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Originally published In Press as doi:10.1074/jbc.M400531200 on March 23, 2004

J. Biol. Chem., Vol. 279, Issue 22, 22875-22882, May 28, 2004
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The Significance of Circulating Factor IXa in Blood*

Saulius Butenas, Thomas Orfeo, Matthew T. Gissel, Kathleen E. Brummel, and Kenneth G. Mann{ddagger}

From the Department of Biochemistry, University of Vermont, Burlington, Vermont 05405-0068

Received for publication, January 16, 2004 , and in revised form, March 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of activation peptides (AP) of the vitamin K-dependent proteins in the phlebotomy blood of human subjects suggests that active serine proteases may circulate in blood as well. The goal of the current study was to evaluate the influence of trace amounts of key coagulation proteases on tissue factor-independent thrombin generation using three models of coagulation. With procoagulants and select coagulation inhibitors at mean physiological concentrations, concentrations of factor IXa, factor Xa, and thrombin were set either equal to those of their AP or to values that would result based upon the rates of AP/enzyme generation and steady state enzyme inhibition. In the latter case, numerical simulation predicts that sufficient thrombin to produce a solid clot would be generated in ~2 min. Empirical data from the synthetic plasma suggest clotting times of 3–5 min, which are similar to that observed in contact pathway-inhibited whole blood (4.3 min) initiated with the same concentrations of factors IXa and Xa and thrombin. Numerical simulations performed with the concentrations of two of the enzymes held constant and one varied suggest that the presence of any pair of enzymes is sufficient to yield rapid clot formation. Modeling of states (numerical simulation and whole blood) where only one circulating protease is present at steady state concentration shows significant thrombin generation only for factor IXa. The addition of factor Xa and thrombin has little effect (if any) on thrombin generation induced by factor IXa alone. These data indicate that 1) concentrations of active coagulation enzymes circulating in vivo are significantly lower than can be predicted from the concentrations of their AP, and 2) expected trace amounts of factor IXa can trigger thrombin generation in the absence of tissue factor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Five vitamin K-dependent proteins serve as precursors for serine proteases, which are essential for normal hemostasis (factors VII, IX, and X, prothrombin, and protein C) (1). The zymogens are converted to fully functional enzymes by the proteolytic removal of corresponding activation peptides. The only exception is factor VII, which is converted to an active enzyme by a single cleavage not followed by the release of an activation peptide (2). For the release of activation peptides from factor X, protein C, and prothrombin, single cleavages are required, whereas the activation peptide of factor IX is generated by two proteolytic cleavages (3). The length of activation peptides varies from 12 amino acids for protein C to 271 amino acids for prothrombin (Fragment 1.2). There are no structural domains observed for activation peptides of factor IX, factor X, and protein C. However, fragment 1.2 of prothrombin contains a Gla domain and two kringle domains. The former one is rich in {gamma}-carboxyglutamic acid residues and facilitates a calcium-dependent binding of prothrombin to the membranes containing acidic phospholipids (1). The kringle domain 1 contains two of three prothrombin glycosylation sites (4).

The detection of activation peptides of the vitamin K-dependent proteins in plasma of human subjects suggests that active serine proteases may circulate in blood either as a result of continuous in situ production ("idling motor") or as a consequence of production during localized coagulation ("historical record"). The relationship between the concentrations of enzymes and their corresponding activation peptides is not clear in part because the source(s) of these peptides is not identified. A possible trigger for the generation of these peptides and corresponding enzymes can be active tissue factor hypothetically circulating in blood (5). Alternatively, they can be generated due to the initiation of the coagulation cascade during phlebotomy and processing of blood. The key problem for the evaluation of active enzymes is that direct assays of these serine proteases are difficult (if possible at all) due to their rapid neutralization by the natural inhibitors present in blood.

To circumvent this obstacle, assays for the activation peptides released during the generation of serine proteases involved in blood coagulation as well as for the reaction and inhibition products of these enzymes have been developed (6–11). Products, including activation peptides of prothrombin, factor X, and factor IX, related to the processes leading to thrombin generation and clot formation have been used as markers for the assessment of the coagulation state of human beings. Elevated levels of activation peptides are observed in patients suffering from disseminated intravascular coagulation (11, 12), deep vein thrombosis (13, 14), coronary heart disease (15, 16), unstable angina, myocardial infarction (17, 18), and other coagulation-related disorders (19). The concentrations of activation peptides are also affected by deficiencies or mutations in procoagulants and coagulation inhibitors (10, 20–24), by smoking (25), and by age (26).

Although there is a strong correlation between the prothrombotic state of individuals and estimated levels of activation peptides, the question of whether these levels reflect concentrations of active enzymes circulating in vivo remains open. The validity of this question can be illustrated by the observation that estimated activation peptide levels are dependent upon the conditions of sample collection (27, 28).

In the current study, we evaluated the capability of thrombin, factor Xa, and factor IXa to trigger thrombin generation and clot formation, when these enzymes are present at concentrations relevant to those reported for their activation peptides in healthy individuals. Three in vitro models of coagulation developed in our laboratory were used to achieve this goal (29–34).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Human coagulation factors VII, X, and IX and prothrombin were isolated from fresh frozen plasma using the general methods of Bajaj et al. (35) and were purged of trace contaminants and traces of active enzymes as described (36). Human factor V and antithrombin III (AT-III) were isolated from freshly frozen plasma (37, 38). Recombinant factor VIII and recombinant tissue factor (TF) (residues 1–242) were provided as gifts by Drs. Shu Len Liu and Roger Lundblad (Hyland Division, Baxter Healthcare Corp., Duarte, CA). Recombinant human factor VIIa was provided as a gift by Dr. Ula Hedner (Novo Nordisk, Denmark). Recombinant full-length tissue factor pathway inhibitor (TFPI) produced in Escherichia coli was provided as a gift by Dr. K. Johnson (Chiron Corp., Emeryville, CA). Factor IXa, factor Xa, and {alpha}-thrombin were provided as a gift by Dr. R. Jenny (Hematologic Technologies, Essex Junction, VT). Corn trypsin inhibitor (CTI)1 was isolated from popcorn as described elsewhere (34). Washed platelets were prepared by the procedure of Mustard et al. (39). Preparation of the TF/lipid reagent was done as described elsewhere (34). 1,2-Dioleolyl-sn-glycero-3-phospho-L-serine (PS) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), and EDTA (Ca2+ quencher) was purchased from Sigma. Phospholipid vesicles (PCPS) composed of 25% PS and 75% PC were prepared as described (40). Spectrozyme TH was purchased from American Diagnostica, Inc. (Greenwich, CT). D-Phe-Pro-ArgCH2Cl (FPRck) and the fluorogenic substrate 6-(D-Val-Pro-Arg)amino-1-naphthalene(n-butyl)sulfonamide (VPRnbs) (41) were synthesized in house. Recombinant hirudin was a gift from Genentech. An enzyme-linked immunosorbent assay thrombin-AT-III (TAT) kit (Enzygnost TAT) was purchased from Behring (Marburg, Germany).

Numerical Simulation
The present effort is based upon prior publications by Jones et al. (29) and Hockin et al. (30). An additional step of factor X activation by factor IXa was incorporated using kinetic constants published by Walsh and co-workers (42) (Km = 1.4 x 10–7 M; kcat = 8.0 x 10–4 s–1).

Synthetic Coagulation Model
The procedure used was a modification of Lawson et al. (31) and van't Veer et al. (36).

Procofactor Solution—4 x 108/ml platelets or 4 µM PCPS were incubated in HBS (20 mM HEPES and 150 mM NaCl, pH 7.4), 2 mM CaCl2 for 10 min at 37 °C. When desired, 10 pM relipidated TF was added (molar ratio of PCPS/TF = 5000). Factor V (40 nM) and factor VIII (1.4 nM) were added prior to the initiation of the reaction.

Zymogen-Inhibitor Solution—Prothrombin (2.8 µM), factors VII (20 nM), VIIa (0.2 nM), X (340 nM), IX (180 nM), and XI (60 nM), TFPI (5 nM), and AT-III (6.8 µM) were preheated in HBS, 2 mM CaCl2 at 37 °C for 3 min. Thrombin, factor IXa, and factor Xa were added at 2x desired concentrations prior to the initiation of the reaction.

The reaction was started by mixing equal volumes of both solutions, resulting in physiological concentrations of the zymogens, pro-cofactors, inhibitors, and platelets (or 2 µM PCPS) and desired concentrations of enzymes. Following initiation of the reaction, at selected time points, 10-µl aliquots were withdrawn from the reaction mixture and quenched in 20 mM EDTA in HBS (pH 7.4) containing 0.2 mM Spectrozyme TH and assayed immediately for thrombin activity. The hydrolysis of the substrate was monitored by the change in absorbance at 405 nm using a Vmax spectrophotometer (Molecular Devices Corp., Menlo Park, CA). Thrombin generation was calculated from a standard curve prepared by serial dilutions of {alpha}-thrombin.

Whole Blood Model
The protocol used was a modification of Rand et al. (33). Two healthy donors were recruited and advised according to a protocol approved by the University of Vermont Human Studies Committee (33, 34), and their consent was obtained. Individuals selected exhibited normal values for the parameters of blood coagulation, protein levels, and platelet counts. Experiments were performed in tubes placed on a rocking table enclosed in a 37 °C temperature-controlled glove box using fresh CTI-inhibited (100 µg/ml CTI) blood. Blood was drawn by venipuncture and immediately delivered into the reagent-loaded tubes.

Time Course—All tubes (four series per experiment) were loaded with CTI. No additional reagents are added to the phlebotomy control series (four tubes). Six tubes were loaded with relipidated TF (TF/PCPS, 5 pM/25 nM) in HBS, 2 mM CaCl2; eight tubes were loaded with 97 pM factor IXa; and another eight tubes were loaded with 97 pM factor IXa, 5.5 pM factor Xa, and 5.3 pM thrombin. The zero-time tube of each series was pretreated with 1 ml of 50 mM EDTA and 10 µl of 10 mM FPRck (diluted in 10 mM HCl). After blood was delivered, the tubes were periodically (2–20 min) quenched with EDTA and FPRck.

Final Levels of the TAT—In these experiments, all tubes were loaded in duplicates with CTI and the desired concentrations of thrombin, factor IXa, and factor Xa (individually or all three together). The TF control tubes are loaded with 5 pM relipidated TF. Tubes were quenched with EDTA and FPRck 20 min after fresh blood was added.

In all experiments, no more than 35 µl of reagents were loaded in each tube. The clotting time was observed visually by two 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 and analyzed for the TAT concentration.

Plasma Preparation for the Fluorogenic Assay
Blood (10 ml) from a healthy volunteer was drawn into a syringe loaded with 10 ml of HBS containing 50 mM EDTA, pH 7.4. The platelet-poor plasma (PPP) was made by centrifugation at 3000 rpm for 20 min followed by the centrifugation at 11,000 rpm (20,000 x g) for 10 min. When kept on ice, plasma did not clot for at least 10 h. One fraction of the PPP was used for the fluorogenic assay immediately and another one was spiked with 1 pM {alpha}-thrombin and kept for 1 h before the assay.

Fluorogenic Thrombin Activity Assay in the PPP
Fluorogenic substrate VPRnbs at a final 50 µM concentration was added to a cuvette containing PPP. This substrate allows the quantitation of {alpha}-thrombin at concentrations as low as 20 fM (41). Change in fluorescence over time representing substrate hydrolysis was monitored in a spectrofluorometer FluoroMax-2 (Jobin Yvon-Spex Instruments S.A., Inc.) at 25 °C for 15 min using {lambda}ex = 350 nm and {lambda}em = 470 nm. Light-scattering artifacts were minimized with a 450-nm cut-off filter in the emission light beam. Hirudin at 1 µM was added to the reaction mixture, and a change in fluorescence was monitored again. Rates of substrate hydrolysis were established from a calibration line constructed using the detecting group (6-amino-1-naphthalene-(n-butyl)sulfonamide).

Estimation of the in Vivo Concentrations of Free Enzymes
The concentration of a given free enzyme in a dynamic system containing stoichiometric inhibitors is determined by the balance between the rate of enzyme generation and the rate(s) of inhibition. Assuming that the concentrations of inhibitors are relatively constant in blood under normal flow, the concentration of free enzyme can be described by the following equation: v = k1[I1]·[Ef] + k2[I2]·[Ef] +... + kn[In]·[Ef], where v is the rate of enzyme generation; k1, k2,... kn are the second order rate constants defining the rates of reactions of inhibitors I1, I2,... In with a particular enzyme (30); and Ef is free enzyme.

The rate of generation of each enzyme was assumed to be equal to the rate of removal of its activation peptide from blood (19). The latter was assumed to be a first order process, and the rate constant (kAP) could be calculated using the reported half-life for each activation peptide (AP) (see Table I). Ongoing rates of activation peptide removal and equal rates of enzyme formation are then defined as the product kAP[AP] using reported steady state concentrations of activation peptides (13–17, 20–26). These rate estimates are used to calculate [Ef] for each enzyme using the above equation and reported concentrations of inhibitors and their second order rate constants of inhibition for the particular enzyme.


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  		TABLE I
Estimated in vivo concentrations of active enzymes

 
Operational Definitions
The initiation phase of thrombin generation was defined as a time interval from the start of the reaction (represented by 0 on the x axis in the figures) to the point of intersection of the x axis and a tangent to the maximum slope of thrombin generation. It is characterized by relatively slow prothrombin activation and the proteolytic processing steps required for the assembly of the vitamin K-dependent protein activating complexes. The propagation phase of thrombin generation is defined as a time interval from the end of the initiation phase to the maximum thrombin concentration. It is characterized by robust prothrombin activation principally by factor Xa·factor Va·membrane (the prothrombinase complex) with factor Xa provided predominantly by the factor IXa·factor VIIIa·membrane complex (the intrinsic factor Xase).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Numerical Simulations Predicting the Influence of Simultaneous Variations of Thrombin, Factor Xa, and Factor IXa on Thrombin Generation in the Absence of TF
Fig. 1 represents thrombin generation over time initiated with the mixture of all three enzymes. Reported concentrations of activation peptides of serine protease zymogens involved in blood coagulation circulating in vivo for healthy individuals varied from 94 pM for factor X to 210 pM for factor IX and to 1.0 nM for prothrombin (F1.2). In the presence of serine proteases at these concentrations, thrombin generation entered the propagation phase after a very short initiation phase (i.e. blood containing these concentrations of enzymes would clot in a few seconds (Fig. 1, solid line). The maximum concentration of active thrombin produced reached almost 1.0 µM, with the maximum rate of generation as high as 28 nM/s. A decrease in the concentration of all three enzymes by 1 order of magnitude (9.4 pM for factor Xa, 21 pM for factor IXa, and 100 pM for thrombin) prolonged the initiation phase to 100 s and decreased both maximum concentration and maximum rate of thrombin generation to 630 nM and 8.5 nM/s, respectively (long dashed line). A further decrease of the enzyme concentrations to 1% of those reported for activation peptides (0.94 pM factor Xa, 2.1 pM factor IXa, and 10 pM thrombin) prolonged the initiation phase to 600 s, decreased maximum concentration of active thrombin to 260 nM, and provided a maximum rate of thrombin generation of 1.1 nM/s (*). No active thrombin was observed in 20 min of the reaction when concentrations of all three enzymes were decreased to 0.1% (94 fM for factor Xa, 0.21 pM for factor IXa, and 1 pM for thrombin) of those detected for their activation peptides (short dashed line).



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  		FIG. 1.
Thrombin generation initiated by factors IXa and Xa and thrombin in the absence of TF (numerical simulations). Coagulation enzymes are present at concentrations identical to the physiologic concentrations of their activation peptides (1.0 nM for thrombin, 94 pM for factor Xa, and 210 pM for factor IXa) (solid line), at 10% (long dashed line), 1% (*), and 0.1% (short dashed line) of those or at concentrations that would occur based upon the rates of enzyme generation and inhibition in a steady state situation (5.3 pM for thrombin, 5.5 pM for factor Xa, and 97 pM for factor IXa) ({square}). Prothrombin, factors V, VIII, VII, VIIa, IX, and X, AT-III, and TFPI are present at their mean physiological concentrations.

 
The enzyme concentrations selected for the initial studies of thrombin generation were either equal to those of activation peptides or decreased in sequential studies by orders of magnitude. However, the free enzymes continuously generated in vivo would be inhibited by the inhibitors present in blood, primarily by AT-III and TFPI. Our calculations based upon activation peptide/enzyme generation and enzyme inhibition rates suggest that the steady state concentrations of coagulation serine proteases for the "idling motor" assumption should be 5.3 pM for thrombin, 5.5 pM for factor Xa, and 97 pM for factor IXa (see "Experimental Procedures" and Table I). At these enzyme concentrations, the initiation phase duration would be 160 s (Fig. 1, {square}). Thus, blood containing these enzyme concentrations would clot in less than 3 min. The maximum concentration of active thrombin achieved was 780 nM, and the maximum rate of thrombin generation was 17 nM/s.

Variations of a Single Enzyme Concentration in Numerical Simulations in the Presence of Two Other Enzymes
In an attempt to determine which one enzyme of the mixture of three potential resident enzymes would have the most significant impact on thrombin generation, the concentration of the selected enzyme was varied, whereas concentrations of the other two were kept at the levels of their activation peptides.

Fig. 2 represents thrombin generation initiated with varying concentrations of thrombin, 94 pM factor Xa, and 210 pM factor IXa (A), with varying concentrations of factor Xa, 210 pM factor IXa, and 1.0 nM thrombin (B) and varying concentrations of factor IXa, 94 pM factor Xa, and 1.0 nM thrombin (C).



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  		FIG. 2.
Titration of a single enzyme in the presence of two others at concentrations identical to those of their activation peptides (100%) in the absence of TF (numerical simulations). A, thrombin titration; B, Factor Xa titration; C, Factor IXa titration. Titrants are present at 100% (1.0 nM for thrombin, 94 pM for factor Xa, and 210 pM for factor IXa) (solid line), 10% (long dashed line), 1% (*), 0.1% (short dashed line), 0.01% (+), and 0% ({circ}). Prothrombin, factors V, VIII, VII, VIIa, IX, and X, AT-III, and TFPI are present at their mean physiological concentrations.

 
In the simulation of thrombin titration (Fig. 2A), thrombin generation during the propagation phase was almost not affected by the amount of this enzyme used as an initiator. The maximum concentration of active thrombin produced (~900 nM) was similar at 1.0 nM (100%) thrombin (solid line) and without any exogenous thrombin ({circ}). The maximum rate of thrombin generation was also little affected by the presence of exogenous thrombin (28 nM/s at 1.0 nM exogenous thrombin and 24.5 nM/s in the absence of it). The initiation phase of thrombin generation was more dependent upon exogenous thrombin, especially at the high end of the range. At 1.0 nM thrombin, the initiation phase was 10 s (solid line). A decrease of the thrombin concentration by 1 order of magnitude (to 100 pM) tripled (30 s) the initiation phase duration (long dashed line). A decrease of exogenous thrombin to 10 pM added 20 s to the initiation phase (i.e. extends it to 50 s) (*). Further decreases in thrombin concentration had almost no effect on the duration of the initiation phase (see short dashed line at 1 pM thrombin and {circ} without exogenous thrombin).

When thrombin generation is initiated with varying concentrations of factor Xa and steady concentrations of exogenous thrombin (1.0 nM) and factor IXa (210 pM) (Fig. 2B), thrombin generation was almost independent of the presence of factor Xa at concentrations tested. Only a limited decrease in thrombin generation rate was observed during the initiation phase and the inception of the propagation phase when factor Xa was omitted (compare solid line for 94 pM (100%) exogenous factor Xa and {circ} without it).

The most pronounced effect on thrombin generation was observed in a hypothetical factor IXa titration at constant concentrations of exogenous thrombin and factor Xa (1.0 nM and 94 pM, respectively) (Fig. 2C). A decrease in exogenous factor IXa concentration from 210 pM (100%) (solid line) to 21 pM (10%) (long dashed line) caused decreases in both the maximum concentration of active thrombin (from 940 to 670 nM) and the maximum rate of thrombin generation (from 28 to 10 nM/s). A decrease in factor IXa concentration to 2.1 pM (1%) (*) led to a further decrease in the maximum thrombin concentration and generation rate (to 380 nM and 2.4 nM/s, respectively). In the presence of 0.21 pM exogenous factor IXa (0.1% of maximum), the highest concentration of active thrombin was decreased to 180 nM, and the maximum rate of thrombin generation was decreased to 1.8 nM/s (short dashed line). No further decrease in the maximum rate of thrombin generation was observed when the factor IXa concentration is decreased below 0.21 pM. Thrombin generation profiles in the presence of 21 fM factor IXa (0.01% of maximum) (+) and in the absence of factor IXa ({circ}) were almost identical. The variations in factor IXa concentration within the tested range (from 0 to 210 pM) had no effect on the initiation phase of thrombin generation.

The data presented in Fig. 2 (A–C) show that any two enzymes present at concentrations identical to those reported for their activation peptides are able to initiate thrombin generation. Decreasing the concentration of exogenous thrombin or factor Xa had little effect (if any) on thrombin generation when the other two enzymes are present as initiators of thrombin generation. In contrast, a decrease in exogenous factor IXa led to pronounced impairments in both maximum thrombin concentration and the generation rate. These data suggest that factor IXa, if present in blood, would play the major role in the initiation of thrombin generation.

Numerical Simulations of Thrombin Generation Initiated with a Single Enzyme in the Absence of Other Enzymes
After it was established in the numerical simulations that the presence of any combination of two enzymes could function as an initiator of thrombin generation, producing amounts of thrombin substantial enough for clot formation, the ability of a single exogenous enzyme in the absence of TF to trigger thrombin generation was tested in three models of coagulation.

The Influence of Thrombin and Factor Xa on Thrombin Generation— Data presented in Fig. 3 show thrombin generation over time at varying concentrations of exogenous thrombin (A), factor Xa (B), and factor IXa (C). No other enzymes were present at the initiation of the reaction.



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  		FIG. 3.
Titration of a single enzyme in the absence of other enzymes and TF (numerical simulations). A, thrombin titration; B, factor Xa titration; C, factor IXa titration. Titrants are present either at 100% concentrations of their activation peptides (1.0 nM for thrombin, 94 pM for factor Xa, and 210 pM for factor IXa) (solid line), 10% (long dashed line), 1% (*), 0.1% (short dashed line), and 0% ({circ}) or at steady state concentrations (5.3 pM for thrombin, 5.5 pM for factor Xa, and 97 pM for factor IXa) ({square}). Prothrombin, factors V, VIII, VII, VIIa, IX, and X, AT-III, and TFPI are present at their mean physiological concentrations.

 
When exogenous thrombin was used as a trigger of thrombin generation in the absence of tissue factor (Fig. 3A), no additional thrombin was produced. Thrombin introduced into the reaction mixture is inhibited by AT-III at a thrombin concentration-dependent rate. The maximum inhibition rate is 18 pM/s when 1.0 nM (100%) thrombin is added (solid line) and 2 pM/s for 0.1 nM thrombin (long dashed line). These data indicate that thrombin alone would not be able to trigger its own generation.

Data of Fig. 3B predict that exogenous factor Xa alone at 94 pM (100%), in the absence of exogenous thrombin and factor IXa, would be able to trigger thrombin generation at concentrations that would yield clot formation (i.e. >10 nM (solid line)). The propagation phase of thrombin generation started after a relatively short (20-s) initiation phase with the maximum rate of thrombin generation of 0.9 nM/s. The concentration of active thrombin reached only 84 nM (compare with almost 1.0 µM in Fig. 1, when all three enzymes are present). Any further thrombin generation was prevented due to the inhibition of factor Xa. When the concentration of exogenous factor Xa was decreased by 1 order of magnitude (to 9.4 pM), thrombin generation was almost completely abolished (long dashed line). These data suggest that the presence of factor Xa in blood at the concentration reported for the factor X activation peptide would cause clot formation in less than 1 min. On the other hand, at concentrations of <=9.4 pM, factor Xa alone would not be able to trigger thrombin generation above the coagulation threshold. It will be inhibited by TFPI and AT-III before producing enough thrombin required for its own amplification.

The Influence of Factor IXa on Thrombin Generation—In contrast to thrombin and factor Xa, factor IXa in the absence of other enzymes could trigger an efficient thrombin generation comparable with that observed in the presence all three enzymes (Fig. 3C). In the presence of 210 pM (100%) exogenous factor IXa, thrombin generation occurred at a maximum 24 nM/s rate, and the maximum concentration of thrombin reached 800 nM (solid line). These parameters of thrombin generation are comparable with those observed when the mixture of all three enzymes at their maximum concentrations is used to trigger thrombin generation (28 nM/s and 940 nM, respectively; see solid line in Fig. 1). The duration of the initiation phase, however, was significantly prolonged (160 s for factor IXa alone versus 20 s for all three enzymes). A decrease in exogenous factor IXa to 10% (21 pM) of the maximum more than tripled the initiation phase (to 500 s) and significantly decreased the maximum rate of thrombin generation (to 6.5 nM/s) (long dashed line). The maximum concentration of active thrombin produced (540 nM) was affected by this decrease in factor IXa concentration to a lesser extent than the initiation phase duration and thrombin generation rate. No active thrombin was observed in 20 min when the reaction was initiated with 2.1 pM (1%) factor IXa (*). When thrombin generation is initiated with the steady state concentration of factor IXa (97 pM), the profile of the process is similar to that observed at 210 pM factor IXa, although the initiation phase is prolonged from 160 to 240 s ({square}).

Empirical Models
The data generated in computer simulations and presented in Figs. 1, 2, 3 suggest that, in the absence of tissue factor, the major trigger of thrombin generation in vivo should be factor IXa if present at concentrations related to the reported amount of factor IX activation peptide. Thrombin and factor Xa played an accessory role to the factor IXa-induced thrombin generation. To test the validity of predictions obtained in computer simulations, two empirical models of blood coagulation were used: synthetic plasma and whole blood.

In the synthetic plasma, reconstituted with washed platelets at mean physiologic concentration (2 x 108/ml) (Fig. 4) and without TF, factor IXa, factor Xa, and thrombin, no thrombin generation was observed over a period of 840 s (). When calculated steady state concentrations of thrombin, factor Xa, and factor IXa (5.3, 5.5, and 97 pM, respectively) were used to initiate the reaction, thrombin generation entered the propagation phase after an initiation phase of 190 s ({square}). Thrombin generation occurred at a maximum 0.9 nM/s rate, with the maximum concentration of active thrombin reaching 130 nM. The omission of thrombin and factor Xa had almost no effect on thrombin generation (i.e. thrombin generation initiated with 97 pM factor IXa alone ({diamondsuit}) was comparable with that observed in the presence of all three enzymes ({square})). The initiation phase was slightly prolonged (from 190 to 210 s), and the maximum rate of thrombin generation and maximum concentration of thrombin were slightly increased (to 1.3 nM/s and 160 nM, respectively).



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  		FIG. 4.
Synthetic plasma in the presence of 2 x 108/ml platelets and in the absence of TF. Thrombin generation is shown in the absence of an initiator (); initiated by 5.3 pM thrombin, by 5.5 pM factor Xa, and 97 pM factor IXa ({square}); and initiated by 97 pM factor IXa alone ({diamondsuit}). Prothrombin, factors V, VIII, VII, VIIa, IX, and X, AT-III, and TFPI are present at their mean physiological concentrations.

 
In an attempt to evaluate the influence of exogenous thrombin, factor Xa, and factor IXa on thrombin generation (evaluated as thrombin-antithrombin III complex (TAT) formation) under conditions approaching those present in vivo, the three enzymes were tested in fresh CTI-inhibited whole blood (Fig. 5A). The propagation phase of thrombin generation initiated in whole blood in the absence of tissue factor with 5.3 pM thrombin, 5.5 pM factor Xa, and 97 pM factor IXa started after the initiation phase of ~4 min (A) ({square}). Thrombin was generated (TAT formation) at a maximum rate of 1.7 nM/s, and the final TAT concentration reached 800 nM. The omission of thrombin and factor Xa had little effect on thrombin generation. When 97 pM factor IXa alone was added to the blood ({diamondsuit}), the initiation phase of thrombin generation was ~3 min, and the maximum rate of thrombin generation and final concentration of the TAT complex were 1.3 nM/s and 720 nM, respectively. For comparison, thrombin generation in CTI-inhibited whole blood induced with 5 pM relipidated TF, with no exogenous enzymes added, entered the propagation phase following the initiation phase of ~4 min ({circ}). No thrombin generation was observed during 20 min of the reaction when neither enzyme(s) nor TF were added to the blood ().



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  		FIG. 5.
Whole blood. A, Thrombin generation (TAT formation) is shown over time in contact pathway-inhibited whole blood (0.1 mg/ml CTI) in the absence of an initiator (), initiated by 97 pM factor IXa ({diamondsuit}), initiated by 97 pM factor IXa, 5.5 pM factor Xa, and 5.3 pM thrombin ({square}) or initiated by 5 pM relipidated TF ({circ}). B, Clotting times (in seconds) are shown at the top of bars (B).

 
Fig. 5B shows whole blood clotting times induced with relipidated TF, thrombin, factor Xa, and factor IXa as well as with the mixture of all three enzymes. When no activator was added to the CTI-inhibited fresh blood, it did not clot in 1200 s (the duration of the experiment). The initiation of the reaction in whole blood with 5 pM relipidated TF causes clot formation in 240 s. Comparable clotting times (119–188 s) were observed when individual enzymes were added at their activation peptide concentrations. However, clotting times were significantly prolonged when either 5.3 pM thrombin or 5.5 pM factor Xa (calculated steady-state concentrations) were added to the CTI-inhibited whole blood (1080 and 860 s, respectively). In contrast, the initiation of whole blood with 97 pM factor IXa led to clot formation in 188 s (i.e. identical to that observed with 210 pM factor IXa and slightly faster than with 5 pM TF). The addition of all three enzymes to whole blood at their activation peptide concentrations simultaneously shortened clotting time to 86 s, whereas the addition of these enzymes at their calculated steady-state concentrations did not decrease the clotting time (220 s) below that observed for factor IXa alone.

The above empirical results together with computer simulations indicate that factor IXa at the estimated steady state in vivo concentration (97 pM) should cause at physiologic conditions a robust thrombin generation leading to a relatively rapid (3–4-min) clot formation. Thrombin and factor Xa at the estimated in vivo concentrations had quite limited (if any) effect on factor IXa-induced thrombin generation.

The Concentration of Active Thrombin in Nonstimulated Whole Blood
All three models of blood coagulation were used so far to evaluate thrombin generation initiated with factor IXa, factor Xa, and thrombin at concentrations estimated from activation peptide measurements as circulating in vivo. The availability of efficient fluorogenic substrates (41) allows direct detection of picomolar amounts of certain coagulation enzymes. One of these substrates, VPRnbs, was used for the detection of enzymatic activity in fresh, EDTA-containing plasma. No detectable substrate hydrolysis was observed over 15 min of the assay, when VPRnbs was added to the PPP. The addition of purified human {alpha}-thrombin to this plasma at a final 1 pM concentration led to substrate hydrolysis at a rate of 0.23 nM/s. The subsequent addition of 1 µM hirudin quenched enzymatic activity completely. A possible explanation for the lack of enzymatic activity in fresh plasma is that active enzymes are inhibited by their endogenous inhibitors during the processing of blood. To address this issue, {alpha}-thrombin (1 pM) was added to plasma and incubated for 1 h (time required to make the PPP). The rate of substrate hydrolysis observed after this incubation was 0.16 nM/s, indicating that ~70% of added thrombin remained active. The results of this experiment suggest that the concentration of active thrombin in whole blood is significantly lower than that estimated for steady state conditions (5.3 pM) on the basis of reported Fragment 1.2 concentration.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Activation peptides of the vitamin K-dependent proteins are reported to be present in the blood of normal individuals (6, 10–26); altered levels of these peptides correlate with various coagulation disorders (11–19) and risk factors related to these disorders (20–26).

One hypothesis ("idling motor") proposes that systemic, continuous activation of serine protease zymogens is a normal feature of the vasculature. Implied in this idea is the presence of active enzymes at levels insufficient to trigger clot formation. Studies in whole blood indicate that enzymatic activity, if present in blood, is below the activation threshold required for coagulation, because unstimulated, contact pathway-inhibited blood remains fluid in vitro for >20 min (33, 34, 43) (present work) with no measurable thrombin observed. The data of the current study show that the addition of three enzymes of coagulation (thrombin, factor Xa, and factor IXa) at estimated steady state concentrations causes solid clot formation in ~200 s. Similar estimated "clotting times" (the duration of the initiation phase) (32, 33) were observed in the other two models of blood coagulation used in this study: empirical (synthetic) and mathematical (computational) (190 and 160 s, respectively). Moreover, the addition of any single enzyme to whole blood at the steady state concentration leads to the clot formation in less than 1200 s with factor IXa showing the highest efficiency (clotting time 190 s). The two other models predict a similar clotting time for this factor IXa concentration (210–240 s). For comparison, whole blood initiated to react with 5 pM relipidated tissue factor clots in 240 s and does not clot for >1200 s in its absence. These data suggest that concentrations of active enzymes (thrombin, factor Xa, and factor IXa) circulating in blood are significantly lower (if present at all) than those estimated either on the basis of reported in vivo concentrations of corresponding activation peptides or on the basis of rates of their generation/clearance and known kinetics of enzyme inhibition by their in vivo inhibitors. Thus, an additional pathway for the clearance of thrombin, factor IXa, and factor Xa has to exist in vivo. It can be related to the binding of these serine proteases to cell-surface receptors with subsequent internalization via endocytosis (44–47). Additionally, the binding of AT-III and TFPI to their target serine proteases could be altered in vivo by proteoglycans present on endothelial cells (48, 49).

Several observations related to the mechanism of thrombin generation can be made based upon the data of the current study. Only factor IXa at the picomolar concentrations relevant to the reported concentration of factor IX activation peptide is able to trigger thrombin generation similar to that observed with the mixture of all three enzymes used as an initiator. Factor IXa at those concentrations generates enough factor Xa for the production of initial amounts of thrombin. The latter amplifies its own generation through the activation of factor V and factor VIII. Although factor Xa is efficiently inhibited by TFPI (50), a relatively poor inhibition of factor IXa by anti-thrombin-III (51) allows generation of factor Xa for a prolonged period of time, keeping prothrombin activation alive. A decrease in factor IXa concentration prolongs the initiation phase of thrombin generation due to the slower factor X activation.

In the reaction system lacking tissue factor (i.e. an essential component of the physiologic activator of factor IX and factor X), factor Xa can trigger thrombin generation above the coagulation threshold (>10 nM) (33, 43) only when present at the highest concentration tested (94 pM). The maximum concentration of thrombin produced, however, is much lower than that observed in the presence of factor IXa alone. At a lower factor Xa concentration (9.4 pM), factor Xa is inhibited by TFPI and AT-III (51) prior to the generation of detectable amounts of thrombin.

In the numerical simulations, thrombin even at the highest concentration used (1 nM) does not trigger its own generation. However, whole blood initiated with 1 nM thrombin in the absence of tissue factor clotted in 119 s, suggesting relatively rapid thrombin generation. This discrepancy between the two models occurs due to the absence of factor XI in the numerical simulations, and, as a consequence, the absence of an efficient pathway of factor IX and factor X activation because the only enzymes present in the system are thrombin and 0.1 nM factor VIIa. The numerical model is an evolving entity, and new proteins, reactions, and their kinetic constants have been and will be added to this model.

The data obtained when two enzymes are used simultaneously as a trigger of thrombin generation show that only the concentration of factor IXa has a pronounced effect on the process. Although factor Xa and thrombin initiate prothrombin activation in the absence of factor IXa, the maximum concentration of thrombin produced is relatively low, resembling that observed in hemophilia (29, 34). The absence of factor Xa when thrombin and factor IXa are present has almost no effect on prothrombin activation, presumably due to the rapid activation of factor V and factor VIII by thrombin (30, 31–33) and, as a result, robust factor Xa generation by the factor IXa-factor VIIIa complex. The absence of initiating thrombin when factor IXa and factor Xa are present as initiators of thrombin generation has almost no effect on rates of prothrombin activation and maximum levels of thrombin achieved. The initiation phase, however, is slightly prolonged in the absence of thrombin. This delay in the onset of the propagation phase is related to the time required to generate initial amounts of thrombin by factor Xa. When produced, thrombin amplifies its own generation by activating factor V and factor VIII.

In conclusion, the data of this study suggest that concentrations of coagulation enzymes circulating in vivo are significantly lower than can be predicted from the reported concentrations of their activation peptides and rates of their inhibition by AT-III and TFPI. These peptides thus probably represent a "historical record" rather than an "idling motor." Our results suggest that in prothrombotic disease states, which are characterized by increased levels of activation peptides, exceptional attention has to be paid to the concentration of the factor IX activation peptide due to the high efficiency of factor IXa in initiation of thrombin generation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 HL34575 and PHS T32 HL07594. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, University of Vermont, Given Bldg., Rm. C401, 89 Beaumont Ave., Burlington, VT 05405-0068. Tel.: 802-656-0335; Fax: 802-862-8229; E-mail: Kenneth.Mann{at}uvm.edu.

1 The abbreviations used are: AT-III, antithrombin III; CTI, corn trypsin inhibitor; PS, 1,2-dioleolyl-sn-glycero-3-phospho-L-serine; PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PCPS, phospholipid vesicle composed of 25% PS and 75% PC; FPRck, D-Phe-Pro-ArgCH2Cl; VPRnbs, 6-(D-Val-Pro-Arg)amino-1-naphthalene(n-butyl)sulfonamide; PPP, platelet-poor plasma; AP, activation peptide(s); TAT, thrombin-AT-III; TF, tissue factor; TFPI, tissue factor pathway inhibitor. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Shu Len Liu and Dr. Roger Lundblad for providing TF and factor VIII; Dr. Kirk Johnson for pro-viding TFPI; Dr. Ula Hedner for providing recombinant factor VIIa; and Dr. R. Jenny for providing thrombin, factor IXa, and factor Xa.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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