(Received for publication, March 5, 1997, and in revised form, May 18, 1997)
From the Department of Biochemistry, University of
Vermont, Burlington, Vermont 05405-0068, § Hemostasis and
Thrombosis Research Center, University Hospital, 2300 RC Leiden, The
Netherlands, and ¶ Institute of Medical Semeiotics, University of
Padua, 35100 Padua, Italy
The effect of the Arg506 Gln mutation in factor VLEIDEN on thrombin generation
was evaluated in a reconstituted system using the purified components
of the tissue factor (TF) pathway to thrombin and the components of the
protein C pathway. Recombinant full-length tissue factor pathway
inhibitor (RTFPI) was included in the system because of a previously
observed synergistic inhibitory effect of TFPI and the protein C
pathway on TF-initiated thrombin generation. Thrombin generation
initiated by 1.25 pM factor VIIa·TF in the absence of the
protein C pathway components occurs following an initiation phase,
after which prothrombin is quantitatively converted to 1.4 µM thrombin. The factor VLEIDEN mutation did
not influence thrombin generation in the reconstituted model in the
absence of the protein C pathway. In the presence of 2.5 nM
TFPI, 65 nM protein C, and 10 nM recombinant
soluble thrombomodulin (Tm), thrombin generation catalyzed by normal
factor V was abolished after the initial formation of 25 nM
thrombin. In contrast, persistent thrombin generation was observed in
the presence of factor VLEIDEN in the same system, although
the rate of thrombin generation was slower compared with the reaction
without protein C and Tm. The rate of thrombin generation with factor
VLEIDEN increased with time and ultimately resulted in
quantitative prothrombin activation. When the TFPI concentration was
reduced to 1.25 nM, thrombin generation is still curtailed
in the presence of normal factor V. In contrast, under similar
conditions using factor VLEIDEN, the protein C pathway
totally failed to down-regulate thrombin generation. The dramatic
effect of a 50% reduction in TFPI concentration on the inhibitory
potential of the protein C pathway on thrombin generation catalyzed by
factor VLEIDEN suggests that the observed synergy between
TFPI and the protein C pathway is directly governed by the TFPI
concentration and by cleavage of the factor Va heavy chain at
Arg506. This cleavage appears to have a dramatic regulatory
effect in the presence of low concentrations of TFPI. Markedly
increased thrombin generation in the presence of both 1.25 nM TFPI and factor VLEIDEN was also observed
when antithrombin-III was added to the system to complete the natural
set of coagulation inhibitors. Protein S (300 nM) had a
minimal effect in the model on the inhibition of thrombin generation by
protein C, Tm, and TFPI, with either normal factor V or factor
VLEIDEN. Protein S also failed to significantly potentiate
the action of the protein C pathway in the presence of antithrombin-III
in reactions employing normal factor V or factor VLEIDEN.
The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the reported interactions of protein S with coagulation factors are not decisive in
the reaction. Altogether the data predict that TFPI levels in the lower
range of normal values are a risk factor for thrombosis when combined
with the Arg506
Gln mutation in factor
VLEIDEN.
The procoagulant reactions leading to the generation of thrombin during the blood clotting process are governed by three vitamin K-dependent enzyme complexes, each of which is composed of a serine protease and a cofactor protein assembled on a membrane surface (1). The serine protease (factor VIIa, factor IXa, or factor Xa) in each case is derived from a plasma zymogen, while cofactors are regulated either by presentation (tissue factor) or proteolytic activation (factor Va or factor VIIIa). The procoagulant process is thought to be initiated when the small amounts of circulating two-chain factor VIIa bind to the tissue factor (TF)1 (2) presented as a consequence of vascular disruption or an inflammatory process. The reaction initiated by the formation of this complex activates the serine protease components of both the intrinsic tenase (factor IXa·factor VIIIa) and prothrombinase (factor Xa·factor Va). The latter complex converts prothrombin to thrombin (for a review on blood coagulation, see Ref. 3). The formation of thrombin is tightly regulated by a system of the stoichiometric inhibitors tissue factor pathway inhibitor (TFPI) (4) and antithrombin-III (AT-III) (5, 6) and by the activated protein C (APC) pathway through which thrombin serves as its own down-regulator by complexing with constitutively expressed vascular thrombomodulin (Tm) (7). The resulting complex activates the plasma zymogen protein C to APC. APC down-regulates coagulation by proteolytic inactivation of the cofactor factor Va.
We have shown in earlier studies that the combination of the stoichiometric inhibitors TFPI, which regulates the factor VIIa·TF complex, and AT-III, an inhibitor of all the procoagulant serine proteases, and the protein C pathway behave synergistically in their attenuation of thrombin generation (8, 9). These studies demonstrated that the system produces threshold-limited bursts of thrombin when the procoagulant reactions proceed in the presence of the combined regulatory systems. The cooperative interactions between procoagulant and anticoagulant systems regulate thrombin generation such that it is limited by the extent of presentation of tissue factor and concentrations of pro- and anticoagulants.
Congenital and acquired alterations of either the procoagulant or
anticoagulant pathways are associated with hemorrhagic disease or
thrombosis. A factor V genetic mutation (factor VLEIDEN)
has recently been described that is present in approximately 5% of the
Caucasian population and appears to be responsible for 20% of familial
venous thrombosis (10, 11). The G A mutation at nucleotide 1691 leads to the substitution of glutamine for arginine at position 506 in
the factor VLEIDEN molecule. The principle regulatory
influence of the protein C pathway is associated with the inactivation
of factor Va (9, 12), the cofactor of the prothrombinase complex. The
normal proteolytic inactivation process involves cleavages of the
factor Va molecule at Arg506, Arg306, and
Arg679 in the heavy chain (13, 14). The substitution of
glutamine for arginine at position 506 slows the inactivation process
and therefore extends the kinetic lifetime of the expression of
prothrombinase complex activity (14). The synergistic relationships
between the stoichiometric inhibitors and the protein C system in the regulation of thrombin generation is a largely kinetic phenomenon (8,
9). We have therefore studied the influence of the slowed inactivation
rate of factor VLEIDEN on the expression of thrombin in a
reconstituted system of procoagulant and anticoagulation reactions
representative of the blood clotting process.
Phosphatidylserine from bovine brain,
phosphatidylcholine from egg yolk, and Hepes were purchased from Sigma
(St. Louis, MO). D-Phenylalanyl-L-arginine
chloromethyl ketone was a gift from Dr. R. Jenny, Hematologic
Technologies Inc. (Essex Junction, VT). Spectrozyme TH was purchased
from American Diagnostica, Inc. (Greenwich, CT). S2366 was obtained
from Chromogenix (Kabi Pharmacia Hepar, Inc.). Blue Sepharose was
obtained from Pharmacia (Uppsala, Sweden). All other reagents were
of analytical grade. The mouse monoclonal antibody HFV-6 was
provided by Dr. William Church, Department of Biochemistry,
Antibody Core Facility, University of Vermont (Burlington, VT).
Human coagulation factors X, IX, and prothrombin
were isolated from freshly frozen plasma using the general methods of
Bajaj et al. (15) and were depleted of trace contaminants
and traces of active enzymes as described (8). Human protein C was
purified by heparin-Sepharose and immunochromatography and treated with D-phenylalanyl-L-arginine chloromethyl ketone
as described (9). Human protein S was a gift from Dr. R. Jenny or was
purified from the protein S pool of the initial ion exchange
chromatography step involved in the purification of the vitamin
K-dependent clotting factors using blue Sepharose
chromatography as described by Dahlbäck (16). The purified
protein S prepared by the method described by Dahlbäck was
depleted of trace amounts of contaminants and treated with
D-phenylalanyl-L-arginine chloromethyl ketone
as described (9). Human factor V was isolated from freshly frozen human
plasma using the method of Nesheim et al. (17). Factor VLEIDEN (Arg506 Gln) was purified from
plasma of patients who were homozygous for the factor
VLEIDEN mutation as described (14). AT-III was purified
according to the method described by Griffith et al. (18).
Recombinant factor VIII and recombinant tissue factor (residues 1-242
of the human sequence) were provided as gifts from Drs. Shu Len Liu and
Roger Lundblad, Hyland division, Baxter Healthcare Corp. (Duarte, CA). Recombinant human coagulation factor VIIa was purchased from NOVO pharmaceuticals. Recombinant soluble thrombomodulin (Solulin) was
provided as a gift by Dr. J. Morser, Berlex (Richmond, CA). Recombinant
full-length TFPI produced in E. coli was provided as a gift
from Dr. K. Johnson, Chiron Corp. (Emeryville, CA). Purified human
factor Xa and purified human activated protein C were gifts from Dr. R. Jenny and Dr. P. Haley, Hematologic Technologies Inc. Tick
anticoagulant peptide (TAP) was provided as a gift by Dr. S. Krishnaswamy, Hematology/Oncology Division, Emory University (Atlanta,
GA). Hirudin was provided as a gift by Genentech (South San Francisco,
CA).
Thrombin generation initiated by factor VIIa·TF in a reconstituted model using normal plasma protein concentrations was studied as described previously (19). TF was relipidated into 400 µM 75% phosphatidylcholine, 25% phosphatidylserine vesicles, for 30 min at 37 °C in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl2, pH 7.4 (Hepes/Ca2+). The relipidated TF was incubated with factor VIIa for 20 min at 37 °C to allow factor VIIa·TF complex formation. Factor V and factor VIII were added in microliter amounts of concentrated stock solutions to the equilibrated factor VIIa·TF mixture (total volume of the addition not exceeding 0.25% of the final reaction volume), and immediately thereafter the reaction was started by the addition of a solution containing factor X, factor IX, and prothrombin, which was prepared in Hepes/Ca2+. The zymogen solution was preheated at 37 °C for 3 min before the addition to the factor VIIa·TF, factor V, and factor VIII mixture. When protein C, TFPI, AT-III, or protein S was included it was added to the factor X, factor IX, and prothrombin mixture. Thrombomodulin was added to the factor VIIa·TF mixture.
The final concentrations of the proteins in the reaction, chosen to
represent mean plasma values, are 160 nM factor X, 90 nM factor IX, 0.7 nM factor VIII, 20 nM factor V, and 1.4 µM prothrombin (19).
When added, protein C (65 nM), TFPI (2.5 nM),
protein S (300 nM), and AT-III (3.4 µM)
(20-23) were also present at their respective plasma concentrations in
the reaction unless indicated otherwise. The final
phosphatidylcholine/phosphatidylserine concentration in the reactions
was 200 µM. Following initiation of the reaction, aliquots were withdrawn from the reaction mixture and quenched in
either 20 mM EDTA, 20 mM Tris, 150 mM NaCl, pH 7.4, to assay for thrombin formation or in 2%
SDS, 0.062 M Tris, 10% glycerol, 0.04% bromphenol Blue,
pH 6.8, for SDS-polyacrylamide gel electrophoresis and immunoblotting.
Assays for thrombin activity were performed using the chromogenic
substrate Spectrozyme TH. The hydrolysis of the substrate was monitored
by the change in absorbance at 405 nm using a Molecular Devices Vmax
spectrophotometer. Thrombin measurements in samples withdrawn from
experiments that included AT-III were performed by rapid quenching of
samples in a solution containing 20 mM EDTA and 0.4 mM Spectrozyme TH, and thrombin generation was measured
immediately as described (8). Thrombin generation was calculated from a
standard curve prepared with serial dilutions of purified human
-thrombin. APC generation was measured using substrate S2366 in
samples that were withdrawn from the reaction mixture and diluted
5-fold into excess hirudin (7 µM), tick anticoagulant
peptide (2.31 µM), and 20 mM
EDTA/Tris-buffered saline. APC generation was calculated from a
standard curve prepared using serial dilutions of purified human APC.
Some residual amidolytic activity was observed in the thrombin activity
assay in the presence of hirudin in samples taken from reactions with
Tm and protein C. This residual amidolytic activity, caused by
generated APC, did not significantly affect the higher thrombin levels
measured in the reactions and never exceeded 20% of the total
amidolytic activity at the lowest thrombin levels measured. The
SDS-quenched samples were analyzed using SDS-polyacrylamide gel
electrophoresis under nonreducing conditions on 4-12% polyacrylamide
gels essentially as described by Laemmli (24). Following
SDS-polyacrylamide gel electrophoresis the proteins were transferred to
nitrocellulose membranes for immunoblot analysis using the techniques
described by Towbin et al. (25). Membranes were blocked for
nonspecific binding with 5% nonfat dry milk in 0.05% Tween,
Tris-buffered saline and incubated for 1.5 h with monoclonal
antibody
HFV-6. This antibody recognizes an epitope on the heavy
chain of factor V between residues 307 and 506 (14). This fragment is a
final product of factor Va cleavage by APC. The products of factor V recognized by this antibody were visualized using peroxidase-conjugated horse anti-mouse IgG and the Renaissance chemiluminescent reagent obtained from DuPont. The membranes were stripped with 0.5 M NaCl, 0.1 M glycine, pH 2.7, to remove bound
antibodies. The stripped membranes were blocked again with 5% nonfat
milk and probed for prothrombin activation products using a polyclonal
burro anti-prethrombin-1 antibody as described (19).
The effect of the factor
VLEIDEN mutation was studied in reactions that were
initiated with 1.25 pM factor VIIa·TF in the presence of
2.5 nM TFPI. TFPI was included in the reaction mixtures to provide the physiological kinetically transient factor VIIa·TF activity and because of a synergistic effect of TFPI and the protein C
pathway observed in this model (9). Thrombin generation with normal
factor V or factor VLEIDEN in the absence of the protein C
pathway occurred following an initial lag phase of 2.5 min, after which
prothrombin was converted into thrombin at 180 nM·min1 during the propagation phase. The
profile was essentially the same for normal factor V and factor
VLEIDEN (Fig. 1, filled
symbols).
In the presence of protein C and 10 nM Tm with normal
factor V (Fig. 1, open circles), thrombin generation
occurred at a reduced rate after a lag time of 2.5 min. At 5 min,
thrombin generation ceased after ~25 nM thrombin was
produced. Thus, at the factor VIIa·TF concentration used (1.25 pM) the protein C pathway eliminated the propagation phase
of the reaction after a limited amount of thrombin was formed (9). In
the case of factor VLEIDEN (Fig. 1, open
squares) the same lag time (2.5 min) was observed; however,
subsequent thrombin generation, while slowed, continued at a rate of
~26 nM·min1. After 10 min, the rate of
thrombin generation increased, ultimately rising at 12 min to the rate
observed in the absence of protein C/Tm. Prothrombin was quantitatively
converted to thrombin (1.4 µM) in the presence of factor
VLEIDEN after 20 min. The rate of APC generation (~3
nM·min
1) was the same in reactions
containing either normal factor V or factor VaLEIDEN (data
not shown). Thrombin generation with normal factor V in the presence of
protein C, 10 nM Tm and 300 nM protein S (Fig. 1, open triangles) results in a nearly identical rate of
thrombin generation as observed in the absence of protein S (Fig. 1,
open circles). Thus, under the conditions used, protein S
does not strongly potentiate the inhibitory effect of the protein C
pathway. Protein S (300 nM) had only minimal influence on
the initial rate of thrombin generation by factor VLEIDEN
(Fig. 1, diamonds) in the presence of the components of the
protein C pathway. The rate of secondary thrombin generation in the
presence of factor VLEIDEN and the protein C pathway is
decreased slightly by protein S (by approximately 30%) but still
results in the quantitative activation of prothrombin. The lack of a
major effect of protein S on thrombin generation under these conditions
in the presence of factor VaLEIDEN and the protein C
pathway does not support a decisive role for protein S in
accelerating the APC-dependent cleavage at
Arg306 and subsequent inactivation of factor
VaLEIDEN as reported by Rosing et al. (26).
Analyses of the proteolytic activation and
inactivation of normal factor V and factor VLEIDEN by
immunoblotting using an antibody reacting with an epitope on the heavy
chain within residues 307-506 (HFV-6) (14) showed that both
cofactors were cleaved during the initial phase (0-1.5 min) of
thrombin generation to generate the heavy chain (Fig. 2). Densitometric data of the factor Va
heavy chain bands of the immunoblots is shown in Fig.
3. No factor V intermediate is observed at Mr = 280,000, indicating that there is no
significant direct inactivation of intact factor V by cleavage of the
procofactor at Arg306 (14). In the presence of 10 nM thrombomodulin and protein C (Fig. 2A),
inactivation of normal factor Va occurs during the initiation phase of
the reaction (1.5 min) due to initial cleavage at Arg506. A
subsequent cleavage at Arg306 gives rise to a
Mr 30,000 product (14) (Fig. 2A).
After 5 min, most of the heavy chain is degraded by APC, yielding the
Mr 30,000 fragment, consistent with the
elimination of prothrombinase activity at 5 min (Fig. 1, open
circles).
Inactivation of factor VaLEIDEN in the presence of protein C/thrombomodulin (Fig. 2B) is observed after 2.5 min by the appearance of the Mr 60,000 reactive band (residues 307-709) and a Mr 54,000 reactive band (residues 307-679) (14). These fragments are the consequence of cleavages at Arg306 and Arg679. Compared with the data obtained with normal factor Va (Fig. 2A), the heavy chain of factor VaLEIDEN is cleaved at a slower rate. Quantitation of the factor Va heavy chain by densitometry (Fig. 3, squares) shows that higher levels of the factor Va heavy chain are obtained in the presence of factor VLEIDEN (squares) compared with normal factor V (circles). The complete proteolysis of the factor VaLEIDEN heavy chain is accomplished only after 18 min. It is noteworthy that, during the initial 20 min of the reaction, the integrated concentration of the factor VaLEIDEN heavy chain is approximately 3-fold greater than that of the normal factor Va heavy chain. The abundant amounts of thrombin and/or factor Xa formed during the course of the reaction cleave the 307-679 fragment resulting in Mr 43,000 and 40,000 fragments (Fig. 2B). Some remaining intact heavy chain is cleaved to generate a Mr 85,000 fragment.
The sustained activity of factor VaLEIDEN in the presence
of the protein C pathway is made obvious by following the analysis of prothrombin activation by immunoblotting. Prothrombin activation in
reactions with factor V or factor VLEIDEN is quantitative
in the absence of the protein C pathway (Fig. 4, A and B) and
evolves consistent with the lag and propagation phases observed for the
generation of thrombin (Fig. 1, filled symbols). Consistent
with the attenuation of thrombin generation catalyzed by normal factor
V in the presence of the protein C pathway, quantitative prothrombin
consumption was not observed under these conditions (Fig.
4C), indicating that the observed inhibition of thrombin
generation is caused by the elimination of prothrombinase activity and
is not the result of conversion of prothrombin to inactive products. In
contrast, under similar conditions, quantitative prothrombin
consumption ultimately occurred when the reaction was catalyzed by
factor VLEIDEN (Fig. 4D).
The Effect of TFPI Concentration on Thrombin Generation Catalyzed by Factor VLEIDEN in the Presence of the Protein C Pathway and Protein S
The lower limit of normal TFPI concentration in
plasma is thought to be ~1.25 nM (22). Experiments
performed with a TFPI concentration of 1.25 nM, initiated
by 1.25 pM factor VIIa·TF, are displayed in Fig.
5. Thrombin generation at 1.25 nM TFPI in the absence of the protein C pathway
(filled symbols) occurs after a shorter lag period (1.5 min)
and proceeds with a slightly higher rate in the propagation phase when
compared with reactions with 2.5 nM TFPI (Fig. 1). No major
difference is observed between reactions with normal factor V
(filled circles) or factor VLEIDEN (filled
squares). In the presence of 1.25 nM TFPI (Fig. 5,
open circles), thrombin generation by normal factor V was
temporarily attenuated up to 10 min in the reaction by the protein C
pathway (65 nM protein C, 10 nM thrombomodulin,
300 nM protein S).
In marked contrast, thrombin generation by factor VLEIDEN in the presence of the protein C pathway components and 1.25 nM TFPI occurred after a lag time of 1.5 min and proceeded explosively (Fig. 5, open squares), resulting in a thrombin generation profile similar to the reaction without protein C and thrombomodulin (filled squares). Thus, in contrast to the effective down-regulation of thrombin generation observed with normal factor V by the protein C pathway at a 50% reduced TFPI concentration, the protein C pathway fails to inhibit thrombin generation by factor VLEIDEN at this concentration of TFPI (Fig. 5, open squares). This is of particular significance, since thrombin generation with normal factor V (under these conditions) is effectively slowed down by the protein C pathway in the complete absence of TFPI (9). These data demonstrate the critical roles of both the cleavage site in factor Va at Arg506 and TFPI in the regulation of TF-initiated thrombin generation by the protein C pathway. The results also indicate that relatively small differences in TFPI concentrations within the range of "normal" plasma levels may have a significant influence on thrombin generation in individuals with the factor VLEIDEN mutation.
Thrombomodulin Titration with Factor VLEIDEN and Normal Factor V in the Presence of AT-III at Varying TFPI ConcentrationsThe dramatic effect of a 50% reduction in TFPI
concentration on the inhibitory potential of the protein C pathway on
thrombin generation catalyzed by factor VLEIDEN suggests
that the synergy observed between TFPI and the protein C pathway is
directly governed by the TFPI concentration and the rate of factor Va
inactivation via initial cleavage at Arg506. This effect
and/or the lack of a major effect of protein S could be a consequence
of the absence of thrombin regulation by other physiological
inhibitors such as AT-III. The effect of the factor VLEIDEN mutation was therefore also evaluated in the
presence of AT-III. Reactions performed with factor V or factor
VLEIDEN in the presence of AT-III (3.4 µM),
protein C (65 nM), and TFPI at normal (2.5 nM)
and 50% plasma concentrations (1.25) at various concentrations of
thrombomodulin are presented in Fig. 6.
Panels A and C show reactions with normal factor
V; panels B and D are with factor
VLEIDEN. In all cases, thrombin generation was initiated
with 5 pM factor VIIa·TF. This concentration of initiator
was chosen because of an observed threshold between 1 and 5 pM factor VIIa·TF for explosive thrombin generation in
the presence of 2.5 nM TFPI and AT-III.
Thrombin generation initiated with 5 pM factor VIIa·TF in the presence of protein C, 2.5 nM TFPI, and 3.4 µM AT-III in the absence of Tm reaches a peak concentration of thrombin of ~195 nM at 5 min (Fig. 6A, filled circles). Subsequently, the thrombin concentration declined to approximately 45 nM at 12 min and reached a plateau value of 10 nM at 20 min. In the absence of Tm, normal factor V and factor VLEIDEN produce similar thrombin generation curves at 2.5 nM TFPI (Fig. 6, A and B, filled circles). Control reactions performed in the absence of protein C (data not shown) resulted in similar thrombin generation curves as observed in the presence of protein C. Tm decreases thrombin generation in the presence of protein C in a concentration-dependent manner without appreciably affecting the initiation phase of the reaction (Fig. 6, A-D). Higher Tm levels are needed to inhibit thrombin generation in the presence of factor VLEIDEN (panels B and D) compared with normal factor V (panels A and C). For example, at 0.25 nM, Tm has no affect on the reaction with factor VLEIDEN (compare Fig. 6B, open and filled circles), whereas for the reaction with normal factor V at this Tm concentration the peak level of thrombin generation is decreased by ~50% (compare Fig. 6A, open and filled circles). A 50% inhibition of thrombin generation in the presence of factor VLEIDEN is observed with 1 nM Tm (Fig. 6B, open squares). These data demonstrate that a 4-fold higher Tm concentration is required to inhibit thrombin generation by 50% in the system comprising factor VLEIDEN when compared with the system containing normal factor V. Similarly, in a reaction with normal factor V, 2.5 nM Tm (Fig. 6A, open triangles) causes a 90% reduction in the peak level of thrombin (20 nM), whereas a 4-fold higher Tm concentration (10 nM) is needed to reduce the peak level of thrombin generation by 90% in the case of factor VLEIDEN (Fig. 6B, open diamonds). Analogous to this, 3-fold higher peak levels of thrombin are observed in the presence of 1-2.5 nM Tm (Fig. 6, A and B) in reactions containing factor VLEIDEN compared with reactions with normal factor V. A similar but exacerbated set of results is seen when the TFPI concentration is reduced to 50% of normal (Fig. 6, compare panels C and D).
Fig. 7 displays the peak levels of
thrombin observed in the reactions shown in Fig. 6 plotted
versus the Tm concentration present in the reaction.
The open symbols are for factor VLEIDEN,
while the filled symbols are for normal factor V. Note the
dramatic effect of halving the TFPI concentration in the case of factor VLEIDEN (open squares) observed for all Tm
concentrations used. Tm not only decreases the peak levels of thrombin
observed in the presence of AT-III but also shortens the time interval
required before a steady state level of ~10-20 nM
thrombin is reached. This thrombin level persists in all reactions over
the 20-min time interval studied (Fig. 6). With 10 nM Tm, a
2-fold higher level of thrombin generation is observed with factor
VLEIDEN compared with normal factor V. The stable level of
thrombin observed in the reactions at the later time points suggests
that thrombin is being generated at a rate equal to its rate of
inhibition, resulting in a steady-state thrombin concentration.
At 1.25 nM TFPI, the rate of thrombin generation with
normal factor V (Fig. 6C) and factor VLEIDEN
(Fig. 6D) is increased when compared with reactions with 2.5 nM TFPI (Fig. 6, A and B). The
reduced TFPI level results in ~1.7-fold higher peak levels of
thrombin generation in the reactions without Tm (filled
circles). In contrast, for normal factor V at Tm concentrations 1 nM, the difference observed in thrombin generation
between the normal and 50% reduced TFPI concentration become almost
negligible (Fig. 7, filled symbols). At 1.25 nM
TFPI and in the presence of factor VLEIDEN, up to a 6-fold
increase in the peak level of thrombin is observed compared with
reactions with normal factor V and 1.25 nM TFPI. This
6-fold increase in peak thrombin level is observed at 1 nM Tm (Fig. 6, compare panels C and D, open
squares). The differences in peak levels of thrombin generation
observed at low thrombomodulin concentrations caused by low TFPI or
factor VLEIDEN are virtually eliminated at 10 nM Tm (Fig. 7, filled circles, filled
squares, and open circles).
The thrombin generation profiles observed in the presence of 10 nM Tm are compared in Fig. 8.
Thrombin generation in the presence of 10 nM Tm evolves
without the display of a peak level of thrombin in the reaction that
contains normal levels of TFPI and normal factor V (Fig. 8,
filled circles). Similar curves are obtained at 10 nM Tm in the presence of either a reduced TFPI level and normal factor V (filled squares) or a normal TFPI
concentration and factor VLEIDEN (open circles).
These reactions show only slight differences in thrombin generation
(2-fold), and all result in persistent levels of thrombin between
approximately 10 and 20 nM (Fig. 8). The isolated potential
thrombotic states do not seem to result in highly increased thrombin
generation when the reaction is strongly down-regulated by high Tm
concentrations. In marked contrast, the combination of low TFPI and
factor VLEIDEN (Fig. 7, open squares) results in
significantly increased levels of thrombin at 10 nM Tm. In
contrast to the other reactions, thrombin generation occurs with a peak
level of 60 nM with 1.25 nM TFPI and factor
VLEIDEN (Fig. 8, open squares), a level that is
6-fold higher compared with the level reached in the reaction with 2.5 nM TFPI and normal factor V.
The reaction with 1.25 nM TFPI and factor VLEIDEN is inhibited by 85% with 10 nM Tm in the presence of AT-III (Fig. 6D, compare open circles and diamonds). In the experiment using the same conditions (1.25 nM TFPI, factor VLEIDEN, 10 nM Tm) and in the absence of AT-III (Fig. 5, open squares), virtually no inhibition is observed. This demonstrates the change in the kinetics of thrombin appearance when the reaction is dampened by AT-III and TFPI (8).
Altogether these results strongly suggest that a 50% decrease in the TFPI concentration will significantly increase the prothrombotic state of individuals bearing the factor VLEIDEN mutation.
Effect of Protein S as Cofactor of the Protein C Pathway on Thrombin Generation by Factor VLEIDEN and Normal Factor V in the Presence of AT-IIIThe lack of a significant effect of
protein S on the progress curves of thrombin generation in the presence
of either normal factor V or factor VLEIDEN (Fig. 1) could
have been the result of the absence of AT-III. Based on the differences
in progress curves of thrombin in the presence of the protein C
pathway, 2.5 nM TFPI, and 3.4 µM AT-III in
the experiment presented in Fig. 6, A and B, a
similar experiment was performed in the presence of 2.5 nM
thrombomodulin with normal factor V and factor VLEIDEN in
the absence or presence of 300 nM protein S. Thrombin
generation with normal factor V was identical in the absence and
presence of protein S (Fig. 9,
open symbols). The progress curves obtained with
factor VLEIDEN (filled symbols)
reached a 2.5-fold higher thrombin level compared with normal factor V. Protein S did not affect the thrombin generation profile by factor
VLEIDEN. These data demonstrate that under the conditions
employed, in the presence of an excess of membrane surface, protein S
has no potentiating effect on the attenuation of thrombin generation by
the protein C pathway.
The effect of the Arg506 Gln substitution in
factor VLEIDEN was evaluated in a reconstituted model which
represents the tissue factor pathway to thrombin in the presence of the
protein C system and TFPI. While thrombin generation by normal factor V
is synergistically down-regulated and terminated by the combination of
protein C, Tm, and TFPI (9), this was not observed for factor
VLEIDEN. TF-initiated thrombin generation in the presence
of factor VLEIDEN continued in the presence of
physiological concentrations of TFPI, protein C, and high (10 nM) concentrations of soluble Tm. A reduction of 50% in
the TFPI concentration essentially eliminated the ability of the
protein C pathway to slow thrombin generation down in
the presence of factor VLEIDEN, while thrombin
generation in the presence of normal factor V was still curtailed by
the protein C pathway in the presence of 50% levels of TFPI. In the
presence of AT-III and high (10 nM) Tm concentrations, a
reduced TFPI level or the presence of factor VLEIDEN results in regulated thrombin generation equivalent
to control values. However, the combination of factor
VLEIDEN with a reduced TFPI level results in a 6-fold
increase of the thrombin level observed in the control in the presence
of the highest Tm concentrations evaluated.
The presented data show that the regulation of the tissue factor pathway by the protein C system is directly governed by the TFPI concentration and the inactivation of factor Va by initial cleavage at Arg506. These results strongly suggest that the combination of homozygosity for factor VLEIDEN and a mild TFPI deficiency would lead to a severe thrombotic state. Thus far no human deficiencies of TFPI have been reported (4). However, Huang et al. (27) have reported the lethal effect of homozygous TFPI deficiency in mice. Heterozygous TFPI-deficient mice appear without symptomatic disease. Our model is consistent with these results, since in the model, with the protein C pathway and AT-III, only marginal differences are observed when the TFPI concentration is at 50% of the mean normal concentration. Based on the profound effects of TFPI in combination with AT-III in our model, we proposed that a homozygous TFPI deficiency would result in a thrombotic status (8). In addition we propose, based on the present data, that the combination of a heterozygous TFPI deficiency with factor VLEIDEN will result in a severe thrombotic tendency.
While the risk for thrombosis is increased 7-fold in individuals heterozygous for the factor VLEIDEN mutation, an 80-fold increased risk of thrombosis is observed in cases of homozygosity for factor VLEIDEN (28). The high prevalence of heterozygous cases of factor VLEIDEN in the normal population results in a significant number of case reports in which this mutation is coinherited with other risk factors such as heterozygous protein C (29), protein S (30), or AT-III (31) deficiency. The greatly increased thrombotic risk with the combination of heterozygous inheritance of factor VLEIDEN with protein C, protein S, or AT-III deficiency explains some of the variation in thrombotic risk associated with heterozygous factor VLEIDEN (29-31). The data presented here suggest that reduced TFPI levels will be another factor that will contribute to variation in the risk of thrombosis when associated with factor VLEIDEN.
The Arg506 Gln substitution leads to partial resistance
to inactivation of factor Va by APC (10, 11). Due to the absence of the
APC cleavage site at Arg506, the inactivation of factor
VaLEIDEN is dependent upon the relatively slower cleavage
at Arg306 (13, 14).
In the reconstituted model, the generation of factor Xa and the factor Va light chain are the rate-limiting factors for the generation of thrombin (9). In a previous report we have demonstrated that in the presence of protein C and high concentrations of Tm, the heavy chain of factor V becomes inactivated by APC before the generation of the light chain is complete (9). Generation of the light chain of factor Va is reported to be essential when cofactor activity is tested in a clotting assay or in a prothrombinase assay using low factor Xa concentrations (32, 33). This means that the relative stability of the heavy chain of factor VLEIDEN in the presence of the protein C pathway results in higher prothrombinase activity because of higher and persistent levels of intact heavy chain. The kinetic stability of the heavy chain associated with increasing concentrations of light chain can lead to high levels of fully active factor Va.
Cleavage of factor Va at Arg506 diminishes the rate of
thrombin generation by decreasing the affinity of factor Xa for factor Va (34). This cleavage is accompanied by a relatively small loss in
cofactor activity when assayed at saturating levels (5-10 nM) of factor Xa (14, 34). However, cleavage at
Arg506 results in significant loss of cofactor activity
when assayed at low concentrations of factor Xa (34). Thus, the
observed increased and ongoing thrombin generation in the reconstituted model in the presence of factor VLEIDEN, bearing the
Arg506 Gln substitution, indicates that the initial
cleavage at Arg506 is necessary for efficient factor Va
inactivation and the resulting arrest of thrombin generation.
Most provocative is the absence of a major effect of protein S in the model. Our data show only a small but significant potentiation of the protein C system by protein S. This observation is in contrast to studies that have reported 1) a cofactor function of protein S for APC in the inactivation of factor Va and factor VIII(a) (35, 36) and 2) a normalization of the APC inactivation of factor VLEIDEN by acceleration of cleavage at Arg306 (26). The protein S used in this study supports increased inactivation of factor VLEIDEN by APC in an isolated reaction performed under conditions identical to those described by Rosing et al. (26).2 Hence, the lack of an effect of protein S in the reconstituted model is not caused by differences in the protein preparations. Based upon previous studies with isolated reactions, we had expected that protein S would become an important player by the elimination of the protection of factor Va by factor Xa from inactivation by APC (37, 38). Under our experimental conditions, however, protein S has no major inhibitory effect. The reported neutralization of the protecting effect of factor Xa by protein S on factor Va inactivation (38) was studied using nanomolar concentrations of factor Xa that were equimolar or higher than the factor Va used. Under the present experimental conditions and during the TF-mediated clotting of whole blood, only subnanomolar concentrations of factor Xa are formed (9, 39). However, nearly complete activation of factor V occurs during the initiation phase of the reaction, resulting in the generation of 20 nM of factor Va heavy chain. Thus, the protective effects of subnanomolar concentrations of factor Xa on the excess factor Va formed, although reproducible, may be insignificant at the concentration present in the biologically relevant reaction.
It should be mentioned that the function of protein S has been proposed to increase the membrane interaction of APC (35). The relatively high concentrations of phospholipids in the present model probably result in saturation of APC with respect to phospholipid binding, eliminating the requirement for optimal functioning of APC on protein S (35). The observations also suggest that the reported protein-protein interactions of protein S with Va or factor Xa (40, 41), which were hypothesized to play a role in the functioning of protein S as an independent prothrombinase inhibitor, do not occur or are of no kinetic significance in this complex model.
The phospholipid for the procoagulant enzyme complexes is presumably principally provided in vivo by platelets. The exposure of the required membrane phospholipid is, however, dependent on stimulation of the platelets; thus, the concentration of phospholipids on which the reactions may occur varies with the accumulation and activation of platelets. The present model, with its relative excess of phospholipids, is more representative for a platelet aggregate with high concentrations of a procoagulant membrane surface. Under these conditions, protein S has no decisive effect on the thrombin generation reaction. The APC-dependent and -independent actions of protein S may be more prominent in reactions that are initiated with quiescent platelets.
Portions of this work were presented at the Thirty-seventh Annual Meeting of the American Society of Hematology, December 1-5, 1995, Seattle, WA (42).
We thank Dr. Kirk Johnson (Chiron Corp.) for
the generous gift of recombinant TFPI, Dr. Shu Len Liu and Dr. Roger
(Lundblad, Hyland Division, Baxter Healthcare Corp.) for providing
recombinant factor VIII and recombinant tissue factor, Dr. John Morser
for the generous gift of recombinant soluble thrombomodulin, Dr.
William Church (University of Vermont, Department of Biochemistry) for providing antibody HFV-6, and Kelly Begin and Neal Golden for technical assistance.