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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2169-2178
By
From the Department of Biochemistry, University of Vermont,
Burlington, VT.
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.
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, 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.
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
Coagulation in whole blood.
The protocol used is a modification of the protocol of Rand et
al.52
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.
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).
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
(
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.
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.
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
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.
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.
1.
Jenny NS, Mann KG:
Coagulation cascade: An overview, in
Loscalzo J,
Schafer AI
(eds):
Thrombosis and Hemorrhage. Baltimore, MD, Williams & Wilkins, 1998, p 3.
2.
Beijering RJR, ten Cate H, ten Cate JW:
Clinical applications of new antitihrombotic agents.
Ann Hematol
72:177, 1996[Medline]
[Order article via Infotrieve]
3.
Verstraete M:
Modulating platelet function with selective thrombin inhibitors.
Haemostasis
26:70, 1996 (suppl 4)
4.
Schumacher WA, Steinbacher TE, Heran CL, Seiler SM, Michel IM, Ogletree ML:
Comparison of thrombin active site and exosite inhibitors and heparin in experimental models of arterial and venous thrombosis and bleeding.
J Pharmacol Exp Ther
267:1237, 1993
5.
Kyrle PA, Mannhalter C, Beguin S, Stumpflen A, Hirschl M, Weltermann A, Stain M, Brenner B, Speiser W, Pabinger I, Lechner K, Eichinger S:
Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene.
Arterioscler Thromb Vasc Biol
18:1287, 1998
6.
Cui J, O'Shea KS, Purkayastha A, Saunders TL, Ginsburg D:
Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V.
Nature
384:66, 1996[Medline]
[Order article via Infotrieve]
7.
Degen SJ, Sun WY:
The biology of prothrombin.
Crit Rev Eukaryot Gene Expr
8:203, 1998[Medline]
[Order article via Infotrieve]
8.
Rosen ED, Chan JC, Idusogie E, Clotman F, Vlasuk G, Luther T, Jalbert LR, Albrecht S, Zhong L, Lissens A, Schoonjans L, Moons L, Collen D, Castellino FJ, Carmeliet P:
Mice lacking factor VII develop normally but suffer fatal perinatal bleeding.
Nature
390:290, 1997[Medline]
[Order article via Infotrieve]
9.
Kundu RK, Sangiorgi F, Wu LY, Kurachi K, Anderson WF, Maxson R, Gordon EM:
Targeted inactivation of the coagulation factor IX gene causes hemophilia B in mice.
Blood
92:168, 1998
10.
Chiu HC, Whitaker E, Colman RW:
Heterogeneity of human factor V deficiency. Evidence for the existence of an antigen-positive variants.
J Clin Invest
72:493, 1983
11.
Poort SR, Michiels JJ, Reitsma PH, Bertina RM:
Homozygosity for a novel missense mutation in the prothrombin gene causing a severe bleeding disorder.
Thromb Haemost
72:819, 1994[Medline]
[Order article via Infotrieve]
12.
Ragni MV, Lewis JH, Spero JA, Hasiba U:
Factor VII deficiency.
Am J Hematol
10:79, 1981[Medline]
[Order article via Infotrieve]
13.
McGraw RA, Davis LM, Lundblad RL, Stafford DW, Roberts HR:
Structure and function of factor IX: Defects in haemophilia B.
Clin Haematol
14:359, 1985[Medline]
[Order article via Infotrieve]
14.
Hoyer LW:
Hemophilia A.
N Engl J Med
330:38, 1994
15.
Trimble M, Bell DA, Brien W, Hachinski V, O'Keefe B, McLay C, Black J:
The antiphospholipid syndrome: Prevalence among patients with stroke and transient ischemic attacs.
Am J Med
88:593, 1990[Medline]
[Order article via Infotrieve]
16.
Ozsoylu S, Ozer FL:
Acquired factor IX deficiency.
Acta Haematol
50:305, 1973[Medline]
[Order article via Infotrieve]
17.
Broekmans AW, Veltkamp JJ, Bertina RM:
Congenital protein C deficiency and venous thromboembolism. A study of three Dutch families.
N Engl J Med
309:340, 1983[Abstract]
18.
Comp PC, Esmon CT:
Recurrent venous thromboembolism in patients with a partial deficiency of protein S.
N Engl J Med
311:1525, 1984[Abstract]
19.
Vigano-D'Angelo S, D'Angelo A, Kaufman CE Jr, Sholer C, Esmon CT, Comp PC:
Protein S deficiency occurs in the nephrotic syndrome.
Ann Intern Med
107:42, 1987
20.
Scott BD, Esmon CT, Comp PC:
The natural anticoagulant protein S is decreased in male smokers.
Am Heart J
122:76, 1991[Medline]
[Order article via Infotrieve]
21.
Sporrong T, Mattsson LA, Samsioe G, Stigendal L, Hellgren M:
Haemostatic changes during continuous oestradiol-progestogen treatment of postmenopausal women.
Br J Obstet Gynaecol
97:939, 1990[Medline]
[Order article via Infotrieve] |
TOP
ABSTRACT
INTRODUCTION
2-macroglobulin, and 
), 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
(
).
), 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.
0.5 minutes). The reduction of TFPI from the mean plasma value to
50% results in a significant shortening (by 
), factor X (
),
factor XI (
), factors V, VIII, IX, and X simultaneously
(
), TFPI (⊠), protein C
(
), and protein S (
).
