(Received for publication, February 20, 1996, and in revised form, December 26, 1996)
From the Department of Biochemistry, University of Vermont, Burlington, Vermont 05405-0068
The effects of the components of the protein C
pathway on thrombin generation were studied in a reconstituted model in
which thrombin is generated by factor VIIa and relipidated tissue
factor (TF) via the activation of the purified coagulation factors X, IX, VIII, V, and prothrombin. The influence of protein C and soluble thrombomodulin on thrombin generation was correlated with factor Xa
generation, factor V(a) and factor VIII(a) formation/inactivation, and
protein C activation. Thrombin generation initiated by low concentrations of factor VIIa·TF (1.25 pM) occurs in an
explosive fashion during a propagation phase which occurs after an
initiation phase of ~1 min in which only traces of thrombin are
formed. In the absence of other inhibitors, protein C (65 nM) in combination with high concentrations of soluble
thrombomodulin (10 nM) resulted in a reduced rate of
thrombin generation during the propagation phase without affecting the
initiation phase; the activated protein C generated failed to
neutralize prothrombinase activity and did not prevent
prothrombin consumption. In the presence of plasma levels of the tissue
factor pathway inhibitor (2.5 nM recombinant TFPI), the
protein C pathway reduced the rate of thrombin generation, initiated by
1.25 pM factor VIIa·TF, and completely eliminated prothrombinase activity at soluble thrombomodulin concentrations of 1
nM. The neutralization of prothrombinase
activity coincided with cleavages at Arg-506 and subsequent
cleavage at Arg-306 of the factor Va heavy chain by activated protein
C. Thus, the protein C pathway combined with TFPI creates a minimal
inhibitory potential required to shut down TF-initiated thrombin
generation. The protein C pathway constituents did not influence factor
Xa generation or factor VIIIa degradation over the interval in which
prothrombinase activity was neutralized. Our data thus
suggest that the protein C pathway regulates thrombin generation solely
by the inactivation of factor Va. At low initiating factor VIIa·TF
(1.25 pM) and high thrombomodulin concentrations (10 nM), the factor Va heavy chain is cleaved before
significant amounts of light chain are generated. The ability of the
protein C pathway to inhibit thrombin generation was greatly reduced
when the reaction was initiated in the presence of factor Va,
supporting the hypothesis that effective down-regulation of thrombin
generation by the protein C pathway, in reactions initiated with the
procofactor, occurs by prevention of the coexistence of the factor Va
heavy and light chains.
The extrinsic pathway of blood coagulation initiated by tissue factor involves the activation of multiple coagulation factors leading to thrombin generation. The reaction starts with the binding of plasma factor VIIa to tissue factor (TF),1 an integral membrane protein that is exposed as a result of either vessel wall injury or cytokine activation of endothelial cells and/or peripheral blood monocytes. The membrane bound factor VIIa·TF enzyme complex activates the zymogens factor X and factor IX by limited proteolysis (1). Factor IXa combines with factor VIIIa on the membrane surface to form the intrinsic tenase that activates factor X. The factor Xa generated associates with factor Va on a membrane surface to form prothrombinase which activates prothrombin to thrombin. (For reviews on blood coagulation and membrane dependent reactions in blood coagulation see, respectively, Refs. 2 and 3). The thrombin initially formed accelerates further thrombin generation by feedback activation of the procofactors factor V and factor VIII. Deficiencies in factors VII, X, IX, V, VIII, or prothrombin are associated with bleeding. Thrombin stimulates platelets, which secrete their granule contents and aggregate, cleaves fibrinogen to generate the fibrin network, and activates the protransglutaminase factor XIII. The fibrin-platelet aggregate, stabilized by factor XIIIa catalyzed cross-links, forms the hemostatic plug which maintains the integrity of the circulatory system after vessel wall damage.
In normal hemostasis, the procoagulant system is in balance with the anticoagulant and fibrinolytic systems. The anticoagulant system consists of several stoichiometric protease inhibitors that include, at a minimum, the tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III), and heparin cofactor-II, and the dynamic protein C pathway that involves thrombin, protein C, protein S, and thrombomodulin.
The dynamic negative feedback mechanism provided by the protein C pathway is initiated by the binding of thrombin to thrombomodulin (for a review on thrombomodulin and protein C see Ref. 4). The binding of thrombin to thrombomodulin changes the specificity of the enzyme, resulting in the loss of procoagulant functions and enhancement of protein C activation. The formation of the thrombin-thrombomodulin complex accelerates the rate of protein C activation approximately 400-fold. Thrombomodulin is constitutively expressed as a transmembrane protein on the surface of vascular endothelial cells, providing an anticoagulant function at the blood/vessel wall interface (4) and is also present on platelets (5). A recent report of an individual suffering from thrombosis associated with a thrombomodulin mutation leading to a defective protein (6) and the embryonic lethality in mice lacking the thrombomodulin gene (7) identify the significance of thrombomodulin as a natural anticoagulant.
Activated protein C (APC) is thought to exert its inhibitory effect on coagulation by proteolytically inactivating the cofactors of prothrombinase and the intrinsic tenase, factor V(a) and factor VIII(a), respectively (8-16). Protein S appears to contribute an accessory function toward the APC inactivation of factor Va and factor VIIIa (14, 17, 18). It has also been reported that protein S directly inhibits prothrombinase activity (19). The importance of both protein C and protein S as anticoagulants is clearly illustrated by the numerous reports of thrombotic complications in either protein C- or protein S-deficient individuals (20, 21).
TFPI is a protease inhibitor that regulates coagulation by inhibition of the factor VIIa·TF complex activity in a factor Xa-dependent manner (for a review on TFPI see Ref. 22). TFPI is also a reversible, active site-directed inhibitor of factor Xa. The TFPI·factor Xa complex binds to the factor VIIa·TF complex resulting in the formation of an inhibited TF·factor VIIa·TFPI·factor Xa quaternary complex (23). Although no human deficiencies have been reported, the in vivo relevance of TFPI is supported by experiments that showed the sensitization of rabbits to TF triggered disseminated intravascular coagulation after immunodepletion of TFPI (24).
In the absence of coagulation inhibitors, thrombin generation in a
reconstituted model (25) initiated by factor VIIa·TF is characterized
by an initiation phase, in which the cofactors factor V and factor VIII
are activated by traces of factor Xa and thrombin initially produced.
After the initiation phase is completed thrombin generation occurs
rapidly during the subsequent propagation phase, in which prothrombin
is quantitatively activated. The rate of thrombin generation during the
propagation phase of reactions initiated by factor VIIa·TF in a
reconstituted model in the absence of inhibitors is only mildly
influenced when the initiator concentration is varied (25). In a
previous report we found that TFPI, at physiological concentrations,
inhibits thrombin generation by inhibition of factor VIIa·TF activity
and by the direct inhibition of factor Xa (26). TFPI thus extends the
initiation phase of thrombin generation and reduces the rate of
thrombin generation during the propagation phase; however, in the
presence of the components of the factor IX pathway, thrombin generation still becomes explosive after the TFPI extended lag phase
(26). The combination of TFPI/AT-III (for reviews on AT-III see Refs.
27 and 28) at their physiological concentrations (2.5 nM
and 3.4 µM, respectively) is effective in neutralizing explosive thrombin generation by factor VIIa·TF concentrations 10 pM. This inhibition is overcome by increasing the
initiator concentration; thus explosive thrombin generation becomes a
threshold-mediated event in the presence of the combination TFPI/AT-III
(26).
The present study describes the effect of components of the protein C pathway, protein C and thrombomodulin on the tissue factor pathway to thrombin, and the combined actions of the protein C pathway and TFPI.
Phosphatidylserine (PS) from bovine brain,
phosphatidylcholine (PC) from egg yolk, and Hepes were purchased from
Sigma. D-Phenylalanyl-L-arginine chloromethyl
ketone (FPR-ck) was a gift from Dr. R. Jenny, Hematologic Technologies
Inc, (Essex Jct., VT). Spectrozyme TH and Spectrozyme Xa were purchased
from American Diagnostica Inc. (Greenwich, CT). S2366 was obtained from
Chromogenix, Kabi Pharmacia Hepar Inc. Q-Sepharose FF and
CNBr-activated Sepharose CL-4B were obtained from Pharmacia (Uppsala,
Sweden). All other reagents were of analytical grade. Mouse monoclonal
antibodies HFV-9,
HFV-17, and
HPC-2 were provided by Dr.
William Church, Thrombosis Center Antibody CORE, Dept. of Biochemistry,
University of Vermont.
Human coagulation factors X, IX, and prothrombin
were isolated from fresh frozen plasma using the general methods of
Bajaj et al. (29) and were depleted of trace contaminants
and traces of the enzyme products as described (26). Human protein C
was purified by heparin-Sepharose and immuno-chromatography (monoclonal antibody HPC-2 coupled to CNBr-activated CL-4B) from the protein C
pool eluted in the NaCl gradient of the DEAE-Sepharose column involved
in the purification of the vitamin K-dependent clotting factors. Purified protein C was treated for 1 h at 22 °C with 20 µM FPR-ck; residual FPR-ck and hydrolysis products
were removed by extensive dialysis in 0.15 M NaCl, 0.05 M Tris, pH 7.4 (TBS). Aliquots of the protein solution were
frozen in liquid nitrogen and stored at
80 °C. Human protein S was
purified from the protein S pool of the DEAE-Sepharose column using
Blue-Sepharose chromatography as described by Dahlbäck (30). The
purified protein S was passed over anti-protein C and anti-factor X
immunoaffinity columns which were previously equilibrated in TBS. The
flow-through of the immunoaffinity columns was concentrated on a 3-ml
Q-Sepharose column that was equilibrated in TBS. Protein S was eluted
from the Q-Sepharose with TBS containing 1 M NaCl. Possible
traces of enzyme activity in the preparation were inhibited by
treatment for 1 h with 20 µM FPR-ck at 22 °C.
Unreacted FPR-ck and hydrolysis products were removed by extensive
dialysis in TBS, and the protein S was stored frozen in aliquots at
80 °C. Human factor V was isolated from freshly frozen human
plasma using the method of Nesheim et al. (31). Recombinant
factor VIII and recombinant tissue factor (consisting of amino acid
residues 1-242 of the human sequence, lacking the cytosolic stack)
were provided as gifts from Drs. Shu Len Liu and Roger Lundblad, Hyland
Division, Baxter Healthcare Corp (Duarte, CA). Factor VIII was
radiolabeled with 125I using the IODO-GEN (Pierce) method
(32) as described previously (25). Factor VIII loses significant
cofactor activity following iodination (25). Control experiments showed
that the use of an excess of radiolabeled factor VIII to obtain the
same amount of active factor VIII in the reaction results in identical
thrombin generation profiles. Thus, the inactive factor VIII does not
inhibit the reaction (25). Recombinant human coagulation factor VIIa was purchased from NOVO Pharmaceuticals. Recombinant soluble
thrombomodulin (Solulin) was provided as a gift from Dr. J. Morser,
Berlex (Richmond, CA). Recombinant full-length TFPI produced in
Escherichia 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. (Essex Jct., VT). TLCK
anticoagulant peptide (TAP) was provided as a gift from Dr. S. Krishnaswamy, Hematology/Oncology Division, Emory University (Atlanta,
GA). Hirudin was provided as a gift from Genentech (South San
Francisco, CA).
Thrombin generation initiated by factor VIIa·TF in a reconstituted procoagulant model using plasma protein concentrations was studied as described previously (25). Tissue factor (TF) was relipidated at 0.5 nM 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 addition not exceeding 0.25% of the final reaction volume), and immediately thereafter the reaction was started by the addition of an equal volume 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 addition to the factor VIIa·TF, factor V, and factor VIII mixture. When protein C, TFPI, or protein S were included, they were added to the factor X, 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 (25).
When added, protein C (65 nM), protein S (300 nM), and TFPI (2.5 nM) were also present at
their respective plasma concentration (33-35) in the reaction. The
factor VIIa concentrations were varied to change the concentration of
the initiator complex, whereas TF was kept constant and in substantial
excess at 0.25 nM. The final PCPS concentration in the
reaction was 200 µM. Following initiation of the
reaction, aliquots were withdrawn from the reaction mixture and
quenched in either 20 mM EDTA/TBS, 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-PAGE and
immunoblotting. Samples quenched in SDS were heated for 5 min at
95 °C and stored at 20 °C, prior to SDS-PAGE analyses with
intact or reduced disulfide bonds (2% 2-mercaptoethanol, 5 min at
95 °C). Assays for thrombin activity were performed using the
substrate Spectrozyme TH. Hydrolysis of the substrate was monitored by
the change in absorbance at 405 nm using a Molecular Devices
Vmax spectrophotometer. Thrombin generation was
calculated from a standard curve prepared using serial dilutions of
-thrombin. Prothrombinase concentrations were calculated
from the rate of thrombin generation using a
kcat of 5016 min
1 (36). Factor Xa
generation was measured in samples quenched in EDTA and excess hirudin
using the substrate Spectrozyme Xa. Assay specificity for factor Xa was
guaranteed by subtraction of the signal observed in the presence of
excess TAP (2.31 µM), a specific factor Xa inhibitor.
Factor Xa concentrations were calculated from a standard curve prepared
by serial dilutions of purified factor Xa. Apparent dissociation
constants (Kd app) for
prothrombinase were calculated using the equation for the equilibrium relation: Kd = [Vafree]·[Xafree]/[Va·Xa]. 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), TAP (2.31 µM), and 20 mM EDTA/TBS. APC generation was calculated from a standard
curve prepared using serial dilutions of purified APC. Expected rates
of enzyme formation were calculated using the Michaelis-Menten
equation: V = (Vmax·[E]·[S])/([S] + KM), in which V is the velocity of
product formation, Vmax the maximum product
formation at saturating substrate concentrations, [E] the
enzyme concentration, [S] the substrate concentration, and
KM the Michaelis constant for the particular
enzyme-substrate conversion. The samples quenched in SDS were subjected
to SDS-PAGE on 4-12% polyacrylamide gels as generally described by
Laemmli (37). Following SDS-PAGE the proteins were transferred to
nitrocellulose membranes for immunoblot analysis using general
techniques described by Towbin et al. (38). Membranes were
blocked for nonspecific binding with 5% nonfat dry milk (Carnation
Co., Los Angeles, CA) (w/v) in TBS containing 0.05% Tween. Generation
and inactivation of the factor Va heavy chain was followed by
incubating the blocked membranes for 1 h with monoclonal antibody
HFV-17. This antibody recognizes an epitope on the heavy chain of
factor V between residues 307 and 506 and reacts with the same
fragments of the factor V heavy chain upon cleavage by APC recognized
by monoclonal antibody
HFV-6, which was described previously (39).
Generation of the factor Va light chain was followed by immunoblotting
using monoclonal antibody
HFV-9, which recognizes an epitope on the
factor Va light chain (40). The reactive bands were visualized using
peroxidase-conjugated goat anti-mouse IgG at a 1:5000 dilution,
Southern Biotechnology Associates, Inc. (Birmingham, AL) using the
chemiluminescent substrate Luminol (Renaissance chemiluminescent
reagent, DuPont NEN). Light emitted from the hydrolysis of the added
Luminol substrate was exposed to Kodak X-Omat film. Films were
developed in a Kodak X-Omat and scanned using a Microscan 1000 scanning
densitometer (TRI, Inc). Products of radiolabeled factor VIII formed in
the reaction were analyzed by SDS-PAGE under reducing conditions as described previously (25).
In some experiments the membranes that were used for immunoblotting factor V products were washed with 0.5 M NaCl, 0.1 M glycine, pH 2.7, to remove bound antibodies and probed for prothrombin products using a polyclonal burro anti-prethrombin-1 (Grettle) antibody as described (25).
The effects of protein C and thrombomodulin
were studied on factor VIIa·TF (1.25 pM)-initiated
thrombin generation as described under "Materials and Methods."
At this concentration of initiating complex, thrombin generation occurs
explosively after approximately 60 s in the absence of inhibitors
(Fig. 1, filled circles). Neither protein C
at its physiological concentration (65 nM) (Fig. 1, open squares) nor thrombomodulin (10 nM)
(open triangles) influenced thrombin generation when added
separately. The absence of an effect of thrombomodulin alone on
thrombin generation indicates that the soluble thrombomodulin used at
concentrations up to 10 nM does not interfere with the
activation of factor V or factor VIII by thrombin, critical steps
during the initiation phase of the reaction (25). Analyses of thrombin
generation in the presence of 65 nM protein C and varying
concentrations of soluble thrombomodulin are displayed in Fig. 1. The
initiation phase of the reaction does not appear to be influenced in
the presence of protein C at thrombomodulin concentrations up to 10 nM (Fig. 1, inverted triangles). No effect on
the rate of thrombin generation during the propagation phase is
observed in the presence of protein C (65 nM) at
thrombomodulin concentrations 1 nM (Fig. 1, filled squares). However, at 2.5 nM thrombomodulin the rate
of thrombin generation is significantly decreased during the
propagation phase as compared with the control (Fig. 1,
stars). At 5 and 10 nM thrombomodulin (Fig. 1,
diamonds and inverted triangles, respectively),
the rate of thrombin generation declines after approximately 100 nM thrombin is formed, resulting in an extended period of
relatively low prothrombinase activity. Subsequently,
thrombin generation increases again. These delayed propagation phases,
however, ultimately result in the quantitative activation of
prothrombin after 20 min. The delayed increase in thrombin generation
is most probably the result of the accumulation of factor Xa activated
by the uninhibited factor VIIa·TF complex and the continuously
generated factor IXa·factor VIIIa complex. The large amount of factor
Xa that accumulates probably produces activity by
prothrombinase complex formation with traces of intact or
partially degraded factor Va. Thrombin is generated at a rate of 100 nM/min in the delayed propagation phase of the reaction,
which, based on a kcat for
prothrombinase of 5016 min
1 (36), indicates
the presence of approximately 20 pM
prothrombinase.
Effect of the Protein C Pathway in the Presence of TFPI
The
influence of the components of the protein C pathway were studied under
conditions in which factor VIIa·TF activity is only expressed
transiently, i.e. in the presence of TFPI (Fig. 2). Thrombin generation initiated by factor VIIa·TF
(1.25 pM) in the presence of 2.5 nM TFPI (Fig.
2, filled circles) occurs after an extended initiation phase
(2.5 min) followed by a propagation phase in which prothrombin is
quantitatively converted to thrombin with a rate 30% that of the
control reaction without TFPI present (open circles).
Studies with increasing concentrations of thrombomodulin from 2.5 to 10 nM in the presence of protein C (65 nM) and
TFPI (2.5 nM) reveal that the protein C pathway does not
affect the initiation phase of the reaction even in the presence of
TFPI (Fig. 2 inset, inverted filled triangles). This
observation is consistent with the notion that the protein C pathway
functions only subsequent to the generation of thrombin and, thus, acts as a dynamic feedback inhibition mechanism.
In the presence of 2.5 nM TFPI, inhibition of thrombin generation during the propagation phase is observed at a thrombomodulin concentration of 0.1 nM (Fig. 2, filled triangles); thus the lowest concentration of thrombomodulin observed to inhibit thrombin generation by the factor VIIa·TF pathway is an order of magnitude lower when TFPI is present (see the reaction with 2.5 nM thrombomodulin in the absence of TFPI, Fig. 1, stars). At 1 nM thrombomodulin and 2.5 nM TFPI, thrombin generation occurs with an approximately 50% decreased rate after the initiation phase (Fig. 2, filled squares) compared with the controls (in the absence or presence of protein C) without thrombomodulin. After 50 nM thrombin is formed, the thrombin generation rate declines and elimination of prothrombinase activity occurs after 6 min, when ~100 nM thrombin has been generated (Fig. 2, filled squares). Thus, under these conditions, the combined actions of the protein C pathway (1 nM thrombomodulin) and TFPI (2.5 nM) quench the reaction during the propagation phase. The lack of an effect of 1 nM thrombomodulin and protein C in the absence of TFPI (Fig. 1), together with the inability of TFPI to prevent quantitative prothrombin consumption (26), and the observation that the combined actions of 1 nM thrombomodulin, protein C, and TFPI are able to eliminate prothrombinase activity lead to the conclusion that the protein C pathway and TFPI act in synergy under conditions present in the model. Increasing the thrombomodulin concentration (2.5, 5 and 10 nM) results in further decreases in the rate of thrombin generation and lower maximal levels of thrombin formed (Fig. 2, stars (2.5 nM), diamonds (5 nM), and inverted filled triangles (10 nM)). These data demonstrate that the combined actions of the protein C pathway and TFPI in the absence of other inhibitors can quench tissue factor-initiated thrombin generation.
APC Generation in the Reconstituted ModelThe generation of
APC (open symbols) measured in reactions initiated by 1.25 pM factor VIIa·TF in the presence of 2.5 nM
TFPI is displayed along with the observed thrombin generation
(filled symbols) in Fig. 3. In the presence
of protein C and the absence of thrombomodulin, detectable APC
generation (0.2 nM) (open circles) is observed
at 5.5 min by which time 350 nM thrombin had been generated
(filled circles). At the point of nearly quantitative prothrombin activation (~10 min) approximately 1.5 nM APC
has been generated. The APC generation rate in the absence of
thrombomodulin is insufficient to affect the thrombin generation rate
(compare thrombin generation in Fig. 2, in the control reaction
(filled circles), and the reaction with protein C alone
(open squares)). In the presence of 1 nM
thrombomodulin, APC activity (open squares) is observed at
the onset of thrombin generation (1.5 min, filled squares)
in the reaction, resulting in a maximal formation rate of ~0.7
nM APC/min. This rate of APC generation is ~4-fold lower than that expected, based upon the kcat of 370 min1 and KM of 7.6 µM
reported for the soluble thrombomodulin-thrombin complex (41). The
thrombin concentration in the reaction increases from 33 to 113 nM in the reaction from 3 to 6 min without a further significant increase in the rate of APC generation, suggesting that the
thrombomodulin is saturated by the thrombin formed early in the
reaction. This saturation appears to be complete when thrombin concentrations are 30 nM or greater. In separate protein C
activation experiments using purified
-thrombin, a derived
Kd app of ~10 nM was
observed for the thrombomodulin-thrombin interaction with the soluble
thrombomodulin used in the experiments. Together the results suggest a
somewhat lower than expected kcat for the complex with the thrombomodulin used in our experiments.
The generation of 1 nM APC (Fig. 3, open squares) in the presence of 1 nM thrombomodulin at 3 min in the reaction results in a decreased rate of thrombin generation (filled squares). In the presence of 10 nM thrombomodulin, APC generation proceeds from 30 s in the reaction with a rate of 3.1 nM APC/min (Fig. 3, open diamonds). This results in the accumulation of ~9 nM APC after 3 min. The accumulation of 9 nM APC occurs prior to the end of the initiation phase in reactions without thrombomodulin. This generation of APC in the presence of 10 nM thrombomodulin terminates thrombin generation in the initiation phase (Fig. 3, filled diamonds).
Effect of the Protein C Pathway on Factor V ProductsFactor V
circulates as a single chain procofactor (Mr = 330,000), which is activated by limited proteolysis by a variety of enzymes. Thrombin and factor Xa are the most likely activators of
factor V under physiological conditions. The fully active product consists of a heavy and a light chain. Cleavage of factor V at Arg-709
results in generation of the heavy chain (residues 1-709, Mr = 105,000). Cleavage at Arg-1018 gives rise
to a light chain precursor (residues 1019-2196). Cleavage at Arg-1545
results in the generation of the light chain (residues 1546-2196,
Mr = 74,000). Factor V and its products formed
during reactions initiated by 1.25 pM factor VIIa·TF in
the presence of 2.5 nM TFPI were followed by
immunoblotting. Fig. 4 displays the generation of the
heavy chain, using antibody HFV-17, and Fig. 5
displays the generation of the factor Va light chain by immunoblotting
with monoclonal antibody
HFV-9.
In the control reaction in the absence of the protein C pathway components, factor V is cleaved to produce the heavy chain (Mr = 105,000) during the initiation phase of the reaction (Fig. 4A). In the absence of protein C the heavy chain (HC) is a stable product throughout the propagation phase (Fig. 4A). The light chain (LC), is generated at a slower rate (Fig. 5A) than the HC and is a relatively stable product in the reaction. In the presence of 65 nM protein C, but in the absence of thrombomodulin, cleavage of the factor Va HC is observed (Fig. 4B). However, this occurs after most of the prothrombin has been activated to thrombin (8 min). The inactivation process is signaled by the appearance of products that result from cleavage of the HC at Arg-506, indicated by the Mr = 75,000 band (Fig. 4B, 6 min), and subsequent cleavage of this product at Arg-306, giving rise to a Mr = 30,000 immunoreactive band (Fig. 4B, 12 min) consisting of residues 307-506 (42, 43) of the factor Va heavy chain.
The lifetime of the intact factor Va heavy chain is shortened in the presence of protein C and increased concentrations of thrombomodulin. At 1 nM thrombomodulin (Fig. 4C) degradation of factor Va is observed by cleavage at Arg-506 (Fig. 4C, 2.5 min) immediately after the initiation phase when thrombin generation starts to accelerate (Fig. 2, filled squares). In an identical experiment approximately 0.6 nM APC was measured in the reaction at this point (Fig. 3, open squares). Arg-306 cleavage following cleavage at Arg-506 is observed at 3.5 min. Complete attenuation of thrombin generation coincides with complete degradation of intact heavy chain at ~6 min (compare Fig. 2, filled squares, and Fig. 4C). Quantitative cleavage of the 1-506 Mr = 75,000 HC fragment of factor Va at Arg-306 is observed at ~8 min (Fig. 4C) at which time ~4 nM APC is measured in an identical experiment (Fig. 3, open squares). Evaluation of the factor Va LC in the presence of 1 nM thrombomodulin (Fig. 5C) showed a decreased rate of LC generation, probably the result of the lower concentration of thrombin available for factor V activation. However, the factor Va LC is observed as a stable product in the reaction, consistent with the lack of a cleavage site for APC in the human factor Va light chain.
In the presence of 10 nM thrombomodulin, cleavage of the factor Va heavy chain at Arg-506 is observed within the 1st min (Fig. 4D). Complete degradation of the heavy chain is observed after 4 min (Fig. 4D), at which point ~10 nM APC has been generated in an identical reaction (Fig. 3, open diamonds). A product is observed at 4 min in the Mr = 54,000-60,000 region of the gel which indicates some initial cleavage of the heavy chain at Arg-306 (43) at the high APC concentration present in the reaction at that time. However, even at these high thrombomodulin concentrations the factor Va HC inactivation occurs predominantly by initial cleavage at Arg-506. The rate of generation of the factor Va light chain is further slowed in the presence of 10 nM thrombomodulin (Fig. 5D).
The quantitative relationships between thrombin generation and the
appearance/disappearance of the heavy and light chains of factor Va are
displayed in Fig. 6. Thrombin generation is indicated by
the filled circles and the relative factor Va HC and LC
concentrations by the open squares and open
circles, respectively. In the control reaction (Fig.
6A) the concentration of fully activated factor Va and
prothrombin activation is limited by the slower generation of the
factor Va LC (Mr = 74,000). The increase in
light chain concentration at the start of the propagation phase
coincides with the increasing rate of thrombin generation (Fig.
6A, filled circles), suggesting that thrombin generation
during the initiation phase is, in part, limited by the concentration
of factor Va LC in the presence of an excess of HC. Thrombin formed in
the reaction as a result of the factor Xa generated feeds back to
activate factor V. Hence, the reaction is initially limited by the
amount of factor Xa and Va. The maximum concentration of factor Va,
judged from the appearance of both heavy and light chains, is reached at approximately 4 min in the reaction. The factor V activation profile
in the reaction in the presence of protein C without thrombomodulin (Fig. 6B) is similar to that of the control reaction;
however, after reaching a maximum factor Va concentration at 4 min, the HC concentration progressively decreases. Quantitative degradation of
the HC is observed after 10 min.
In the reaction with 1 nM thrombomodulin and protein C
(Fig. 6C), the maximum cofactor concentration observed is
decreased compared with the control reactions with and without protein
C (Fig. 6, A and B). In the early phase of the
reaction the factor Va concentration is limited by the appearance of
the LC and in the later phase by the HC disappearance (Fig.
6C), the latter being complete at 6 min. A maximum rate of
thrombin generation is observed at 3 min in the reaction, when the HC
is present at approximately 40% and the light chain at 20% of their
respective maximum concentrations, when compared with the maximum
densities observed in the control reaction (Fig. 6A). This
maximum level of factor Va, as judged from the relative concentration
of HC and LC in the reaction, coincides with the maximal observed rate of thrombin generation. With initially 20 nM factor V
present, the 20% level of LC would correspond to a maximum
concentration of approximately 4 nM factor Va. This
conclusion assumes that the LC is generated in the same molecule that
possesses an intact HC. However, if the intact heavy and light chains
are randomly distributed, the resulting maximal concentration of fully
active factor Va would be approximately 1.6 nM. Altogether
these data suggest that the greatest cofactor activity existent in the
reaction would range from 1.6 to 4 nM. Since only ~0.15
nM factor Xa (presented later in Fig. 9) is present in the
reaction at this point, it might be concluded that factor Va is not the
limiting component for the kinetic expression of
prothrombinase activity at this point. However, in the
dynamics of the reaction, the factor Va concentration becomes a
limiting component for prothrombinase activity. The
neutralization of prothrombinase activity during the
reaction (Fig. 6C, filled circles) occurs at 6 min
coincidental with quantitative degradation of intact heavy chain (Fig.
6C, open squares).
At 10 nM thrombomodulin (Fig. 6D, filled circles) prothrombinase formation is further limited by an earlier onset of HC degradation and a further decrease in the rate of LC generation compared with the reaction in the presence of 1 nM thrombomodulin (Fig. 6D, open circles and open squares). The increased rate of disappearance of the HC and decreased rate of light chain formation are such that the coexistence of the heavy and light chain during the reaction is nearly prevented at 10 nM thrombomodulin. At a maximum 10% of the light chain and 15% of the heavy chain are present in the reaction at 2.5 min. Since 20 nM factor V was added to the reaction, the maximum level of factor Va reached could be maximally 2 nM. However, in the more likely case of a random HC-LC distribution only 10% of the intact heavy chains would be associated with a light chain, resulting in only 0.3 nM factor Va at the maximum. These data demonstrate how the slowed appearance of the light chain enables the protein C pathway to prevent active cofactor accumulation by cleavage of the HC before the LC is generated.
Analyses of prothrombin activation products reveal that the complete inhibition of thrombin generation in the presence of protein C and 1 or 10 nM thrombomodulin (Fig. 2, squares and diamonds) is associated with a lack of prothrombin consumption (results not shown). The continued presence of intact prothrombin demonstrates that the inhibitory effect of the protein C pathway on thrombin generation is due to inhibition of prothrombinase activity and not due to substrate depletion.
Effect of the Protein C Pathway on Reactions Initiated in the Presence of Factor VaThe effect of the protein C pathway on
thrombin generation initiated in the presence of factor Va was
evaluated to examine the effects when the heavy and light chains of the
active cofactor are both present at the start of the reaction. This
experiment was performed to test whether the profound inhibition of
thrombin generation, observed in the presence of TFPI and 10 nM thrombomodulin (Fig. 6D), is caused by the
prevention of the coexistence of the heavy and light chains under these
reaction conditions. Reactions initiated by 1.25 pM factor
VIIa·TF in the presence of TFPI and factor Va (Fig. 7)
display a shorter initiation phase (filled symbols) when
compared with reactions initiated in the presence of the procofactor
(open symbols); however, the rate of thrombin generation
during the propagation phase of the reaction is not increased by
preactivation of factor V. In the presence of protein C and 1 nM thrombomodulin, the thrombin generation curves of the reactions with factor Va and factor V are similar after the onset of
thrombin generation (Fig. 7, open and filled
squares, respectively), and preactivation of factor Va only leads
to a 20% increase in the maximal level of thrombin generation. In
contrast, in the presence of 10 nM thrombomodulin and
protein C, a 15-fold higher thrombin concentration is generated when
the cofactor is activated (Fig. 7, filled and open
triangles). These data show a dramatic loss of effectiveness of
the protein C pathway in inhibiting thrombin generation when the
reaction is started with the fully active cofactor. These data support
the hypothesis that inhibition of the thrombin generation pathway at
limiting factor Xa concentration is dependent upon both the slowed
formation of the light chain coupled with inactivation of the heavy
chain by APC.
Effect of the Protein C Pathway on Factor VIII Products
Factor VIII consists of a light chain (Mr = 80,000) which is associated via a Ca2+-dependent interaction with a heavy chain that may vary in size (Mr = 110,000-200,000) (10). Activation of factor VIII occurs by cleavage of the heavy chain species at Arg-740 to initially generate the A1-A2 fragment (Mr = 90,000) (10, 44) containing residues 1-740 of factor VIII (13). Cleavage at Arg-372 bisects the A1 (Mr = 50,000) and the A2 (Mr = 40,000) domains (10, 16, 44). Generation of the active species of the light chain occurs by cleavage at Arg-1689 resulting in the generation of a Mr = 74,000 light chain containing the carboxyl terminus of factor VIII. Active factor VIIIa is thus composed of the A2 domain noncovalently associated with the A1/light chain heterodimer. The A2 domain is associated with the A1/light chain dimer via a binding site contained within residues 337-372 of the A1 domain (45). Proteolytic inactivation of factor VIIIa by APC or factor Xa has been reported to occur by cleavage at Arg-336 in the A1 domain (13).
The factor VIII products generated during reactions (initiated with
1.25 pM factor VIIa·TF in the presence of 2.5 nM TFPI) with and without the components of the protein C
pathway were followed using radiolabeled factor VIII (Fig.
8). Thrombin generation curves using radiolabeled factor
VIII showed identical rates of thrombin generation under the different
conditions when compared with reactions with unlabeled factor VIII
(results not shown). In the control reaction factor VIII is activated
by cleavage of the heavy chain species at Arg-740 after 1 min to
generate the Mr = 90,000 intermediate (A1-A2
domains) (Fig. 8A, lane 3). Cleavage by thrombin at Arg-372
and generation of the Mr = 50,000 fragment (A1
domain) and the Mr = 40,000 fragment (A2 domain)
becomes apparent at 1.5 min (Fig. 8A, lane 4). Cleavage of
the light chain of factor VIII (Mr = 80,000)
also occurs at 1.5 min to generate the active species
(Mr = 74,000). Near quantitative activation of
factor VIII is observed at 4.5 min when virtually all light chain is converted to the Mr = 74,000 species. The A1
domain is the reported target of proteolytic inactivation by APC,
factor Xa, or thrombin by cleavage at Arg-336 (13). Cleavage at this
site results in a peptide that migrates on SDS-PAGE at
Mr = 43,000 just above the A2 domain (46). The
A1 fragment seems relatively stable in the reaction, although some
product is lost between 7 and 20 min. This may be the result of
cleavage of the A1 domain by thrombin at Arg-226 (47) which results in
low molecular weight fragments such as observed at the bottom of the
gel. The Mr = 74,000 light chain and the A2
domain appear as stable products in the reaction (Fig. 8A).
The density of the B domain decreases after 4.5 min accompanied by the
appearance of a low molecular weight triplet at the bottom of the gel.
Thus, the low molecular weight fragments seem to be derived from
proteolysis of the B domain and the A1 domain by the high
concentrations of thrombin generated in the reaction.
The generation of factor VIII products in reactions containing 10 nM thrombomodulin and protein C (Fig. 8B) showed a slower activation rate of factor VIII, associated with the lower rate of thrombin generation as a result of factor Va inactivation under these conditions. In contrast to the control reaction, no significant cleavage of the A1 domain is observed with time. A stable density is observed in the A1 domain region in the presence of the protein C pathway at this high thrombomodulin concentration (10 nM) (Fig. 8B). No new cleavages in the other products are observed in the presence of the protein C pathway compared with the control reaction (compare Fig. 8, A and B), an observation consistent with the lack of any cleavages by APC in factor VIIIa besides the A1 domain. Thus, the protein C pathway does not result in an accelerated inactivation of factor VIIIa by proteolysis. The reduced level of thrombin formed in the presence of the protein C pathway results in the persistence of the B domain compared with the control reaction (Fig. 8A); consequently, a decreased rate of appearance of the low molecular weight triplet is observed.
Protein S has been reported to facilitate inactivation of factor VIIIa
by APC (14, 18, 46). However, in a reaction in the presence of 300 nM protein S and the protein C pathway (65 nM
protein C, 10 nM thrombomodulin), the A1 domain appeared as a stable product up to 12 min in the reaction (Fig. 8C).
Some density is lost in the A1 domain region between 12 and 20 min, accompanied by increasing density at Mr = 43,000 (indicated by * in Fig. 8C), indicating that cleavage at
Arg-336 (13, 44-46) occurs in the presence of protein S. However,
quantitative conversion of the A1 domain is not observed in the
presence of protein S over the 20-min period evaluated. No other
cleavages are observed in the presence of protein S (compare Fig.
8, B and C). It is noteworthy to mention that the
lack of inactivating cleavages of factor VIII during the experiments
does not mean that a stable level of factor VIIIa activity is reached.
Factor VIIIa at physiological concentrations (0.7 nM) loses
most of its activity as a consequence of dissociation of the A2 domain
from the A1/light chain heterodimer (16, 44, 48). These data
demonstrate that in the present model the protein C pathway, even in
the presence of protein S, does not result in the proteolytic
inactivation of the factor VIIIa molecule in the model within the
interval in which factor Va is completely inactivated by APC (4 min,
Fig. 4D), and prothrombin activation is totally
suppressed.
The lack of proteolytic inactivation of factor VIIIa in the presence of the protein C pathway components suggested that factor VIIIa cofactor activity in the activation of factor X by the factor IXa·factor VIIIa complex is not altered by APC proteolysis. Under the experimental conditions (low factor VIIa·TF in the presence of TFPI), thrombin generation during the propagation phase is virtually dependent upon factor IXa·factor VIIIa activity (26). The effect of the protein C pathway (10 nM thrombomodulin) on factor Xa generation in reactions initiated by 1.25 pM factor VIIa·TF in the absence or presence of 2.5 nM TFPI is displayed in Fig. 9. Factor Xa generation in the control without inhibitors is observed after 30 s in the reaction, increases to a rate of 0.2 nM/min after 30 s, and is linear up to 8 min in the reaction (Fig. 9, filled circles). Addition of protein C and 10 nM thrombomodulin did not influence factor Xa generation at any stage in the reaction (Fig. 9, filled squares). In the presence of 2.5 nM TFPI, factor Xa generation is observed at 1.5 min and proceeds with a maximal rate of 0.062 nM/min (Fig. 9, open circles). In the presence of TFPI the factor Xa generation rate is not further affected by protein C and 10 nM thrombomodulin (Fig. 9, open squares). Thus the lack of factor VIIIa cleavage by APC (Fig. 8B) is consistent with the lack of an effect of the protein C pathway upon factor Xa generation. Overall these data lead to the conclusion that the effects of the protein C pathway on thrombin generation in this model are solely explained by the inactivation of factor Va in the reaction and not by inactivation of factor VIIIa cofactor activity.
Correlation of the Prothrombinase Components in a Reaction Containing 1 nM ThrombomodulinA representative
correlation of the prothrombinase concentration, with the
concentrations of the prothrombinase components, factor Xa
and factor Va, for a reaction inhibited by 1 nM
thrombomodulin and protein C in the presence of TFPI, is displayed in
Fig. 10. The prothrombinase concentration
was calculated from the change in thrombin generation (filled
circles). The fully active factor Va concentration (open
squares) was deduced from the concentration of the limiting
component, the light chain or heavy chain. As can be seen in Fig. 10,
the factor Xa (open triangles) concentration exceeds the
concentration of prothrombinase (filled diamonds) during the reaction in the presence of substantial excess of factor Va
until 6 min. At the peak of prothrombinase activity at 4 min when 65 pM prothrombinase is formed, the
measured factor Xa concentration is 130 pM. The assembly of
factor Xa and factor Va may occur almost instantaneously under the
conditions used (excess negatively charged phospholipid (49)). When
entered in the equilibrium relation for prothrombinase, the
concentrations measured at 4 min in the reaction of factor Va (3.3 nM), factor Xa (130 pM), and
prothrombinase (65 pM) suggest a
Kd app of ~3.2 nM for
prothrombinase. This observed
Kd app for prothrombinase as
estimated in the complex reaction is in reasonable agreement with the
measured Kd of ~1 nM for
prothrombinase (49). It should be mentioned that the factor
Xa generation in the reaction measured by the chromogenic assay may
give an overestimation of the free concentration of factor Xa because
of some dissociation of the formed factor Xa·TFPI complexes in the
presence of the competing substrate. Furthermore, a number of different
factor V species are present at 4 min in the reaction, and it is
uncertain at the present time whether they bind factor Xa or not. It is
thus possible that some factor Xa is bound to factor V species that do
bind factor Xa but lack or have reduced cofactor activity in
prothrombinase. The drop in factor Va concentration after 4 min correlates with declining prothrombinase activity, and
this activity ceases when factor Va is depleted from the reaction after
6 min.
Effect of the Protein C Pathway at Varying Factor VIIa·TF Concentrations
An experiment in which thrombin generation was
evaluated at varying factor VIIa·TF concentrations in the presence of
TFPI (2.5 nM) and protein C (65 nM) with or
without 10 nM thrombomodulin is displayed in Fig.
11. In the presence of TFPI and protein C, thrombin
generation is observed over a range of 0.05-100 pM factor VIIa·TF, showing prolonged initiation phases and decreased rates of
thrombin generation during the propagation phase with decreasing factor
VIIa·TF concentrations (Fig. 11A). The rate of thrombin generation in the propagation phase decreases 20-fold over a range of
100-0.05 pM factor VIIa·TF. At 0.05 pM
factor VIIa·TF thrombin generation occurs after a lag phase of 10 min
in the presence of TFPI and protein C (Fig. 11A, inverted
triangles). Despite this long initiation phase explosive thrombin
generation still occurs, indicating the lack of any significant protein
C activation by traces of thrombin in the absence of
thrombomodulin.
When the reaction is initiated in the presence of 10 nM
thrombomodulin, protein C, and TFPI, a completely different profile is
observed (Fig. 11B). In the presence of 10 nM
thrombomodulin/protein C/TFPI (Fig. 11B, diamonds) factor
VIIa·TF concentrations 5 pM fail to provide explosive
thrombin generation. Initiation with 25 pM factor VIIa·TF
in the presence of 10 nM thrombomodulin results in the
sudden attenuation of prothrombinase activity after the explosive generation of 200 nM thrombin (Fig. 11B,
squares). In contrast, at 100 pM, factor VIIa·TF
overcomes the threshold raised by the protein C pathway components and
TFPI, and the result is the quantitative activation of prothrombin
(Fig. 11B, triangles). The presence of 10 nM thrombomodulin prevents explosive thrombin generation at
low initiating factor VIIa·TF concentrations by stopping the
formation of active factor Va (Fig. 6D). The increased rates
of formation of catalysts in the presence of high concentrations of
initiator overcome the inhibitory potential of the protein C pathway.
The rapid procoagulant response at high initiator concentrations does
not allow the protein C pathway time to suppress the thrombin generation reaction. Of interest is the threshold for explosive thrombin generation in the presence of 10 nM thrombomodulin
(~10 pM factor VIIa·TF) is similar to the threshold for
explosive thrombin generation in the presence of the combination of
physiological concentrations of TFPI and AT-III (26). It appears that
the threshold concentration of initiating factor VIIa·TF required to
overcome the inhibition of explosive thrombin generation will be
dependent upon the thrombomodulin concentration present at the
beginning of the reaction.
The effects of the protein C pathway on thrombin, factor Xa, factor Va, factor VIIIa, and APC generation initiated by the extrinsic (factor VIIa·TF) pathway of coagulation were studied in reconstituted reactions. The data demonstrate the following. 1) The isolated protein C pathway (thrombomodulin and protein C) reduces the rate of thrombin generation initiated by pM concentrations of factor VIIa·TF, without influencing the duration of the initiation phase of thrombin generation. 2) The protein C pathway and TFPI inhibit thrombin generation in a synergistic manner; the combination of these inhibitors has the potential to neutralize prothrombinase activity initiated by the tissue factor pathway. 3) The effects of the protein C pathway on thrombin generation are solely correlated with the proteolytic inactivation of factor Va by APC. 4) The generation of the factor Va heavy chain is completed during the initiation phase of the reaction. The onset and rate of the propagation phase of thrombin generation coincides with cleavage of factor V at Arg-1545 to generate the light chain. In the presence of protein C and up to 10 nM thrombomodulin, no intermediates of factor V inactivation of a molecular weight of Mr = 280,000 are observed (39), demonstrating that there is no significant inactivation of intact factor V by cleavage at Arg-306. 5) The inhibition of tissue factor-initiated prothrombinase activity by the protein C pathway is correlated with cleavage of the factor Va heavy chain. The cleavage of the heavy chain occurred by initial cleavage at Arg-506 followed by cleavage at Arg-306 (Fig. 4) over a range of 0-10 nM thrombomodulin in the present model. Thus, the mechanism of inactivation under dynamic conditions, in the presence of the other coagulation factors and in situ generated APC, is similar to that observed in a system using the isolated reaction (42, 43).
These conclusions are based on results of experiments performed at saturating phospholipid concentrations, and therefore the results do not exclude other effects of the protein C pathway components on platelets or at lower phospholipid concentrations.
Although the protein C anticoagulant pathway theoretically has the potential to shut down factor VIIa·TF-triggered thrombin generation in the absence of other inhibitors, this is not observed in the reconstituted model. The rate of factor Xa generation is unaffected by the presence of the protein C pathway, and factor VIIIa cleavage by APC does not occur within a period relevant to the propagation phase of thrombin generation. However, nearly quantitative cleavage of the factor Va heavy chain by the protein C pathway (results not shown) in the absence of other inhibitors results in slower thrombin generation (Fig. 1). The relatively high concentrations of factor Xa, present as a consequence of uninhibited factor VIIa·TF and factor IXa·factor VIIIa, apparently saturate traces of active or partially active factor Va still present resulting in significant prothrombinase activity (20 pM).
When TFPI is the only inhibitor, the reaction proceeds through factor
Xa generation via the factor IXa·factor VIIIa pathway after the
factor VIIa·TF complex is inhibited by TFPI·factor Xa (26). The
actions of TFPI and the protein C pathway are complementary, and these
inhibitors behave in a synergistic manner. TFPI inactivates the factor
VIIa·TF complex and allows the formation of only limited amounts of
factor Xa and factor IXa. In the presence of TFPI and the protein C
pathway the limited amount of factor Xa that escapes TFPI inhibition is
deprived of its cofactor factor Va because of the inactivation of the
latter by APC. The level of thrombin generated and the rate at which
the initial thrombin is formed are governed by the concentration of
initiating factor VIIa·TF and the concentration of thrombomodulin.
Quenching of the thrombin generation reaction in the presence of 10 nM thrombomodulin and TFPI is observed for reactions
initiated by factor VIIa·TF concentrations 20 pM.
In the present model proteolytic processing of factor VIIIa does not contribute to the neutralization process nor is factor Xa generation influenced by the protein C pathway constituents. Hence, the inhibitory effects of the protein C pathway do not correlate with either factor VIIIa cleavage or reduced factor Xa generation. Protein S enhanced factor VIIIa cleavage in the model; however, even in the presence of protein S and the protein C pathway factor VIIIa inactivation occurred only slowly in the model. These observations are consistent with previous data of our laboratory on the inactivation of factor VIIIa by APC in the presence of protein S (46). However, the present data do not predict a significant role for the synergistic cofactor effect of protein S and factor V on the inactivation of factor VIIIa by APC (50).
Based on these results we suggest the regulatory model depicted in Fig.
12. TFPI regulates the initiation phase of factor
VIIa·TF-initiated thrombin generation by inhibition of factor
VIIa·TF and factor Xa. The protein C pathway does not affect the
initiation phase or the factor Xa generation by factor IXa·factor
VIIIa activity required to drive the propagation phase in the presence
of TFPI. The acute inhibition of the propagation phase of thrombin
generation by the protein C pathway is solely dependent on the
inactivation of factor Va, indicated by the negative feedback arrow
from APC to factor Va. The termination of thrombin generation is
dependent on the synergistic inhibitory action of TFPI and the protein
C pathway.
The physiological concentration of thrombomodulin is defined by the endothelial cell surface/volume ratio. The concentration of thrombomodulin may exceed 10 nM in the microcirculation where the surface to volume ratio is high (51). Hence, the effects of thrombomodulin observed in the present model are potentially physiologically relevant. Moreover, intact relipidated thrombomodulin is more efficient as a cofactor in the activation of protein C when compared with the soluble form (41). Furthermore, the soluble thrombomodulin used in the present experiments lacks chondroitin sulfate, a regulatory moiety on thrombomodulin involved in the association of thrombin, which may be present on thrombomodulin in vivo (52). Taken together it is anticipated that the observed effects of soluble thrombomodulin on APC generation in the present model may occur at lower concentrations of the natural form.
TFPI is present in different forms and states in the circulation (reviewed in Ref. 53). Several COOH-terminal truncated, less potent, forms of TFPI exist in plasma, and a major portion of TFPI in plasma is associated with lipoprotein particles, either by noncovalent interactions or by disulfide linkage to the apolipoprotein moieties in the lipoprotein particles. A large pool of full-length TFPI is presumably bound in vivo to endothelial cell exposed heparan sulfate (54-57). Displacement of this TFPI pool from the endothelium by heparin treatment leads to increases in plasma TFPI levels up to 2-6-fold. Thrombomodulin is constitutively expressed as a transmembrane protein on the surface of vascular endothelial cells (4). Hence, our data provide a rationale for the apparent co-localization of the complementary anticoagulant functions of full-length TFPI and thrombomodulin at the blood/vessel wall interface.
Evaluation of clot formation and thrombin generation in a tissue factor-activated whole blood clotting model revealed that the fibrin clot is formed by the action of only 2.5 nM thrombin (58) and that most thrombin generated is formed after clot formation. Thus, the thrombin levels observed in the present model, even in the presence of high thrombomodulin concentrations, would result in significant fibrin formation. Fibrin clots may, however, be lysed by the fibrinolytic system. Fibrin clots can be stabilized against fibrinolytic attack by the action of factor XIII and the thrombin activable fibrinolysis inhibitor. Both factor XIII (59) and thrombin activable fibrinolysis inhibitor (60) are zymogens that are specifically activated by thrombin. Thus, the reduced amount of thrombin formed by the action of the protein C pathway will limit the concentration of thrombin available for the stabilization of fibrin clots. This may result in a clot predisposed to solubilization by the fibrinolytic system.
We thank Dr. Kirk Johnson (Chiron Corp.) for
the generous gift of rTFPI; Dr. Shu Len Liu and Dr. Roger Lundblad
(Hyland Division, Baxter Healthcare Corp.) for providing us with
recombinant factor VIII and recombinant tissue factor; Dr. John Morser
(Berlex) for the generous gift of recombinant soluble thrombomodulin;
and Dr. William Church (University of Vermont, Dept. of Biochemistry) for providing antibodies HFV-17 and
HFV-9; Dr. R. Jenny
(Hematologic Technologies, Inc.) for providing human factor Xa and APC;
Dr. S. Krishnaswamy (Hematology/Oncology Division, Emory University) for providing recombinant TAP; and Genentech for providing recombinant hirudin.