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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Biochemistry, University of
Vermont, College of Medicine, Burlington, VT.
Tissue factor-induced blood coagulation was studied in 20 individuals, for varying periods of time during 54 months, in contact pathway-inhibited whole blood at 37°C and evaluated in terms of the
activation of various substrates. After quenching over time with
inhibitors, the soluble phases were analyzed for thrombin-antithrombin III (TAT) complex formation, prothrombin fragments, platelet activation (osteonectin release), factor Va generation, fibrinopeptide (FP) A and
FPB release, and factor XIII activation. TAT complex formation, for 35 experiments, showed an initiation phase (up to 4.6 ± 0.6 minutes) in
which thrombin was generated at an average rate of 0.93 ± 0.3 nM/min
catalyzed by about 1.3 pM prothrombinase yielding approximately 26 nM
thrombin. During a subsequent propagation phase, thrombin was generated
at a rate of 83.9 ± 3.8 nM/min by about 120 pM prothrombinase,
reaching ultimate levels of 851 ± 53 nM. Clot time, determined
subjectively, occurred at 4.7 ± 0.2 minutes and correlated with the
inception of the propagation phase. The thrombin concentrations
associated with the transitions to rapid product formation are
510 ± 180 pM for platelet activation (1.9 ± 0.2 minutes),
840 ± 280 pM for factor XIII activation and factor Va generation
(2.2 ± 0.6 minutes), 1.3 ± 0.4 nM for FPA release (2.5 ± 0.7
minutes), 1.7 ± 0.5 nM for FPB release and prethrombin 2 (2.8 ± 0.8 minutes), 7.0 ± 2.2 nM for thrombin B chain
(3.6 ± 0.2 minutes), and 26 ± 6.2 nM for the propagation phase of
TAT formation (4.6 ± 0.6 minutes). These results illustrate that the
initial activation of thrombin substrates occurs during the initiation
phase at less than 2 nM thrombin (0.2%). Most thrombin (96%) is
formed well after clotting occurs.
(Blood. 2002;100:148-152) Blood coagulation is best described as a complex,
threshold-limited intertwined set of processes that includes physical,
cellular, and biochemical events. Each of the subprocesses leading to
thrombin generation can be operationally described by initiation,
propagation, and termination phases.1 When the vascular
endothelium is damaged, blood factor VIIa comes in contact with
exposed/expressed tissue factor (TF) and forms the vitamin K-dependent
extrinsic tenase complex, which activates factor X and factor
IX.2 Factor Xa on a membrane activates a small amount of
prothrombin to thrombin, which activates factor V and factor VIII to
their respective active products. During this initiation phase of
thrombin generation, the subnanomolar amounts of thrombin produced
amplify thrombin production by platelet, factor V, and factor VIII
activation, begin to cleave fibrinogen, and activate the
protransglutaminase factor XIII. Vigorous thrombin generation occurs
during a propagation phase when the kinetically efficient intrinsic
tenase (factor IXa-factor VIIIa, Ca++, membrane) activates
increased levels of factor X generating the major burst of
prothrombinase and thrombin. Thrombin generation is attenuated and
terminated by a collection of stoichiometric and enzymatic inhibitors
(antithrombin III, tissue factor pathway inhibitor, and the protein
C pathway).
Prothrombin is converted to the serine protease The present study was undertaken to evaluate the enzymatic selection
and activation of thrombin-sensitive substrates during the dynamic
process of TF-initiated coagulation of whole blood.
Materials
Patients
Whole blood coagulation Blood was collected by venipuncture at the Clinical Research Center, Fletcher Allen Health Care (Burlington, VT) and aliquoted (1 mL) into tubes containing CTI (50-100 µg/mL) and TF (nominally 12.5-25 pM; functionally ~5 pM) relipidated in PCPS (1:2000 protein/lipid) as previously described.16,19,20 The TF concentration was changed during the course of the 54 months due to different preparation methods. Based on a functional assay (see below) the concentration of active TF used in whole blood was ~5 pM. The TF/PCPS concentrations used were chosen to give clot times in the range of 4 to 5 minutes. Samples were quenched at intervals over the course of 20 minutes with a cocktail of inhibitors: 50 mM EDTA and 20 mM benzamidine-HCl in HEPES-buffered saline (HBS), pH 7.4, and 10 µL 10 mM FPRck in 10 mM HCl. The zero time point contained the inhibitors prior to the addition of blood. A control tube, containing CTI and no TF, was used each time. Clot time was determined visually (2 observers). After quenching, samples were centrifuged for 15 minutes at 2000 rpm and clot material separated from the solution phase. Solid and solution phases were stored at 80°C for further analysis.
Determination of active TF in TF/PCPS preparations Relipidated TF (TF/PCPS: 200 pM/400 nM) and varying concentrations of factor VIIa (0-600 pM) were incubated at 37°C in HBS/2 mM CaCl2/0.1% polyethylene glycol (PEG) containing 10 µM PCPS for 10 minutes (total volume = 250 µL). This was followed by the addition of 170 nM factor X. Aliquots (20 µL) were removed every 30 seconds for 5 minutes and quenched into 160 µL HBS containing 20 mM EDTA. The rate of substrate hydrolysis was observed on addition of 20 µL 2 mM Spectrozyme Xa. Factor Xa generation rate was evaluated from a standard curve using serial dilutions of purified factor Xa. The concentration of active TF was evaluated using a Scatchard plot (factor VIIa bound versus bound/free ratio) assuming 1:1 stoichiometry for TF and factor VIIa.Immunoassays and HPLC The ELISAs for TAT, osteonectin, and FPA were performed according to the manufacturers' protocols in duplicate or triplicate using a minimum of 5 standards as previously described.16 The operational sensitivity of the TAT ELISA is about 40 pM. Results were analyzed on a Vmax microtiter plate reader equipped with Softmax version 2.35 (both from Molecular Devices, Menlo Park, CA).The FPA analyses were also performed using HPLC methods. FPA and FPB were isolated and quantitated using reverse-phase (C18 Bakerbond, 4.6 × 250 mm) HPLC methodology.20 Peptide samples were eluted by using linear gradients of H2O/CH3CN/0.05% TFA. The peptides were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (linear model, PE Applied Biosystems). Data presented for FPA analysis are obtained from the combination of ELISA and HPLC quantitation. Measure of free thrombin The rate of FPA generation used to estimate active thrombin concentration was calculated using a modified Michaelis Menten equation that accounts for competitive inhibition as described by Rand et al19 and shown below:
 -thrombin and fibrinogen are 7.2 µM and 84/s.21 For
product inhibition, KI = Km is equal to the
free inhibitory product, FPA concentration at time t. [S] is the
concentration of free substrate (total fibrinogen-FPA) at time t.
Western analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) was performed using a modified Laemmeli procedure.19,22 Factor XIIIAa, factor Va light chain,-thrombin B chain, and a prethrombin 2-like product were
analyzed by 5% to 15% SDS-PAGE under either nonreducing or reducing
conditions as specified. The actual sequence of the prethrombin 2-like
product from whole blood has not been identified, but migrates along
with the purified and characterized prethrombin 2 standard. We will
refer to this as "prethrombin 2" in the text. The proteins from the
electrophorogram were transferred to nitrocellulose membranes using a
semidry method followed by blotting with the primary antibodies rabbit
-factor XIII (7.5 µg/mL), mouse -factor Va#9 (7.5 µg/mL), and
burro -prethrombin 1 (7.5 µg/mL). Secondary horseradish
peroxidase-conjugated goat -rabbit, -mouse, and -horse
antibodies were used at dilutions of 1:12 000, 1:15 000, and
1:7500, respectively. Time courses of factor XIII activation, factor Va
light chain generation, -thrombin B chain, and prethrombin 2 formation were analyzed and quantified by densitometry of immunoblots
on a Hewlett-Packard Scanjet 4C/T (Hewlett-Packard, Palo Alto, CA).
Concentrations were determined from horizontal band density comparison
of standard protein dilutions. Relative concentrations were determined
by normalizing the data with regard to the maximum.
TAT complex formation and prothrombinase concentration Whole blood from 35 experiments (16 individuals, 10 men and 6 women) was studied over the course of 54 months. The time course of TAT complex formation ( , TAT [nM] versus time [minute]) is shown as
the mean ± SEM with a clot time of 4.7 ± 0.2 minutes (Figures
1 and 3A). Complex formation proceeded
initially at a rate of 0.93 ± 0.3 nM/min for a duration of
4.6 ± 0.6 minutes. Subsequently, thrombin formation proceeded at a
rate of 83.9 ± 3.8 nM/min and reached a maximum level of 851 ± 53
nM (Table 1).
Prothrombinase concentrations were estimated from the rate of thrombin
generation using a modified form of the Michaelis-Menten equation
([E]t = Platelet activation Release of-granule osteonectin from activated platelets
was analyzed in quenched whole blood samples in 18 experiments from 10 individuals (5 men/5 women). Osteonectin ( ) release is shown in Figure 1 as the mean ± SEM of osteonectin versus time with a
baseline value of 6.9 ± 1.2 nM. By 1.9 ± 0.2 minutes platelet release occurred at a rate of 6.8 ± 0.4 nM/min, with maximum levels of osteonectin release of 44.6 ± 6.7 nM. The initial baseline and
maximum level reached are consistent with published results of
osteonectin levels present in plasma prior to and on platelet activation.23
Fibrinopeptide release The cumulative results for FPA release (26 experiments; 13 individuals, 6 men/7 women) are shown in Figure 2 as the mean ± SEM. Rapid FPA release ( ) was observed at 2.5 ± 0.7 minutes and increased to
maximum levels of 15.3 ± 1.3 µM by 8 minutes at a rate of
2.7 ± 0.2 µM/min.
The release of FPB from fibrinogen/fibrin is initially seen followed by
the cleavage of the COOH terminal arginine (des-Arg FPB) by an
unidentified carboxypeptidase B-like enzyme (most likely TAFI).20 The sum of FPB and des-Arg FPB from 9 experiments
(7 individuals, 4 men/3 women) detected by HPLC are shown ( Factor XIII activation Factor XIII concentrations detected by Western immunoblots for 9 experiments (6 individuals, 4 men/2 women) is plotted in Figure 2 ( ). Factor XIIIa is detected slightly prior to FPA release, occurring at 2.2 ± 0.6 minutes. Factor XIIIa is generated at a rate
of 11.4% ± 1.0%/min (~10.3 ± 0.9 nM/min, based on a mean concentration of 90 nM) reaching maximum levels of 62.5% ± 3.8% (~56.3 ± 3.4 nM).
Factor Va generation The concentration of factor Va light chain was determined from 5 experiments (4 individuals, 2 men/2 women). Light chain was detected coincidental with factor XIIIa at 2.2 ± 0.4 minutes. The concentration of the light chain increased at a rate of 22.4% ± 6.3%/min (~4.5 ± 1.3 nM/min, based on a mean concentration of 20 nM). Maximum product level present by 20 minutes was determined to be about 62% (~12 nM).Prethrombin 2 and thrombin B chain formation The concentration of prothrombin fragments,-thrombin B chain
( ), and prethrombin 2 ( ) were detected by Western immunoblots (Figure 3A) with quantitation by
comparison to a standard density curve derived from known amounts of
purified standard. Data were collected from 8 experiments (7 individuals, 4 men/3 women) for the prothrombin fragments. The
prethrombin 2 was first detected at 2.8 ± 0.8 minutes and increased
to maximum levels of 549 ± 86 nM at a rate of 52.1 ± 2.0 nM/min.
-thrombin B chain formation was first detected after a slightly
longer period, at 3.6 ± 0.2 minutes and increased at a comparable
rate of 56.9 ± 2.1 nM/min, reaching maximum levels of 494 ± 66 nM
by 20 minutes. TAT complex formation ( ) reaches maximum levels of
851 ± 53 nM. This number corresponds to approximately 61% of
prothrombin conversion to thrombin.
The 3 principal species of thrombin are irreversibly inhibited,
reversibly inhibited, and free thrombin. The major depot for thrombin
product is TAT and the significant amount of B chain seen in SDS
electropherograms must come from dissociation of TAT. Therefore, the
immunochemically determined ELISA of TAT complex formation in the
native state at the time of quenching can be expressed as
TATTOTAL = TATIRREVERSIBLE + TATREVERSIBLE (Figure 3A). The Western immunoblot detection
of thrombin B chain after denaturation is then derived from
TATREVERSIBLE broken down under SDS conditions and free
thrombin where the amount of free thrombin is
negligible (Figure 4). The amount of
TATIRREVERSIBLE can then be calculated from
TATTOTAL-thrombin B chain, assuming all of the reversible
complex is dissociated. The amount of irreversible complex formed is
about 308 nM by 20 minutes (Figure 3B). The negative values seen prior
to 7 minutes probably represent excess thrombin.
Thrombin product formation The thrombin concentrations extant at the inception of major product formation for each of the substrates, the prothrombin fragments, prethrombin 2, and-thrombin B chain are presented in
Figure 4. These values calculated from the first 6 minutes of TAT
generation are shown on a logarithmic scale of TAT (nM) versus time
(minute) with the percent of the maximum TAT observed shown in
parentheses. Platelet activation based on osteonectin release is first
observed at 1.9 ± 0.2 minutes and 510 ± 180 pM TAT (arrow in
Figure 4, OSN). Rapid activation of factor XIII (2.2 ± 0.6 minutes)
and factor Va (2.2 ± 0.4 minutes) occur next at 840 ± 280 pM TAT.
The rapid releases of fibrinopeptides, FPA and FPB, are observed at
2.5 ± 0.7 minutes and 2.8 ± 0.8 minutes, respectively,
corresponding to TAT concentrations of 1.3 ± 0.4 nM or 1.7 ± 0.5
nM. Prethrombin 2 is observed simultaneously with FPB at 2.8 ± 0.8
minutes at 1.7 ± 0.5 nM TAT. Thrombin B chain is observed in the
denatured samples at 3.6 ± 0.2 minutes when 7.0 ± 2.2 nM TAT
complex is present. Rapid thrombin generation signaling the transition
to the propagation phase occurs at 4.6 ± 0.6 minutes corresponding
to 26 ± 6.2 nM TAT. The thrombin concentrations associated with
activation of its substrates are less than 1% of maximum thrombin
observed (as TAT).
Active thrombin estimated from the rate of FPA generation during the initiation phase of the reaction is presented in Figure 4 as a dashed line. At clot time, the thrombin concentration calculated from FPA release is approximately 2 nM and remains fairly constant. The maximum concentration and product yields are shown in Table 1. On average about 61% of prothrombin is converted to thrombin (0.851 µM). Similar yields are observed for factor XIII and factor V. Although 15.3 µM FPA is released, only 5.8 µM FPB is released (~39%).
These data describe the concentrations of thrombin associated with the onsets of rapid activation for several processes. Relatively minute concentrations of thrombin are generated during the initiation phase of blood coagulation, primarily due to the factor Xa generated from the extrinsic tenase complex. These levels, in the range of 0.51 to 2 nM are sufficient for the initiation of rapid activation of platelets, factor XIII, factor V, and fibrin formation. All of these processes occur prior to the major burst of thrombin generation during the propagation phase of the reaction. The small amount of thrombin generated (26 ± 6.2 nM) at approximately clot time (4.6 ± 0.6 minutes) corresponds to a prothrombinase concentration of only about 1.3 pM. Platelet activation (~0.5 nM thrombin) is followed by factor XIII and factor V activation (~0.8 nM thrombin), both of which precede fibrinopeptide release (~1.5 nM thrombin). The advent of formation of major catalysts is signaled by the detection of appreciable amounts of prothrombin activation products by about 4 minutes. At clot time, approximately 4.7 minutes, 25% to 50% of the procoagulant substrates are cleaved and 60% of platelets are activated. These activation processes occur with less than 1% of the total amount of thrombin ultimately produced. This value, calculated from TAT concentration, is an overestimate of the free thrombin available. The amount of free thrombin available at any given time in the reaction is somewhere between that estimated from the rate of FPA generation and the amount of reversible thrombin from the TAT complex. Thrombin calculated from the rate of FPA generation shows about 2 nM thrombin is present at clot time. Because only 2 nM thrombin is involved in fibrinogen cleavage, the amount of free thrombin is somewhere between that and the approximate 540 nM thrombin in reversible complex with antithrombin III. The prothrombin product prethrombin 2 is not predicted by analysis of
prothrombin activation in vitro. In our studies in whole blood the
prothrombin fragment prethrombin 2 is detected by Western immunoblotting and plotted as concentration versus time in Figure 3A.
This product has previously been detected in whole blood using the same
antibody.19 In purified systems, generation by factor Xa-membrane prethrombin 2 (272-579) is a precursor to
The data presented here are consistent with our earlier report of the
simultaneous event of A In summary, these results show that it is during the initiation phase of thrombin generation that is crucial to the activation of the procoagulant substrates. When a clot is visually seen about 25% to 60% of the reactions have already occurred and about 96% of thrombin is still being generated. The reason for the overabundance of thrombin generation after clot time has yet to be elucidated.
We the authors would like to thank Heather Kovich and Shyla L. Tessmer for their technical assistance and Dr Thomas Orfeo for assistance in the preparation of the manuscript.
Submitted October 31, 2001; accepted February 25, 2002.
Supported in part by Program Project Grant no. HL 46703 (Project 1) from the National Institutes of Health (to K.G.M.) and by Training Grant no. PHST32HL07594-15 from the US Public Health Service (to K.E.B.).
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.
Reprints: Kenneth G. Mann, Department of Biochemistry, 89 Beaumont Ave, University of Vermont, Given Building, Room C401, Burlington, VT 05405; e-mail: kmann{at}zoo.uvm.edu.
1. Mann KG. Normal hemostasis Kelley WN, ed). Textbook of Internal Medicine. Philadelphia, PA: Lippincott; 1992:1240-1245.
2.
Krishnaswamy S, Field KA, Edgington TS, Morrissey JH, Mann KG.
Role of the membrane surface in the activation of human coagulation factor X.
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Krishnaswamy S, Mann KG, Nesheim ME.
The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction.
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Monoclonal antibodies to human coagulation factor V and factor Va.
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19.
Rand MD, Lock JB, van't Veer C, Gaffney DP, Mann KG.
Blood clotting in minimally altered whole blood.
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1996;88:3432-3445
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Brummel KE, Butenas S, Mann KG.
An integrated study of fibrinogen during blood coagulation.
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1999;274:22862-22870
21.
Higgins DL, Lewis SD, Shafer JA.
Steady state kinetic parameters for the thrombin-catalyzed conversion of human fibrinogen to fibrin.
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Stenner DD, Tracy RP, Riggs BL, Mann KG.
Human platelets contain and secrete osteonectin, a major protein of mineralized bone.
Proc Natl Acad Sci U S A.
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© 2002 by The American Society of Hematology.
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D. A. Lane, H. Philippou, and J. A. Huntington Directing thrombin Blood, October 15, 2005; 106(8): 2605 - 2612. [Abstract] [Full Text] [PDF]
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S. Butenas, B. A. Bouchard, K. E. Brummel-Ziedins, B. Parhami-Seren, and K. G. Mann Tissue factor activity in whole blood Blood, April 1, 2005; 105(7): 2764 - 2770. [Abstract] [Full Text] [PDF]
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M. Nesheim Thrombin and Fibrinolysis Chest, September 1, 2003; 124(3_suppl): 33S - 39S. [Abstract] [Full Text] [PDF]
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M. Kalafatis, D. O. Beck, and K. G. Mann Structural Requirements for Expression of Factor Va Activity J. Biol. Chem., August 29, 2003; 278(35): 33550 - 33561. [Abstract] [Full Text] [PDF]
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W. L. Chandler and T. Velan Estimating the rate of thrombin and fibrin generation in vivo during cardiopulmonary bypass Blood, June 1, 2003; 101(11): 4355 - 4362. [Abstract] [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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V. Schroeder, H. P. Kohler, K. E. Brummel, and K. G. Mann Factor XIII activation by thrombin depends on FXIIIVal34Leu genotype Blood, January 1, 2003; 101(1): 371 - 371. [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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Abstract
Introduction![[E<SUB>t</SUB>]=&ngr;(K<SUB>m</SUB>+K<SUB>m</SUB>[P]/K<SUB>I</SUB>+[S])/k<SUB>cat</SUB>[S]](/content/vol100/issue1/fulltext/148/img001.gif)
(Km + [S])/kcat[S]) as previously described.
1. During the initial phase
of thrombin generation, product formation was catalyzed by
prothrombinase levels of about 1.3 pM. Thrombin generation subsequently
increased, during the propagation phase, produced by prothrombinase
levels of 120 pM. These values are similar to those previously reported
for a single individual (7 pM and 155 pM) by Rand et al.
in the fibrinogen/fibrin clot.
