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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the University of Vermont, College of Medicine,
Burlington.
The ability of factor VIIa to initiate thrombin generation and clot
formation in blood from healthy donors, blood from patients with
hemophilia A, and in anti-factor IX antibody-induced ("acquired") hemophilia B blood was investigated. In normal blood, both factor VIIa-tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 that of the factor VIIa-TF complex. In congenital hemophilia A blood
and "acquired" hemophilia B blood in vitro, addition of 10 to 50 nM
factor VIIa (pharmacologic concentrations) corrected the clotting time
at all TF concentrations tested (0-100 pM) but had little effect on
thrombin generation. Fibrinopeptide release and insoluble clot
formation were only marginally influenced by addition of factor VIIa.
TF alone had a more pronounced effect on thrombin generation; an
increase in TF from 0 to 100 pM increased the maximum thrombin level in
"acquired" hemophilia B blood from 120 to 480 nM. Platelet
activation was considerably enhanced by addition of factor VIIa to both
hemophilia A blood and "acquired" hemophilia B blood. Thus,
pharmacologic concentrations of factor VIIa cannot restore normal
thrombin generation in hemophilia A and hemophilia B blood in vitro.
The efficacy of factor VIIa (10-50 nM) in hemophilia blood is dependent
on TF.
(Blood. 2002;99:923-930) The blood-coagulation cascade is initiated when
cryptic tissue factor (TF) is expressed and exposed to circulating
blood and binds plasma factor VIIa. The resulting factor VIIa-TF
complex activates the serine protease zymogens factor IX and factor X. The factor Xa that is initially produced generates picomolar amounts of
thrombin, which activates platelets and cleaves procofactors factors V
and VIII. Factor VIIIa forms a complex on a membrane surface with
serine protease factor IXa and activates factor X at a 50- to 100-fold
higher rate than the factor VIIa-TF complex. The factor Xa produced,
in complex with its cofactor, factor Va, and an appropriate membrane
surface forms the prothrombinase complex, which is the
primary activator of prothrombin. The thrombin produced amplifies its
own generation by activating factor XI and completing the activation of
platelets and procofactors. Thrombin cleaves fibrinogen and activates
factor XIII to form the insoluble isopeptide cross-linked fibrin clot.
The coagulation cascade is down-regulated by the stoichiometric
inhibitors antithrombin III (AT-III) and tissue factor pathway
inhibitor (TFPI) and by the dynamic protein C system.1
Genetic and acquired deficiencies in coagulation proteins lead to
hemorrhagic syndromes.2-6 The most common bleeding
disorders result from deficiencies of factor VIII (hemophilia A) or
factor IX (hemophilia B) coagulant activity. In the past, the principal treatments for hemophilia relied on partially purified concentrates of
coagulation factors.7,8 These concentrates, however, have been associated with thromboembolic complications and viral
infections.9-11 During the past decade, plasma-derived,
monoclonal antibody-purified factor VIII12 and factor
IX13 concentrates that contain negligible amounts of
other coagulation factors, as well as recombinant factors VIII14,15 and IX,16 have become available. In
addition, substantial progress in the treatment of hemophilias has been
achieved in animal models by using gene therapy,17-19 and
clinical trials employing this approach in patients have
begun.20,21
In a considerable proportion of patients with hemophilia receiving
replacement therapy, inhibitory antibodies directed against the missing
factor develop,15,22-25 and this complicates further administration of the deficient protein. For patients with antibodies against factor VIII or factor IX, an alternative treatment using supraphysiologic concentrations of factor VIIa was
suggested.26,27 During the past decade, recombinant factor
VIIa has been used successfully for hemophilia treatment in patients
with and without inhibitors,28-32 especially those
undergoing surgical procedures.30-32 Moreover, recombinant
factor VIIa has been suggested for treatment of almost all bleeding
disorders and for enhancement of normal hemostasis in patients without
coagulation defects.33 The mechanism by which hemostasis
is established by high doses of factor VIIa is not known, although it
has been hypothesized that factor VIIa can trigger thrombin generation
in a TF-independent manner.34,35
Several models of TF-initiated blood coagulation have been developed in
our laboratory.36-38 In all these models, thrombin generation can be divided into 2 phases.39 The initiation
phase follows addition of TF and is characterized by generation of
thrombin at low nanomolar concentrations, which leads to activation of platelets and almost quantitative proteolysis of factors V and VIII.
Femtomolar to picomolar amounts of factors VIIa, IXa, Xa, and XIa are
produced during this phase, the duration of which is regulated
primarily by the factor VIIa-TF complex and
TFPI.36,37,40,41 Subsequently, a propagation phase occurs,
which is characterized by rapid activation of prothrombin; increased
rates of activation of factors VII, IX, X, and XI; and formation of
solid clots. The rate of thrombin generation during the propagation
phase depends primarily on the factor Xa produced by the factor
IXa-factor VIIIa complex.39 As a consequence, thrombin
generation during the initiation phase is virtually unaffected by the
absence of factor VIII or factor IX.36,37,39 Thrombin
generation during the propagation phase is significantly suppressed in
hemophilia A and hemophilia B because of decreased generation of factor
Xa.36,37,41 Addition of factor VIII to a physiologic
concentration (0.7 nM) to hemophilia A blood restores normal thrombin
generation.41 In this study, we evaluated the ability of
high concentrations of factor VIIa alone and in the presence of TF to
generate thrombin in blood from healthy donors, blood from patients
with hemophilia A, and "acquired" hemophilia B blood and in a
synthetic blood coagulation model.
Materials
Human donors
The donor with hemophilia A, a 21-year-old man, tested positive for hepatitis C. However, he had normal values for fibrinogen (2.5 mg/mL), platelet count (2.1 × 108/mL), PT (12.8 seconds), and INR (1.0), although he had a severe deficiency of factor VIII (VIII:C < 1%). His levels of factor IX were elevated (209%); those of other coagulation proteins were in the normal range. He had a history of bleeding and joint pain and routinely self-administered recombinant factor VIII products when symptomatic. Blood was drawn 1 week after his most recent factor VIII injection, and his factor VIII levels on the days of the experiments were less than 2%. There was no evidence of inhibitors (eg, anti-factor VIII antibodies). "Acquired" hemophilia B blood The equivalent of acquired hemophilia B was induced in fresh CTI-inhibited normal whole blood in vitro by adding 50 µg/mL -IX-91. At this concentration, the antibody prolonged the activated partial thromboplastin time (APTT) of normal plasma from 38 to 115 seconds. The APTT for commercial factor IX-deficient plasma (< 1% of
factor IX; George King Biomedical, Overland Park, KS) was 112 seconds.
The titer of the -IX-91 at 50 µg/mL was 27 Bethesda units.48
Synthetic blood coagulation model The procedure used was a modification of those described by Lawson et al36 and van't Veer et al.40Procofactor solution. Relipidated TF (50 pM; omitted when desired) was incubated with 4 µM PCPS (omitted in platelet experiments) in HEPES-buffered saline (HBS; 20 mM HEPES and 150 mM sodium chloride) and 2 mM calcium chloride (CaCl2) for 10 minutes at 37°C. Factor V (40 nM), factor VIII (1.4 nM; omitted when desired), and 4 × 108/mL platelets (in platelet experiments only) were added to the relipidated TF before initiation of the reaction. Zymogen-inhibitor solution. Prothrombin (2.8 µM), factor VII (20 nM), factor VIIa (0.2 nM), factor X (340 nM), factor IX (180 nM), TFPI (5 nM), and AT-III (6.8 µM) were preheated in HBS and 2 mM CaCl2 at 37°C for 3 minutes. In some experiments, additional amounts of factor VIIa (10-240 nM) were added. The reaction was started by mixing equal volumes of both solutions, which resulted in physiologic concentrations of the proteins and platelets and a final TF concentration of 25 pM. After initiation of the reaction, 10-mL aliquots were withdrawn from the reaction mixture at selected time points, placed in 20 mM EDTA in HBS (pH 7.4) containing 0.2 mM Spectrozyme TH, and assayed immediately for thrombin activity. Hydrolysis of the substrate was monitored by the change in absorbance at 405 nm by using a Vmax spectrophotometer (Molecular Devices, Sunnyvale, CA). Thrombin generation was calculated from a standard curve prepared by serial dilutions of-thrombin.
TF-initiated clotting of fresh human blood Single-tube clotting time test. The protocol used was that described by Holmes et al.49 Fresh human blood (1 mL) was added to a tube containing 100 µg/mL CTI (CTI prevents the contact pathway of blood coagulation by inhibiting factor XIIa) and various concentrations (0-40 µg/mL) of antibody 5G9 and recombinant factor VIIa (0-60 nM) in the absence or presence of 1 pM or 10 pM TF. The tube was placed in a Hemochron activated-coagulation-time instrument (International Technidyne, Edison, NJ). The time to clot was detected by displacement of a magnet within the rotating tube by formation of fibrin strands. Multiple-tube experiments. The protocol used was a modification of that of Rand et al.38 Experiments were done in 32 tubes placed on a rocking table enclosed in a temperature-controlled (37°C) glove box. Fresh CTI-inhibited (100 µg/mL CTI) blood was used after venipuncture and immediate delivery into reagent-loaded tubes. Thirty of 32 tubes (2 series/experiment; 16 tubes/series) were loaded with CTI and 12.5 pM relipidated TF in HBS and 2 mM CaCl2. Two phlebotomy control tubes (1 tube/series) contained no TF. Recombinant factor VIIa (10 or 50 nM; all tubes, experiment series only) and an equivalent volume of factor VIIa dilution buffer (HBS and 2mM CaCl2; all tubes, control series only) were loaded. The zero-time tube for each series was pretreated with 1 mL 50 mM EDTA and 10 µL 10 mM FPRck (diluted in 10 mM hydrochloric acid). After blood was delivered, the tubes were periodically (1-20 minutes) quenched with EDTA and FPRck. In the "acquired" hemophilia B experiments, all tubes were loaded in duplicate with CTI and various concentrations of TF (0-100 pM) and factor VIIa (0, 10, and 50 nM). All tubes in experiment series were loaded with 50 µg/mL-IX-91; those in control series were loaded
with -IX-91 dilution buffer (HBS and 2 mM CaCl2). Tubes
were treated with EDTA and FPRck 10 minutes after solid clot was observed.
In all multiple-tube experiments, no more than 35 µL of reagents was
loaded in each tube. The clotting time was observed visually by 2 observers and was recorded when clumps were observed on the side of the
tube. After the experiment, tubes were centrifuged and the supernatants
were aliquoted for further analyses. ELISAs for TAT (thrombin
generation) and osteonectin (platelet activation) were done. FPA and
FPB (clot formation) were assessed by high-performance liquid
chromatography and fibrinogen and fibrin by Western blot analysis.47 Solid clots were lyophilized, weighed,
solubilized, and analyzed by gel electrophoresis.47
Factor VIIa and TF in normal blood Thrombin generation in the synthetic blood coagulation model.
Figure 1A (
When PCPS was replaced by washed platelets (2 × 108/mL; Figure 1B), the initiation phase in the reaction initiated by 25 pM relipidated TF lasted almost 5 minutes (Figure 1B;  ). The maximum thrombin level (120 nM) was achieved 9 minutes after initiation of the reaction. In the absence of TF, factor
VIIa at a concentration of 30 nM (Figure 1B;  ) did not produce
detectable amounts of thrombin for 20 minutes. At the highest factor
VIIa concentration tested (120 nM), the initiation phase of thrombin
generation lasted 8 minutes and the maximum thrombin level was 80 nM.
Thus, in the presence of either platelets or phospholipids, factor VIIa
in the absence of TF was able to trigger thrombin generation. However, the efficiency of the factor VIIa-TF complex was about
104-fold higher than that of factor VIIa alone. This
observation is in agreement with the finding by Komiyama et
al51 indicating that TF increases the factor IX- and
factor X-activating efficiency of factor VIIa by 3 to 4 orders of magnitude.
Clotting assays of CTI-inhibited whole blood.
In the presence of 100 µg/mL CTI, fresh, otherwise untreated human
blood clotted in more than 1000 seconds (Figure
2;
Factor VIIa and TF in hemophilia Thrombin generation in the synthetic hemophilia A model.
Thrombin generation initiated by 25 pM TF in the presence of
2 × 108/mL platelets is shown in Figure
3. In a complete system (Figure 3;
"Acquired" hemophilia B blood.
In the absence of TF, CTI-inhibited normal blood clotted in 18 minutes
(Figure 4A;
In normal blood, maximum thrombin levels were independent of TF concentration at concentrations of this protein above 20 pM (Figure 4B;  ). In acquired hemophilia B blood, in either the absence or presence
of additional factor VIIa at concentrations of 10 and 50 nM, increases
in TF concentration cause raised the maximum thrombin levels at all
concentrations of TF tested. In the absence of TF and exogenous factor
VIIa (Figure 4B; ), 120 nM thrombin was generated after 40 minutes
of reaction. In contrast, addition of 10pM TF produced thrombin levels
of 210 nM, and at 100 pM TF, almost 500 nM thrombin was
generated. Addition of 10 or 50 nM factor VIIa to "acquired"
hemophilia B blood only slightly increased thrombin levels at all TF
concentrations tested (0-100 pM).
Platelet activation in normal blood was complete after 11 to 28 minutes
at all TF concentrations tested (Figure 4C; ). In "acquired" hemophilia B blood in the absence of TF and
exogenous factor VIIa (Figure 4C; ), about 25% of
osteonectin was released after 40 minutes of reaction. Addition of 10 pM TF to the "acquired" hemophilia B blood drove platelet
activation to completion. In the absence of TF and the presence of 10 or 50 nM exogenous factor VIIa (Figure 4C; * and  , respectively),
platelet activation in "acquired" hemophilia B blood was complete
at the end of the experiment (37 minutes and 24 minutes, respectively).
Congenital hemophilia A blood.
The influence of 10 nM factor VIIa on clotting of normal
(contemporaneous control) and hemophilia A blood is shown in Figure 5. In all experiments, clotting of normal
and hemophilia A blood was initiated with 12.5 pM TF. In the absence of
exogenous factor VIIa, normal blood clotted in 3.25 minutes (Figure 5;
Thrombin generation in normal blood occurred at a maximum rate of 1.1 nM/second (Figure 5A; ). The maximum level of the TAT complex
observed at the end of the experiment (20 minutes) was 660 nM. The
concentration of thrombin at clotting was between 10 and 20 nM.
Addition of 10 nM factor VIIa to normal blood (Figure 5A;  )
increased both the maximum rate of generation and the level of thrombin
(3.1 nM/second and 750 nM, respectively). Approximately 5 nM thrombin
was observed at clotting. An increase in factor VIIa concentration to
50 nM had no further effect on thrombin generation rate, with an
increase in maximum thrombin level (to almost 1.0 µM). The thrombin
concentration at clotting was about 5 nM.
Thrombin generation in hemophilia A blood (Figure 5A; ) occurred
after an extended initiation phase (~ 6.5 minutes) and at a maximum
of 0.27 nM/second rate during the propagation phase. The maximum
thrombin level was 20% that of the normal value (150 nM). The
concentration of thrombin at clotting was about 10 nM. Addition of 10 nM factor VIIa to hemophilia A blood (Figure 5A; ) in vitro slightly
decreased the duration of the initiation phase, increased the maximum
rate of thrombin generation (to 0.72 nM/second), and slightly increased
the maximum thrombin level (to 200 nM). The thrombin level at clotting
was less than 5 nM. Factor VIIa at a concentration of 50 nM did not
increase the maximum rate of thrombin generation further (0.58 nM/second) but did increase the maximum thrombin level 360 nM. The
thrombin concentration at clotting was less than 3 nM.
FPA release in normal blood (Figure 5B; ) was observed before clot
formation, with about 70% (11 µM) of the FPA released at clotting.
Addition of 10 nM factor VIIa to normal blood (Figure 5B; ) had
almost no effect on FPA release. However, clotting occurred with less
than 1 µM FPA released. In the hemophilia A blood (without exogenous
factor VIIa; Figure 5B; ), about 50% (7 µM) of the FPA was
released at clotting. Addition of 10 nM factor VIIa to this blood
(Figure 5B; ) slightly accelerated FPA release; however, no FPA was
detected at clotting. FPA release occurred at a similar maximum rate
(5-8 µM/minute) and was complete in all experiments.
Release of FPB in normal blood (Figure 5C; ) started just before
clot formation, with 1.8 µM (~ 25% of all FPB released) released
at clotting (3.25 minutes). Addition of 10 nM factor VIIa to normal
blood (Figure 5C; ) slightly shortened the inception of FPB release;
however, no FPB was detected at clotting. In normal blood, FPB release
occurred at a maximum rate of 4.1 µM/minute and the maximum level of
this peptide was 7.4 µM. Addition of 10 nM factor VIIa to this blood
had almost no effect on these parameters of FPB release. In hemophilia
A blood without exogenous factor VIIa (Figure 5C; ), about 25% (0.8 µM) of the FPB was released at clotting. The rate of FPB release was
less than 10% that observed in normal blood (0.35 vs 4.1 µM/minute).
The maximum level of FPB (3.0 µM) was about 40% that in normal blood
(7.4 µM). Addition of 10 nM factor VIIa to hemophilia A blood (Figure 5C; ) increased both the rate of FPB release (to 1.3 µM/minute) and the final concentration of this peptide (to 3.7 µM) but did not
restore these to normal levels. Thus, addition of factor VIIa to normal
or hemophilia A blood affected clotting time more than it influenced
release of FPA or FPB.
Analyses of the platelet-activation data based on osteonectin release
(Figure 5D) showed that at the clotting of normal blood (Figure 5D;
), platelet activation was almost complete. Addition of 10 nM factor
VIIa to normal blood (Figure 5D; ) caused a notable acceleration in
platelet activation, which was comparable to the decrease in clotting
time, ie, platelet activation was almost complete at this point. In
hemophilia A blood without factor VIIa (Figure 5D; ), clotting was
observed when less than 20% (16 nM) of platelet osteonectin was
released. The maximum rate of osteonectin release in hemophilia A blood
was 20% that in normal blood (0.1 nM/second vs 0.5 nM/second).
Addition of 10 nM factor VIIa to hemophilia A blood (Figure 5D; )
corrected the maximum rate of platelet activation.
Analyses of soluble fibrinogen and solubilized fibrin clots (Figure
6) showed that at the clotting of normal
blood (Figure 6A; arrow), fibrinogen was almost depleted from the
solution (Figure 6A; upper lane) and a solid cross-linked clot was
formed (lower panel). Addition of 10 nM factor VIIa to normal blood had
almost no effect on the time course of fibrinogen depletion and
solid-clot formation (data not shown). In hemophilia A blood (Figure
6B), fibrinogen depletion from the solution was delayed and residual amounts of this protein remained until the 12-minute time point (ie,
for approximately 6 minutes after solid clots were observed), whereas
in normal blood, no soluble fibrinogen was detected within 1 minute
after clotting (Figure 6A). Addition of 10 nM factor VIIa to hemophilia
A blood (Figure 6C), although it corrected clotting time to normal
(arrows in Figure 6A and Figure 6C), did not restore the time course
for fibrinogen depletion or solid-clot formation (no solid clots were
formed for > 1 minute after clotting) observed in normal blood.
Fibrinogen was present in the solution phase for about 4 minutes after
clotting. Thus, the pattern of soluble fibrinogen depletion and
solid-clot formation observed in hemophilia A blood with factor VIIa
addition resembled that in hemophilia A blood without factor VIIa
addition and proceeded more slowly than in normal blood.
The average clot weight derived from 11 experiments conducted in normal blood in which clotting was initiated by TF was 1.5 ± 0.2 mg (Figure 7; bar 1). Clots formed in hemophilia A blood (2 experiments with the same patient's blood done 20 months apart) at the same TF concentration were smaller and weighed an average of 1.0 mg (Figure 7; bar 2). Addition of factor VIIa at concentrations of 10 and 50 nM to hemophilia A blood in which clotting was initiated by TF had little effect on clot weights (Figure 7; bar 3). They were similar to those observed in hemophilia A blood without addition of factor VIIa (compare bars 2 and 3 in Figure 7). On the other hand, addition of 0.7 nM recombinant factor VIII (mean physiologic concentration) to the same hemophilia A blood (contemporaneous control) increased the clot weight to normal values (Figure 7; bar 4).
A safe and efficacious hemostatic agent must act only at the site of a vascular lesion. The success of in vivo treatment of bleeding episodes with recombinant factor VIIa, particularly episodes related to hemophilia A and hemophilia B, indicates that this enzyme meets the above requirement. The mechanism by which factor VIIa establishes normal hemostasis in factor VIII and factor IX deficient blood is unknown. The findings of this study indicate that in both normal blood and the synthetic model representing normal blood, factor VIIa alone, in the absence of TF, can initiate thrombin generation. In hemophilia A blood and "acquired" hemophilia B blood, however, pharmacologic concentrations of factor VIIa could not restore normal thrombin generation or formation of solid cross-linked clots. In the normal synthetic system, the concentrations of factor VIIa required to provide thrombin generation profiles comparable to those observed in TF-initiated reactions were approximately 104-fold higher. In whole blood (with approximately 0.1 nM factor VIIa), similar clotting times were observed when coagulation was initiated by 10 pM TF or by 60 nM factor VIIa. In the synthetic model in the presence of 2 µM phospholipids or 2 × 108/mL platelets, factor VIIa concentrations had to be increased above 120 nM to show the efficiency provided by 25 pM factor VIIa-TF complex. These data suggest that the factor VIIa-TF complex is approximately 5 to 6 × 103-fold more efficient than factor VIIa as an initiator of the blood-coagulation cascade. This ratio is based on an assumption that all TF added to the reaction is in a complex with factor VIIa. However, the decrease in clotting time of normal blood caused by addition of factor VIIa in the presence of TF suggests that a fraction of TF is free and that 5-6 × 103-fold is an underestimate. Steady-state data derived by Komiyama et al51 showed that TF increases the efficiency of factor VIIa in the proteolyses of its natural substrates factor IX and factor X by approximately 104-fold. This increase in efficiency toward individual substrates is similar to that observed in the current study for the entire coagulation cascade. Addition of excess factor VIIa to whole blood in the presence of limiting TF affected clotting time until the saturation of TF was achieved. Further increases in factor VIIa had no additional effect on clotting time. The concentration of factor VIIa required to produce the shortest clotting time was inversely dependent on the TF concentration. For example, at 1 pM TF, the shortest clotting time was at 20 nM factor VIIa, whereas at 10 pM TF, the shortest clotting time was at 10 nM factor VIIa. These data suggest that in the presence of TF, the role of factor VIIa is kinetic saturation of TF. Additional excesses of factor VIIa had limited (if any) role in the blood-coagulation process. The superior role of factor VIIa in complex compared with that of factor VIIa acting alone in thrombin generation and clot formation was also observed in congenital hemophilia A blood and "acquired" hemophilia B blood. During the past 2 decades, recombinant factor VIIa has been tested in the treatment of a variety of bleeding disorders.26-33,52-59 It has primarily been used to treat hemophilia patients with acquired inhibitors.28-32 The in vivo concentrations of factor VIIa for this treatment range from 3 to 20 nM29,60-63 (based on 50 000 units/mg specific activity of recombinant factor VIIa63). The mechanism by which factor VIIa at these concentrations maintains normal hemostasis is unknown. In reports from one laboratory, it was postulated that factor VIIa alone, in the absence of TF, can generate factor IXa and factor Xa on the surface of activated platelets at concentrations sufficient to provide normal hemostasis in hemophilia.34,35 The data of this study, however, indicate that factor VIIa at pharmacologic concentrations is a potent hemostatic agent only in the presence of TF. Addition of factor VIIa to the synthetic blood-coagulation model in the absence of factor VIII slightly increased thrombin generation but never raised it to levels observed with factor VIII. The initiation phase remained prolonged, and thrombin generation during the propagation phase was significantly suppressed. Increasing the concentration of factor VIIa from 10 to 40 nM did not enhance thrombin generation. These observations were valid for experiments conducted with phospholipids or platelets. The results suggest that the limited positive effect caused by supraphysiologic factor VIIa concentrations was due to overcoming factor VII competition and the kinetic saturation of TF in the reaction.64 The efficiency of factor VIIa observed in the studies in Roberts' laboratory34,35 reflects differences in either experimental design or protein preparations. Even negligible amounts of enzymes in the synthetic reaction mixtures containing platelets may cause a "spontaneous" generation of thrombin in the absence of both factor VIIa and TF. Similarly, in the hemophilia A blood and "acquired" hemophilia B blood, addition of factor VIIa had little effect on thrombin generation, ie, the duration of the initiation phase and the rate of thrombin generation during the propagation phase. The maximum levels of thrombin were only marginally affected by this addition. An increase in factor VIIa concentration from 10 to 50 nM had almost no additional effect on the parameters of thrombin generation. Pharmacologic concentrations of factor VIIa had a negligible effect on clot formation in hemophilia A blood. Addition of factor VIIa slightly decreased the time for FPA and FPB release, slightly increased the rate of formation and final levels of FPB, and slightly accelerated solid-clot formation and cross-linking. However, none of these variables was corrected to levels observed in blood from healthy donors. Additionally, the smaller clot weights observed in hemophilia blood were not increased by addition of factor VIIa. The only process that was considerably altered by addition of factor VIIa to normal or hemophilia blood was platelet activation. Addition of 10 nM factor VIIa led to more rapid platelet activation in hemophilia A blood and acquired hemophilia B blood, producing rates observed in normal blood. The mechanism by which factor VIIa addition accelerates platelet activation is unknown. One of the possibilities is that high concentrations of factor VIIa can activate platelets directly. More likely, addition of factor VIIa slightly accelerates thrombin generation due to the kinetic saturation of TF and, as a result, increases thrombin during the initiation phase. The additional amount of thrombin cannot alter the entire process of coagulation and clot formation substantially but can accelerate platelet activation. This assumption is supported by our recent study indicating that subnanomolar concentrations of thrombin are required for relatively rapid platelet activation in whole blood.65
We thank Joshua D. Dee and Shyla L. Tessmer for their technical assistance; Dr Shu Len Liu and Dr Roger Lundblad for providing TF and factor VIII; Dr Kirk Johnson for providing TFPI; and Dr Thomas Edgington for providing anti-TF antibody.
Submitted July 9, 2001; accepted September 30, 2001.
Supported by grants R01 HL34575, P01 HL46703, and PHS T32 HL07594 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Presented in part at the 42nd Annual Meeting of the American Society of Hematology, December 1-5, 2000, San Francisco, CA. Reprints: Kenneth G. Mann, Dept of Biochemistry, University of Vermont, Given Bldg, Room C401, 89 Beaumont Ave, Burlington, VT 05405-0068; e-mail: kmann{at}zoo.uvm.edu.
1. Jenny NS, Mann KG. Coagulation cascade: an overview. In Loscalzo J, Schafer AI, eds. Thrombosis and Hemorrhage. Baltimore, MD: Williams and Wilkins; 1998:3-27. 2. Chiu HC, Whitaker E, Colman RW. Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants. J Clin Invest. 1983;72:493-503. 3. Poort SR, Michiels JJ, Reitsma PH, Bertina RM. Homozygosity for a novel missense mutation in the prothrombin gene causing a severe bleeding disorder. Thromb Haemost. 1994;72:819-824[Medline] [Order article via Infotrieve]. 4. Ragni MV, Lewis JH, Spero JA, Hasiba U. Factor VII deficiency. Am J Hematol. 1981;10:79-88[Medline] [Order article via Infotrieve].
5.
Hoyer LW.
Hemophilia A.
N Engl J Med.
1994;330:38-47 6. Ozsoylu S, Ozer FL. Acquired factor IX deficiency. A report of two cases. Acta Haematol. 1973;50:305-314[CrossRef][Medline] [Order article via Infotrieve].
7.
Kekwick RA, Wolf P.
A concentrate of human antihaemophilic factor 8. Gilchrist GS, Ekert H, Shanbrom E, Hammond D. Evaluation of a new concentrate for the treatment of factor IX deficiency. N Engl J Med. 1969;280:291-295. 9. Schulman S, Wiechel B. Hepatitis, epidemiology and liver function in hemophiliacs in Sweden. Acta Med Scand 1984;215:249-256[Medline] [Order article via Infotrieve]. 10. Thompson AR. Factor IX concentrates for clinical use. Semin Thromb Hemost. 1993;19:25-36[Medline] [Order article via Infotrieve]. 11. Chorba TL, Holman RC, Clarke MJ, Evatt BL. Effects of HIV infection on age and cause of death for persons with hemophilia A in the United States. Am J Hematol. 2001;66:229-240[CrossRef][Medline] [Order article via Infotrieve].
12.
Fulcher CA, Zimmerman TS.
Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody.
Proc Natl Acad Sci U S A.
1982;79:1648-1652 13. Kim HC, McMillan CW, White GC, Bergman GE, Saidi P. Clinical experience of a new monoclonal antibody purified factor IX: half-life, recovery, and safety in patients with hemophilia B. Semin Hematol. 1990;27:30-35[Medline] [Order article via Infotrieve]. 14. Schwartz RS, Abilgaard CF, Aledort LM, et al. Human recombinant DNA-derived antihemophilic factor (factor VIII) in the treatment of hemophilia A. Recombinant Factor VIII Study Group. N Engl J Med. 1990;323:1800-1805[Abstract].
15.
Bray GL, Gomperts ED, Courter S, et al.
A multicenter study of recombinant factor VIII (Recombinate): safety, efficacy, and inhibitor risk in previously untreated patients with hemophilia A. The Recombinate Study Group.
Blood.
1994;83:2428-2435 16. White GC, Beebe A, Nielsen B. Recombinant factor IX. Thromb Haemost. 1997;78:261-265[Medline] [Order article via Infotrieve]. 17. Snyder RO, Miao CH, Patijn GA, et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat Genet. 1997;16:270-276[CrossRef][Medline] [Order article via Infotrieve].
18.
VandenDriessche T, Vanslembrouck V, Goovaerts I, et al.
Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice.
Proc Natl Acad Sci U S A.
1999;96:10379-10384
19.
Arruda VR, Hagstrom JN, Deitch J, et al.
Posttranslational modifications of recombinant myotube-synthesized factor IX.
Blood.
2001;97:130-138 20. Kay MA, Manno CS, Ragni MV, et al. Evidence for gene transfer and expression of factor IX in hemophilia B patients treated with an AAV vector. Nat Genet. 2000;24:257-261[CrossRef][Medline] [Order article via Infotrieve]. 21. High KA. Gene therapy: a 2001 perspective. Haemophilia. 2001;7(suppl 1):23-27.
22.
Nilsson IM, Berntorp E, Zettervall O.
Induction of split tolerance and clinical cure in high-responding hemophiliacs with factor IX antibodies.
Proc Natl Acad Sci U S A.
1986;83:9169-9173 23. Nuss R, Jacobson L, Hathaway WE, Manco-Johnson M. Evidence for antiphospholipid antibodies in hemophilic children with factor VIII inhibitors. Recombinate PUP Study Group. Thromb Haemost. 1999;82:1559-1560[Medline] [Order article via Infotrieve]. 24. Parquet A, Laurian Y, Rothschild C, et al. Incidence of factor IX inhibitor development in severe haemophilia B patients treated with only one brand of high purity plasma derived factor IX concentrate. Thromb Haemost. 1999;82:1247-1249[Medline] [Order article via Infotrieve].
25.
Chaney JD, Nielsen VG.
Considerations for the hemophiliac patient with inhibitors to factor VIII.
Anesth Analg.
2001;92:785-786 26. Hedner U, Kisiel W. The use of human factor VIIa in the treatment of two hemophilia patients with high-titer inhibitors. J Clin Invest. 1983;71:1836-1841. 27. Hedner U, Glazer S, Pingel K, et al. Successful use of recombinant FVIIa in a patient with severe haemophilia A during synovectomy. Lancet. 1988;2:1193[CrossRef][Medline] [Order article via Infotrieve]. 28. Lusher JM, Roberts HR, Davignon G, et al. A randomized, double-blind comparison of two dosage levels of recombinant factor VIIa in the treatment of joint, muscle and mucocutaneous haemorrhages in persons with haemophilia A and B, with and without inhibitors. rFVIIa Study Group. Haemophilia. 1998;4:790-798[CrossRef][Medline] [Order article via Infotrieve]. 29. Shapiro AD, Gilchrist GS, Hoots WK, Cooper HA, Gastineau DA. Prospective, randomised trial of two doses of rFVIIa (NovoSeven) in haemophilia patients with inhibitors undergoing surgery. Thromb Haemost. 1998;70:773-778. 30. Shapiro AD. Recombinant factor VIIa in the treatment of bleeding in hemophilic children with inhibitors. Semin Throm Hemost. 2000;26:413-419[CrossRef]. 31. Negrier C, Hay CRM. The treatment of bleeding in hemophilic patients with inhibitors with recombinant factor VIIa. Semin Thromb Hemost. 2000;26:407-412[CrossRef][Medline] [Order article via Infotrieve]. 32. Ingerslev J. Efficacy and safety of recombinant factor VIIa in the prophylaxis of bleeding in various surgical procedures in hemophilic patients with factor VIII and factor IX inhibitors. Semin Thromb Hemost. 2000;26:425-432[CrossRef][Medline] [Order article via Infotrieve]. 33. Hedner U. Recombinant activated factor VII as a universal haemostatic agent. Blood Coagul Fibrinolysis. 1998;9(suppl 1):S147-S152. 34. Monroe DM, Hoffman M, Oliver JA, Roberts HR. Platelet activity of high-dose factor VIIa is independent of tissue factor. Br J Haematol. 1997;99:542-547[CrossRef][Medline] [Order article via Infotrieve]. 35. Hoffman M, Monroe DM, Roberts HR. Activated factor VII activates factors IX and X on the surface of activated platelets: thoughts on the mechanism of action of high-dose activated factor VII. Blood Coagul Fibrinolysis. 1998;9(suppl 1):S61-S65.
36.
Lawson JH, Kalafatis M, Stram S, Mann KG.
A model for the tissue factor pathway to thrombin. I. An empirical study.
J Biol Chem.
1994;269:23357-23366
37.
Jones KC, Mann KG.
A model for the tissue factor pathway to thrombin. II. A mathematical simulation.
J Biol Chem.
1994;269:23367-23373
38.
Rand MD, Lock JB, van't Veer C, Gaffney DP, Mann KG.
Blood clotting in minimally altered whole blood.
Blood.
1996;88:3432-3445
39.
Butenas S, van't Veer C, Mann KG.
Evaluation of the initiation phase of blood coagulation using ultrasensitive assays for serine proteases.
J Biol Chem.
1997;272:21527-21533
40.
van't Veer C, Mann KG.
Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin-III, and heparin cofactor-II.
J Biol Chem.
1997;272:4367-4377
41.
Cawthern KM, van't Veer C, Lock JB, DiLorenzo ME, Branda RF, Mann KG.
Blood coagulation in hemophilia A and hemophilia C.
Blood.
1998;91:4581-4592 42. Bajaj SP, Rapaport SI, Prodanos C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep Biochem. 1981;11:397-412[Medline] [Order article via Infotrieve].
43.
Katzmann JA, Nesheim ME, Hibbard LS, Mann KG.
Isolation of functional human coagulation factor V by using a hybridoma antibody.
Proc Natl Acad Sci U S A.
1981;78:162-166
44.
Griffith MJ, Noyes CM, Church FC.
Reactive site peptide structural similarity between heparin cofactor II and antithrombin III.
J Biol Chem.
1985;260:2218-2225 45. Mustard JF, Perry DW, Ardlie NG, Packham MA. Preparation of suspension of washed platelets from humans. Br J Haematol. 1972;22:193-204[Medline] [Order article via Infotrieve]. 46. Lawson JH, Krishnaswamy S, Butenas S, Mann KG. Extrinsic pathway proteolytic activity. Methods Enzymol. 1993;222:177-195[Medline] [Order article via Infotrieve].
47.
Brummel KE, Butenas S, Mann KG.
An integrated study of fibrinogen during blood coagulation.
J Biol Chem.
1999;274:22862-22870
48.
Lossing TS, Kasper CK, Feinstein DI.
Detection of factor VIII inhibitors with the partial thromboplastin time.
Blood.
1977;49:793-797
49.
Holmes MB, Schneider DJ, Hayes MG, Sobel BE, Mann KG.
Novel, bedside, tissue factor-dependent clotting assay permits improved assessment of combination antithrombotic and antiplatelet therapy.
Circulation.
2000;102:2051-2057 50. Butenas S, Branda RF, van't Veer C, Cawthern KM, Mann KG. Platelets and phospholipids in tissue factor-initiated thrombin generation. Thromb Haemost. 2001;86:660-667[Medline] [Order article via Infotrieve]. 51. Komiyama Y, Pedersen AH, Kisiel W. Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor. Biochemistry. 1990;29:9418-9425[CrossRef][Medline] [Order article via Infotrieve]. 52. Berntorp E. Recombinant FVIIa in the treatment of warfarin bleeding. Semin Thromb Hemost. 2000;26:433-435[CrossRef][Medline] [Order article via Infotrieve]. 53. Muleo G, Santoro R, Iannaccaro PG, et al. Small doses of recombinant factor VIIa in acquired deficiencies of vitamin K dependent factors. Blood Coagul Fibrinolysis. 1999;10:521-522[Medline] [Order article via Infotrieve]. 54. Friederich PW, Wever PC, Briet E, Doorenbos CJ, Levi M. Successful treatment with recombinant factor VIIa of therapy-resistant severe bleeding in a patient with acquired von Willebrand disease. Am J Hematol. 2001;66:292-294[CrossRef][Medline] [Order article via Infotrieve]. 55. Hunault M, Bauer KA. Recombinant factor VIIa for the treatment of congenital factor VII deficiency. Semin Thromb Hemost. 2000;26:401-405[CrossRef][Medline] [Order article via Infotrieve]. 56. Moisescu E, Ardelean L, Simion I, Muresan A, Ciupan R. Recombinant factor VIIa treatment of bleeding associated with acute renal failure. Blood Coagul Fibrinolysis. 2000;11:575-577[CrossRef][Medline] [Order article via Infotrieve]. 57. Udvardy M. New possibilities in the management of hemorrhagic diathesis caused by factor deficiency and thrombocytopenia: recombinant active factor VII concentrate [in Hungarian]. Orv Hetil. 1998;139:2255-2258[Medline] [Order article via Infotrieve]. 58. Bernstein D. Effectiveness of the recombinant factor VIIa in patients with the coagulopathy of advanced child's B and C cirrhosis. Semin Thromb Hemost. 2000;26:437-438[CrossRef][Medline] [Order article via Infotrieve]. 59. Poon MC, d'Oiron R. Recombinant activated factor VII (NovoSeven) treatment of platelet-related bleeding disorders. International Registry on rFVIIa and Congenital Platelet Disorders Group. Blood Coagul Fibrinolysis. 2000;11(suppl 1):S55-S68. 60. Mauser-Bunschoten EP, de Goede-Bolder A, Wielenga JJ, Levi M, Peerlinck K. Continuous infusion of recombinant factor VIIa in patients with haemophilia and inhibitors. Experience in The Netherlands and Belgium. Neth J Med. 1998;53:249-255[CrossRef][Medline] [Order article via Infotrieve]. 61. Scarragi FA, De Mitrio V, Marino R, et al. Treatment with recombinant activated factor VII in a patient with hemophilia A and an inhibitor: advantages of administration by continuous infusion over bolus intermittent injections. Blood Coagul Fibrinolysis. 1999;10:33-38[Medline] [Order article via Infotrieve]. 62. Key NS, Aledort LM, Beardsley D, et al. Home treatment of mild to moderate bleeding episodes using recombinant factor VIIa (Novoseven) in haemophiliacs with inhibitors. Thromb Haemost. 1998;80:912-918[Medline] [Order article via Infotrieve]. 63. Schulman S. Continuous infusion of recombinant factor VIIa in hemophilic patients with inhibitors: safety, monitoring, and cost effectiveness. Semin Thromb Hemost. 2000;26:421-424[CrossRef][Medline] [Order article via Infotrieve].
64.
van't Veer C, Golden NJ, Mann KG.
Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa.
Blood.
2000;95:1330-1335 65. Mann KG, Paradis SG, Butenas S, Branda RF, Brummel KE. Thresholds for thrombin functions during tissue factor-induced blood coagulation [abstract]. Thromb Haemost. 2001;86(suppl 1):P2874.
© 2002 by The American Society of Hematology.
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 L. J. Bowman, W. E. Uber, M. R. Stroud, L. R. Christiansen, J. Lazarchick, A. J. Crumbley III, J. M. Kratz, J. M. Toole, F. A. Crawford Jr, and J. S. Ikonomidis Use of Recombinant Activated Factor VII Concentrate to Control Postoperative Hemorrhage in Complex Cardiovascular Surgery Ann. Thorac. Surg., May 1, 2008; 85(5): 1669 - 1677. [Abstract] [Full Text] [PDF]
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F. Szlam, T. Taketomi, C. A. Sheppard, C. L. Kempton, J. H. Levy, and K. A. Tanaka Antithrombin Affects Hemostatic Response to Recombinant Activated Factor VII in Factor VIII Deficient Plasma Anesth. Analg., March 1, 2008; 106(3): 719 - 724. [Abstract] [Full Text] [PDF]
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G. A. Allen, E. Persson, R. A. Campbell, M. Ezban, U. Hedner, and A. S. Wolberg A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia Arterioscler Thromb Vasc Biol, March 1, 2007; 27(3): 683 - 689. [Abstract] [Full Text] [PDF]
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V. B. Louvain-Quintard, E. P. Bianchini, C. Calmel-Tareau, M. Tagzirt, and B. F. Le Bonniec Thrombin-activable Factor X Re-establishes an Intrinsic Amplification in Tenase-deficient Plasmas J. Biol. Chem., December 16, 2005; 280(50): 41352 - 41359. [Abstract] [Full Text] [PDF]
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H. R. Roberts, D. M. Monroe, and G. C. White The use of recombinant factor VIIa in the treatment of bleeding disorders Blood, December 15, 2004; 104(13): 3858 - 3864. [Abstract] [Full Text] [PDF]
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T. Lisman, J. Adelmeijer, H. F. G. Heijnen, and P. G. de Groot Recombinant factor VIIa restores aggregation of {alpha}IIb{beta}3-deficient platelets via tissue factor-independent fibrin generation Blood, March 1, 2004; 103(5): 1720 - 1727. [Abstract] [Full Text] [PDF]
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M. Tranholm, K. Kristensen, A. T. Kristensen, C. Pyke, R. Rojkjaer, and E. Persson Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A Blood, November 15, 2003; 102(10): 3615 - 3620. [Abstract] [Full Text] [PDF]
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D. M. Monroe and H. R. Roberts Mechanism of Action of High-Dose Factor VIIa: Points of Agreement and Disagreement Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 8 - 9. [Full Text] [PDF]
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K. G. Mann and S. Butenas Response: Mechanism of Action of High-Dose Factor VIIa Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 10 - 10. [Full Text] [PDF]
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K. G. Mann, S. Butenas, and K. Brummel The Dynamics of Thrombin Formation Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 17 - 25. [Abstract] [Full Text] [PDF]
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S. Butenas, K. E. Brummel, S. G. Paradis, and K. G. Mann Influence of Factor VIIa and Phospholipids on Coagulation in "Acquired" Hemophilia Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 123 - 129. [Abstract] [Full Text] [PDF]
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K. G. Mann and M. Kalafatis Factor V: a combination of Dr Jekyll and Mr Hyde Blood, January 1, 2003; 101(1): 20 - 30. [Full Text] [PDF]
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 S. R. Deitcher, T. L. Carman, and K. Kottke-Marchant Simultaneous Deep Venous Thrombosis and Acquired Factor VIII Inhibitor Clinical and Applied Thrombosis/Hemostasis, October 1, 2002; 8(4): 375 - 379. [Abstract] [PDF]
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D. M. Monroe, M. Hoffman, and H. R. Roberts Platelets and Thrombin Generation Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1381 - 1389. [Abstract] [Full Text] [PDF]
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M. Hoffman, D. M. Monroe, H. R. Roberts, K. G. Mann, and S. Butenas Platelet-dependent action of high-dose factor VIIa Blood, June 17, 2002; 100(1): 364 - 366. [Full Text] [PDF]
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Top
Abstract
Introduction
), the initiation phase was extended
(~ 5 minutes) relative to that observed in the presence of 25 pM TF
and 0.1 nM factor VIIa. Thus, an equivalent maximum rate of thrombin
generation (2.7 nM/second) in the absence of TF was achieved only with
a 1200-fold increase (to 120 nM) in factor VIIa.
), and 120 nM (
its use in six cases of haemophilia.
Lancet.
1957;1:647.