musial, K. G. Mann, and A. Szczeklik
Simvastatin Depresses Blood Clotting by Inhibiting Activation of Prothrombin, Factor V, and Factor XIII and by Enhancing Factor Va Inactivation
Circulation,
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[Abstract]
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U. Seligsohn and A. Lubetsky
Genetic Susceptibility to Venous Thrombosis
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S. Solymoss
Risk factors for thromboembolism: pathophysiology and detection
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E. Castoldi, P. Simioni, M. Kalafatis, B. Lunghi, D. Tormene, D. Girelli, A. Girolami, and F. Bernardi
Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT 20210G/A) affecting the prothrombinase complex in a thrombophilic family
Blood,
August 15, 2000;
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[Abstract]
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O. Hiller, A. Lichte, A. Oberpichler, A. Kocourek, and H. Tschesche
Matrix Metalloproteinases Collagenase-2, Macrophage Elastase, Collagenase-3, and Membrane Type 1-Matrix Metalloproteinase Impair Clotting by Degradation of Fibrinogen and Factor XII
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A. R. Rezaie
Partial Activation of Antithrombin without Heparin through Deletion of a Unique Sequence on the Reactive Site Loop of the Serpin
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TOP
ABSTRACT
INTRODUCTION