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Blood Clotting in Minimally Altered Whole Blood
By Matthew D. Rand, Jennifer B. Lock, Cornelis van't Veer, Donald P. Gaffney, and Kenneth G. Mann
The sequences of events regulating thrombin generation
during tissue factor-initiated clotting in whole blood at 37°C
in which the contact pathway was suppressed with corn
trypsin inhibitor are studied using quantitative Western blotting of factor V, prothrombin, platelet factor 4, antithrombin
Ill, and fibrinogen. In addition, fibrinopeptide A (FPA), thrombin-antithrombin 111 (TAT) complex formation, and prothrombin fragment 1.2 (F1.2) were measured via commercially
available enzyme-linked immunosorbent assays (ELISAS).In
a typical experiment initiated with 40 pmol/L recombinant
tissue factor, visual clot time (4.5 minutes), was preceded
by significant cleavage of factor V resulting in 65% factor Va
heavy-chain generation but only 10% light-chain formation.
At this point, 50% of the platelet factor 4 is released, suggesting that half (approximately 700 pmol/L) of the platelet
prothrombinase sites available have been generated. At clot
time, approximately 15 nmol/L thrombin B-chain is present;
however, analyses of FPA release demonstrate that only 15%
of the thrombin is acting on fibrinogen. This thrombin is
produced by the action of 7 pmol/L prothrombinase. The
maximum rate of thrombin production is reached well after
clot time and is consistent with the presence of approximately 150 pmol/L prothrombinase (at about 7 minutes).
These results suggest that factor Xa is the limiting factor
for thrombin generation. After 60 minutes, 75% of the initial
prothrombin (1.24 pmol/L) is consumed yielding 440 nmol/
L prethrombin 2 and 360 nmol/L thrombin (B-chain) products. The sum of these values (800 nmol/L) is similar t o the
(corrected) F1.2 concentration determined by ELISA. The incomplete cleavage of prothrombin indicates both the prothrombinase complex and the formation of prothrombinase
are inhibited in the reaction. TAT complex measured by
ELISA is almost equivalent t o B-chain concentration, but sodium dodecyl sulfate stable thrombin-antithrombin 111 complexes are not observed until well after clot formation and
are never equivalent t o ELISA-TAT values. At the point of
clot formation, 80% of the fibrinogen is depleted from the
fluid phase, whereas only 3540t o 4546 of the FPA is released,
suggesting a significant incorporation of uncleaved fibrinogen into the initial clot formed.
0 1996by The American Society of Hematology.
T
The proteins thought to be essential to clotting have been
extensively purified and characterized. Analyses of the activities of zymogens, enzymes, procofactors, cofactors, and cellular components, as isolates and as participants in reconstituted mixtures, have defined the current paradigms of the
coagulation process. In current theory, the minimally required procoagulant proteins are factor VIWIIa, tissue factor, factor X, factor IX, factor VIII, factor V, prothrombin,
fibrinogen, and factor XIII, whereas the blood-derived cellular elements implicated include monocytes and platelets."'
Because of the high variability in bleeding tendency of factor
XI-deficient individuals, the inclusion of factor XI in the
list of essential coagulation factors remains controversial.' '
Plasma contains approximately 1% of the factor VI1 in the
activated two-chain form, factor VIIa.'' In the absence of
tissue factor this protein is not subject to the stoichiometric
inhibitors because of its poor catalytic properties.I3However.
when tissue factor is exposedexpressed, the factor VIIa
tissue factor complex can activate both factor IX and factor
X.14.tS The resulting factor IXa and factor Xa form complexes
with factor VIIIa and factor Va that are probably initially
derived from their circulating procofactors, factor V and
factor VIII, by the actions of the companions in their respective enzyme c~mplexes.'~.'~
Factor Xa phospholipid is an
efficient activator of factor VII," leading to further accumulation of factor VIIa, which can function only in complex
with tissue factor, which is expressed by cytokine activation
of endothelial cells or monocytes" or tissue damage.*' The
initial factor Xa produced can also contribute to factor IX
activation through the formation of the intermediate factor
IX-a, which is converted to factor IXab by the factor VIIatissue factor complex.2' Following these initial activation
steps, factor Xa is potentially formed by two activation pathways: tissue factor * factor VIIa and factor VIIIa * factor IXa,
the latter being the more efficient. The relative contributions
of these two pathways is dependent upon the tissue factor
concentration exposed to the blood and upon TFPI, because
the factor VIIa. tissue factor complex is downregulated by
HE BLOOD coagulation process requires coordinated
interactions between plasma-derived enzymes, zymogens, cofactors, inhibitors, and the blood and vascular cells.
These processes serve to initiate and localize catalytic events
to injured vascular surfaces. The procoagulant process culminates in the generation of a-thrombin, which activates the
plasma procofactors, factor V and factor VIII, aggregates
platelets, cleaves fibrinogen, activates factor XIII, and serves
numerous other physiological processes.' The enzymes of
the procoagulant pathway are neutralized by a variety of
stoichiometric protein inhibitors including antithrombin I11
(AT-III),' the tissue factor pathway inhibitor (TFPI),' heparin
cofactor I1 (HC-11): and a 2 macroglobulin (a2M).' The
platelet provides sites for catalyst assembly and expres~ion.',~
The plasma cofactors, factor Va and factor VIIIa, are
subject to thrombin-induced negative feedback regulation by
the dynamic protein C pathway mediated by thrombomodulin,' which is supplied principally by the vascular endothelium. Other cells in blood and the vessel wall may also
contribute to procoagulant and anticoagulant activities.'
From the University of Vermont College of Medicine, Burlington.
Submitted February 14, 1996; accepted June 17, 1996.
Supported by research Grant No. POIHL.46703 from the National
Institutes of Health, a TALENT-stipendium from the Netherlands
Organization for Scient$c Research, and the Department of Biochemistry, University of Vermont, College of Medicine.
A preliminary account of this work was presented at the 1995
annual meeting of the American Society of Hematology in Sun Diego,
California, and published in abstract form (Blood 86:187a, 1995).
Address reprint requests to Kenneth G. Mann, PhD, Department
of Biochemistry, Given Building, University of Vermont College of
Medicine, Burlington, VT 05405-0068.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8809-0022$3.00/0
3432
-
Blood, Vol 88, No 9 (November 11, 1996:pp 3432-3445
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3433
BLOOD CLOTTING IN MINIMALLY ALTERED WHOLE BLOOD
-
a complex interaction of the TFPI factor Xa complex with
the factor VIIa tissue factor complex.22
On an activated cell membrane, factor Xa forms a complex
with factor Va, prothrombinase, which activates prothrombin to thr~mbin.’~
Once thrombin is generated, feedback
activations of factor V and factor VI11 accelerate the overall
reaction pro~ess.’~
Additional thrombin feedback contributes
to the activation of factor XI, which may accelerate the
thrombin generation process by activation of factor IX. ”,”
Thrombin acts on the platelet membrane recepto?6 to induce
platelet aggregation and upon fibrinogen to produce fibrinopeptide A and fibrin I and subsequently fibrinopeptide B
and fibrin 11. The fibrin monomers polymerize to form the
~ i ~ t . ~ ~ - ~ ~
The extrinsic pathway, involving the factor VIIa. tissue
factor complex, has strong evidential support as the physiological initiator of in vivo clotting event.^,'^.^'‘^^ whereas the
intrinsic p a t h ~ a y ,which
~ ~ . ~requires
~
the activation of factor
XI1 and factor XI in reactions supported by high molecular
weight kininogen, prekallikrein, and negatively charged surfaces, has a questionable role in normal hemostasis. The
observations of apparently normal hemostasis in individuals
deficient in factor XII, high molecular weight kininogen, and
prekallikrein suggest that the intrinsic pathway is not required for normal hemostatic function; however, this pathway may play a significant role during pathologic state^.^^.^^
In any event, in vitro, surface-dependent contact activation
is initiated when blood comes in contact with virtually any
artificial container. Thus, the intrinsic pathway must be suppressed if the tissue factor-mediated reactions are to be
studied.
Recently, we reported a model system that employs the
procoagulant components of the extrinsic pathway, with each
component reconstituted at its average physiological concentrati~n.~’
In this model system, the activations of prothrombin, factor V, factor VIII, factor IX, and factor X in the
presence of Ca“ and synthetic phospholipid vesicles were
monitored in response to reactions initiated by factor VIIa
tissue factor. Less than 1% of factors IX and X were converted to active enzyme during the interval in which quantitative prothrombin conversion occurred. In this purified system, no novel products of proteolytic processing of the
zymogens and procofactors were observed. In addition,
thrombin generation over time was characterized by an apparent “lag” followed by a rapid propagation phase. It was
also observed that varying the initiator concentration (factor
VIIa-tissue factor) did not significantly alter the rate of
thrombin generation in the thrombin propagation phase of
the reaction but primarily influenced the lag time before the
propagation phase. Similar reconstructed systems have since
been reported that incorporate AT-111, TFPI, and the protein
C-thrombomodulin components.39-4
Most studies aimed at the evaluation of coagulant processes in quasi-physiological systems using plasma and
blood commonly employ Ca2+ chelators such as citrate or
EDTA. However, because many Ca2*-dependentprocesses,
such as vitamin K-dependent protein folding and cellular
Ca’’ transients, occur with rates relevant to those of clot
formation, the chelation-recalcification processes may influ-
‘
ence underlying cellular and enzymatic m e c h a n i ~ m s Fi.~~
nally, most kinetic evaluations of blood or plasma clotting
are limited to analyses of an end point involving clot formation, although a recent study by Kessels et
uses chromogenic peptide substrates to monitor thrombin generation in
a minimally altered whole blood system.
The present study was conducted to evaluate the relationships between protease enzymology and platelet activation
that occur during the tissue factor-induced clotting of minimally altered whole blood.
MATERIALS AND METHODS
HEPES, Tris-HC1, rabbit brain thromboplastin, I-palmitoyl-2oleoyl-phosphatidyl serine (PS), I-palmitoyl-2-oleoyl-phosphatidyl
choline (PC), and 2-mercaptoethanol were purchased from Sigma
Chemical Co (St Louis, MO). Benzamidine-HC1was purchased from
Aldrich, Inc (Milwaukee, WI). Activated partial thromboplastin time
reagent was purchased from Organon Technica (Durham, NC). Nitrocellulose was purchased from Biorad (Hercules, CA). Horseradish
peroxidase-labeled goat antirabbit IgG, antimouse IgG, and antihorse IgG antibodies were purchased from Southem Biotech (Birmingham, AL). Film was purchased from Dupont (Boston, MA).
D-phenylalanylprolyarginyl-chloromethylketone (FPR-ck), rabbit
antihuman a-thrombin, human platelet factor 4 (PF4), and human
antithrombin 111 (AT-111) were gifts from Dr Richard Jenny of
Haematologic Technologies, Inc (Essex Junction, VT). Antihuman
tissue factor antibody 5G9 was a gift from Dr Thomas Edgington
of Scripps Research Institute (La Jolla, CA). Prothrombin and factor
V were purified as previously desc~ibed.~-~’
Recombinant tissue
factor (residues 1 to 242) was a gift from Dr Hissids of Genentech,
Inc and was relipidated in 25% phosphatidylserine, 75% phosphatidylcholine (PCPS) vesicles by a previously described p r o t o ~ o l ~ ~ , ~ ~
at 10 pmol/L PCPS and tissue factor ranging from 2.5 to 300 nmoU
L. Trypsin inhibitor from com was purchased from Fluka (Ronkonkoma, NY) and was stored as a 5 mg/mL stock in 20 m m o n
HEPES, 150 mmolR. NaC1, pH 7.4 at -20°C. Burro antihumanAT-I11 IgG and antihuman-PF4 IgG were prepared as previously
de~cribed.~’
Monospecific anti-AT-I11 IgG was absorbed to AT-IIISepharose and eluted with 0. I m o m glycine, pH 2.5, and neutralized
with 0.1 mom Tris-HC1, pH 9.0. IgG was precipitated by 70%
saturation with ammonium sulfate and stored in 50% glycerol at
-20°C. The IgG fraction of anti-PF4 serum was used as primary
antibody in Western blotting. The monoclonal antibodies anti-HFV6 and anti-HFV-9 are antibodies specific for the human factor Va
heavy chain and light chain, respectively, and have been described
previo~sly.~
Monoclonal
’~~~
antibodies, anti-fibrinogen-3A, antiHFV-6 and anti-HFV-9, were provided by Dr William Church
(University of Vermont). Molecular weight standards for sodium
dodecyl sulfate (SDS)-gel electrophoresis were purchased from
GIBCO-BRL (Gaithersburg, MD). A11 other reagents were of analytical grade.
Whole blood clotting experiments. Blood was drawn from donors with informed consent and according to the guidelines of the
human subjects research committee at the University of Vermont.
Subjects analyzed for whole blood clotting in this study were tested
for fibrinogen, prothrombin time, activated partial thromboplastin
time, and a hemogram was obtained. These tests were conducted by
the outpatient laboratory at Fletcher Allen Health Care, Burlington,
Vermont, under the supervision of Dr Edwin Bovill and Mr Randy
Messier. All of the subjects studied were in the normal range of
prothrombin time, activated partial thromboplastin time, platelets,
and fibrinogen.
Clotting of whole blood was carried out in 12 x 75-mm polystyrene culture tubes purchased from VWR Scientific (product no.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3434
60818-270). The tubes were modified by drilling a 3/16-in hole in
the side at the midpoint. The tubes were then capped with twoposition polypropylene caps (VWR 60819-047) and positioned on
their sides with the hole facing up on a Thermolyne speci-mix rocker
table (Sybron, Dubuque, IA) and enclosed in a 37°C temperaturecontrolled glove box. Blood was drawn from healthy, nonsmoking,
non-aspirin-using volunteers using 19-gauge butterfly needles. The
first 3 mL of blood was discarded. Subsequently, 40 mL of blood
was drawn directly with an Eppendorf repeater pipet equipped with
a 50-mL syringe barrel. One milliliter aliquots of the nonanticoagulated blood was rapidly distributed (over 5 1 minute) into 32 reagentloaded polystyrene tubes via the hole in the side of the tube. The
tubes were rocked continuously and clot times were determined by
the obvious formation of “clumps” on the sides of the tube. Clot
times are expressed as the average of duplicate determinations and
time zero is taken as the time the blood contacts the tube. The effects
of corn trypsin inhibitor (CTI), tissue factor-PCPS, PCPS alone, and
anti-tissue factor antibodies were monitored by preloading the tubes
with various concentrations of the effector(s) in 20 mmol/L HEPES,
150 mmoVL NaCI, 5 mmol/L CaCI2, pH 7.4 in a volume not exceeding 29 pL.
Analyses of whole blood clotting were performed with final
plasma concentrations of 32 pg/mL CTI, 40 pmol/L tissue factor
and 80 nmol/L PCPS. The clotting reactions were quenched at various times by the addition of 1.O mL of 50 mmol/L EDTA, 10 mmol/
L benzamidine in HEPES-buffered saline followed by 10 pL of 5
mmol/L FPR-ck in 0.01N HCI. The hole in the tube was sealed with
tape, and the tubes were rapidly vortexed for 15 seconds to thoroughly mix the enclosed material and permit exchange of entrapped
and bound elements with the fluid phase. Samples were stored on
ice for a maximum of 30 minutes until they were centrifuged and
the supematant (fluid phase) was collected and analyzed. Aliquots
of the fluid phase were stored at -20°C for analysis by enzymelinked immunosorbent assay (ELISA) techniques and gel electrophoresis.
Gel electrophoresis and Western blotting of Juid phase blood
samples. Samples for SDS-polyacrylamide gel electrophoresis
(PAGE) were prepared by diluting 120 pL of fluid phase serum into
380 pL of 31 moln Tris, pH 6.8, 5% glycerol, 1% SDS, 0.05%
bromophenol blue (SDS-PAGE buffer). Each sample was divided
into two aliquots: nonreducing and reducing (1% 2P-mercaptoethanol). The samples were heated for 5 minutes at 95°C and either
analyzed immediately or stored at -20°C. Samples equivalent to
0.38 to 0.75 pL of undiluted fluid blood were analyzed by SDSPAGE.53A separate gel was prepared for each antigen analyzed.
Transfer of proteins to nitrocellulose was performed as previously
described.54Membranes were blocked with 5% dried milk in 20
mmol/L Tris-HC1, 150 mmom NaCI, 0.02% Tween-20 pH 7.4
(TBST) for 1 hour. Westem blotting was performed using the primary antibodies: anti-cy-thrombin, anti-HFV-6, anti-HFV-9, and
anti-fibrinogen-3A (all used at 5 pg/mL), anti-AT-111 (at 2 pg/mL),
and anti-PF4 (at 20 pg/mL) in TBST for a 90-minute incubation.
Peroxidase-conjugated secondary antibodies (antimouse, antirabbit,
or antihorse, as appropriate) were used at 1/2,500 dilution in TBST
for a 60-minute incubation. Membranes were rinsed after each antibody incubation with 4 x 100 mL TBST and a final 30-minute to
16-hour rinse in TBST was performed before development. Blots
were developed using the Renaissance chemiluminescent reagent
(Dupont, Boston, MA) for varying intervals, depending on the analyte. Film was developed in a Kodak X-Omat and scanned using a
Microscan 1000 scanning densitometer (TRI, Inc).
Serial dilution of prothrombin, thrombin, factors V and Va, PF4,
AT-111, and AT-111-thrombin were prepared in SDS-PAGE sample
buffer as intemal standards for quantitation. Quantitation of prothrombin, prethrombin 2, thrombin B-chain, and platelet factor 4 was
RAND ET AL
accomplished using intemal standards of these proteins at various
concentrations, which were applied to the same gel used to analyze
reactants and products. For fibrinogen and factor V, which are quantitatively converted to their end products in the reaction, quantitation
was based on the maximum level of the component present in the
gel. Because analyte concentrations are changing with time, different
exposure times were used to quantitate various analytes, thus
avoiding either lack of sensitivity or film saturation. Comparisons
of immunoblots by densitometry is legitimate with respect to the
horizontal dimension in analyses of the appearance of the same
component over time. Vertical comparisons of the densities of product-precursor relationships are not legitimate because the overall
intensity of an immunostained band will depend on the quality of
transfer (which is size dependent) and on the quantitative expression
of the relevant epitope in precursor and products.
To optimize the resolutiodquantitation of a protein band of interest and to avoid difficulties caused by some of the more abundant
plasma proteins either reducing or nonreducing conditions were employed. IgG interferes in the 150,000 molecular weight range in
nonreduced gels and in the 50,000 and 25,000 molecular weight
ranges on reduced gels. Albumin interferes in the range of 50,000
to 60,000 on both types of gels. Judicious choices of reducing and
nonreducing conditions are therefore required to visualize certain
products obtained from factor V and prothrombin activation. In addition, the immunoblotting technique, either through the primary or
secondary antibody reactions, will display “nonspecific” reactivity
with other constituents in the gel. For example, most antimouse and
antihorse antibodies used as the secondary stains will react with the
large amounts of human IgG present in the gel. Nonspecific reactants
are identified as such by nonimmune controls and their appearance in
unaltered form throughout the time course of the reaction (horizontal
dimension). Bands of interest were positively identified by (1) reactivity with specific antibodies, (2) change in density over time (ie,
disappearance of a zymogen or appearance of the product or enzyme), and (3) the detection of bands of expected mobility compared
with the known purified protein activation products.
Zmmunoassays offibrinopepride A, prothrombin fragment 1.2, and
fhrombin-AT-ZIZ. Fibrinopeptide A (FPA) was measured in the
fluid phase samples using the Asserachrom FPA assay kit from
Diagnostica Stago (American Binproducts, New Jersey) according
to the manufacturer’s instructions. Serial dilutions (up to 1: 1,000)
of the samples were performed to accommodate the standard curve.
Assays were performed in duplicate.
Prothrombin fragment 1.2 (F1.2) was measured using the Enzygnost F1.2 microimmunoassay kit from Behring according to manufacturer’s instructions. For samples obtained after clot formation, a
100- to 1,000-fold dilution was performed to give values on the
standard curve. We noted a significant discrepancy in assays of our
purified samples of F1.2 using this assay. We thus performed the
following analyses: Prothrombin ( I .4 pmoVL) was quantitatively
activated to a-thrombin with factor Xa ( 5 nmol/L), factor Va (10
nmol/L), 5 pmol/L PCPS, 5 mmoVL Ca2’ in TBS buffer containing
100 pmol/L DAPA. At various time points aliquots were quenched
in 100 pmol/L FPR-cW20 mmoVL EDTA, and samples were analyzed by the F1.2 immunoassay and Westem blotting after SDSPAGE. FI .2 was quantitatively and exclusively produced (equimolar
with a-thrombin) by I minute and remained constant for 15 minutes.
The value reported by the Enzygnost assay was 1.96 pmol/L F1.2.
This value is 40% in excess of the true value (1.4 pmol/L) FI .2 in
the sample. The values obtained using the standards provided with
the immunoassay kit were therefore multiplied by 0.713 to give
corrected values for F1.2. Western blots of the kit standards displayed no reaction with monoclonal anti-F2 antibodies and displayed
reaction products significantly smaller than F1.2 when probed with
a polyclonal anti-prethrombin-l antibody.
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3435
BLOOD CLOTTING IN MINIMALLY ALTERED WHOLE BLOOD
Thrombin-AT-III complex (TAT) was measured in the fluid phase
samples using the Enzygnost TAT microimmunoassay kit from Behring according to manufacturer's instructions. For samples after clot
formation, samples were diluted up to 111,OOO in normal plasma to
give values within the standard curve supplied with the assay. Values
in diluted samples were corrected for the TAT concentration of 2.76
ng/L in the normal plasma used for the dilution.
Kinetic analyses. The thrombin concentration at any time was
estimated from densitometry of the thrombin B-chain and by the
rate of FPA generation. The progression of FPA concentration in
fluid phase over the first 7 minutes was fit to a fourth degree polynomial using the program Microsoft Excel version 5. The cleavage of
fibrinogen by thrombin has been reported by Mihalyis5 to follow
first-order kinetics at all concentrations of fibrinogen. This empirical
process can be described mathematically by product inhibition with
a Ki equal to K,. Therefore, thrombin concentrations were calculated
using the following Michaelis-Menten expression that accounts for
competitive product inhibition:
where [E], is the thrombin concentration at time t , [PI is the concentration of free inhibitory product, and [SI is the concentration of free
substrate. The inhibitory product is reported to be the a-chain of
fibrin I, which is essentially equivalent in concentration to P A ? '
The free product concentration [PI was taken as the total product
concentration (FPA concentration) at time t, because the thrombin
concentration is less than 1.0%of product concentration over the
range of fibrinogen cleavage considered. Similarly, [SI is taken as
the fibrinogen Aa-chain concentration (total fibrinogen minus FPA)
at time t. The data were analyzed to the point of 90% FPA release.
The constants K, = 7.2 pmoVL and ka,= 84 s-' were used as
reported for human a-thrombin and human fibrinogen by Higgins
et a1.s6 The K,was made equivalent to K, for the purposes of these
calculations and in accordance with Mihal~i.'~
It has been reported
by other investigators using this assay that FPA levels go to a maximum and then are seen to decrease?' We observed a similar phenomenon using the Asserachrom FPA assay (American Bioproducts).
For kinetic analyses, we have Calculated molar concentration of FPA
generation using the maximum FPA level from the generation curve.
Estimates of prothrombinase concentration were calculated from
the rates of thrombin B-chain production determined by densitometry
and by F1.2 and TAT complex formation rates determined by ELISAs. The rate of thrombin B-chain production at clot time was
estimated by a hyperbolic fit of the densitometry data between 180
and 420 seconds with Stanford Graphics version 3 (Visual Numerics,
Houston, TX). The maximum rate of B-chain production was estimated by point-to-point interpolation between 390 and 420 seconds
performed with Stanford Graphics version 3. The prothrombin concentration was calculated using the following form of the MichaelisMenten equation:
where [E], equals the prothrombinase concentration at time t and
[SI equals the free prothrombin Concentration. Initial prothrombin
concentration was measured by densitometry of Westem blots. Prothrombin at time t was estimated by subtracting the thrombin Bchain concentration at time t from the initial prothrombin concentration. The [SI was taken as the total prothrombin at time t since
prothrombinase was less than 0.1%of the prothrombin concentration
over the course of the reaction analyzed. The constants K, = 0.82
pmol/L and kat= 19 s-' were used as reported for prorhrombinase
at 22" on platelets by Tracy et aL9
Table 1. Effect of Corn Trypsin Inhibitor on Prothrombin Time,
Diluted Thromboplastin Time, and Activated Partial
Thromboplastin Time Assays
Clot Time (sl
Prothrombin time
Normal plasma
20 pg/mL CTI
100 pg/mL CTI
Diluted thromboplastin time*
Normal plasma
20 pg/mL CTI
100 pS/mL CTI
Activated partial thromboplastin time
Normal plasma
20 pg/mL CTI
100 pg/mL CTI
14.8 t 0.4
15.3 t 0.1
15.3 t 0.2
80.4 t 1.7
84.7 t 2.3
83.4 t 5.3
46.6 t 1.5
89.5 t 1.6
124.0 t 1.9
Values are means t SD.
Abbreviation: CTI, corn trypsin inhibitor.
*Thromboplastin was used at a 1/1,000 dilution.
RESULTS
The effect of CTI on the plasma prothrombin time and
the activated partial thromboplastin time were evaluated.
Biggs and Nosse15' demonstrated that at dilute thromboplastin concentrations the plasma clot time becomes dependent
on factor IXaNIIIa activity. Therefore, dilute thromboplastin time assays were performed with a 1/1,000 dilution of
thromboplastin and carried out in polystyrene tubes. A pool
of citrated plasma from 6 normal donors was used. CTI was
added to final concentrations of 20 and 100 pg/mL, and the
plasma was incubated o n ice for 10 minutes before assay.
The results are seen in Table 1. N o significant prolongation
of the prothrombin time and dilute thromboplastin time was
observed, demonstrating that CTI has n o effect on thrombin,
factor Xa, factor VIIa, and factor IXa. The activated partial
thromboplastin time is significantly prolonged by increasing
amounts of CTI, indicating inhibition of the contact pathway.
These observations are consistent with previous reports of
CTI specificity for factor XIIa.*'
Blood with no additions added to polystyrene tubes clotted
between 7 and 10 minutes for all subjects. Anti-tissue factor
antibody 5G9 was used to examine the contribution of the
tissue factor-initiated reaction to this spontaneous clotting
process. This antibody a t 10 pg1mL did not prolong whole
blood clot time. Because this concentration of the antibody
has been previously shown to neutralize tissue factor-dependent clotting in vivo,6*"' the spontaneous clotting process
was most likely contact mediated. Increasing concentrations
of CTI added to whole blood gave a dose-dependent prolongation of clot time. For subject BT, prolongation of clot time
from 8.5 minutes to greater than 25 minutes was achieved
at 32 pg CTUmL of plasma. Increasing amounts of the inhibitor did not further extend this interval.
The addition of tissue factor-PCPS to CTI-inhibited whole
blood produced a concentration-dependent decrease in clot
time, most likely, as a result of complexation of the added
tissue factor with factor VIIa already present in the blood.12
The addition of 40 pmol/L TFl80 nmol/L PCPS reduced the
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3436
clot time from 23 to 4.5 minutes. Eighty nmol/L PCPS alone
added to CTI-inhibited blood (platelet count = 1.9 to 3.4 x
108/mL blood) had negligible (<10 seconds) influence on
the clot time. In range-finding experiments increasing the TF
concentrations by fivefold from 60 to 300 pmoVL produced a
1S-fold decrease in clot time.
Initial experiments performed with blood without added
CTI gave end products of factor V and prothrombin activation that were identical in apparent mobility as those observed in the presence of CTI and TF-PCPS. The SDS and
Triton X-100 extracts of cellular-clot (solid) phase material
contained significantly less prothrombin and thrombin than
that observed in the fluid phase when normalized to the total
blood volume and no enrichment of reactants or products,
indicating that no significant irreversible compartmentalization of these products to the insoluble phase (excepting fibrin) occurred.
Whole blood clotting. The product-precursor data illustrated are for a single experiment performed with a subject
identified as BT and are representative of results seen for 8
different normal subjects in 14 experiments. All data are
expressed as concentrations in the plasma volume. Subject
BT had a hematocrit of 40%, normal prothrombin time (12.6
seconds) and activated partial thromboplastin time (29 seconds), a platelet count of 1.9 X 10' plateletslml, and a
fibrinogen concentration of 2.41 mg/mL (7.09 pmoVL).
Figure 1 displays the temporal processes of product formation in CTI-inhibited blood initiated to clot with 40 p m o K
tissue factor and 80 n m o m PCPS. In the experiment illustrated, SDS-PAGE gels and ELISA analyses were performed
on samples taken at 30-second intervals for the first 8 minutes. Longer intervals were selected for later time points
extending to 1 hour. The clot time (indicated by the arrow
in lane 10) occurred at 4.5 minutes.
Figure 1 displays transverse sections, obtained from multiple immunoblot analyses from individual gel analyses for
the analytes fibrinogen, factor V, factor Va heavy chain,
factor Va light chain, prothrombidthrombin, antithrombin
111, and platelet factor 4. The immunoreactive products are
identified explicitly when the electrophoretic mobility corresponds to that of a known product expected from the reactant. When there is uncertainty with respect to the identification of a reaction product (apart from its immunoreactivity
and its appearance over time), either lower case letters (a,
b, c, or d e ) or a prime (') are used to identify the reaction
products.
In addition to the quantitation of the analytes displayed
in Fig 1, samples taken from the same aliquots were analyzed
using commercial assays for FPA, TAT, and prothrombin
fragment 1.2 (with the latter values corrected as described
in Materials and Methods).
Fibrinogen cleavage and removal from the fluid phase.
The initial products of thrombin cleavage of fibrinogen are
Fibrinogen and I T A are soluFPA and fibrin I m~nomer.*'~*~
ble components that are readily detectable in the fluid phase
of blood. Fig 1A demonstrates fibrinogen in the fluid phase
of clotting blood at various time points. Fibrinogen is clearly
observed (M, = 340,000) for the first 4 minutes, before clot
time. Densitometry of this blot (Fig 2) reveals that at clot
RAND ET AL
time (4.5 minutes), 80% of the fibrinogen is depleted from
and does not reequilibrate with, the fluid phase, suggesting
that it is incorporated into the soluble clot. The appearance
of FPA in the fluid phase increases rapidly, reaching an
apparent maximum of 21 pmoVL at 7.0 minutes. This maximum is followed by a decrease to 15 pmol/L. These latter
values remain essentially unchanged (Fig 2). These observations suggest that either the FPA product initially formed is
more immunoreactive than the stable final product or that
there is some artifact associated with the assay itself. Treatment of blood from subject BT with 0.8 pmol/L a-thrombin
for 5 minutes resulted in a measured value of 15.2 pmol/L
FPA in the fluid phase. Additional incubation of the thrombin
treated blood at 37°C for 10 minutes also resulted in a measured value of 15.2 pmollf, FPA in the fluid phase. However,
prolonged incubation of serum has been reported to give an
apparent reduction in FPA values as determined with this
assay:' consistent with the initial FPA product being more
immunoreactive. At clot time (4.5 minutes), 9.5 pmoVL FPA
is observed, which corresponds to 45% of the apparent maximum in FPA (21 pmollf,). If one uses the stable value of
FPA (15.2 pmoVL), the conclusion would be that 6.9 pmoll
L FPA is released at clot time, or 45.5%. In either case it
appears that substantial amounts of intact fibrinogen may
be incorporated into the initial clot, regardless of the FPA
uncertainty.
Factor V activation. Factor V and activation products
were detected by two monoclonal antibodies: one reactive
toward residues 307 to 506 on the heavy chain (anti-HFV6) and the other raised against the light chain of factor Va
(anti-HFV-9). Anti-HFV-6 has also been shown to react
with platelet-derived factor V, meaning that the observed
immunoreactivity by this antibody is the sum of plasma and
platelet-derived heavy chain.G2The concentration of factor
V contained in platelets is described to be approximately
20% of the blood concentration. Therefore, the observed
bands of factor Va heavy and light chain may consist of
20% of platelet-derived factor V after maximum platelet
release (7 minutes). Staining with aHFV-6 after resolution
on a nonreducing gel is seen in Fig 1B; factor V is observed
migrating at M, = 330,000 and rapidly disappears over the
first 5 minutes, indicating near complete cleavage of factor
V at clot time (FV, Fig lB, lanes 1 through 11). The concomitant appearance of the heavy chain is observed migrating
as a doublet at M, = 105,000/103,000 (FVaHc, Fig lC, lanes
5 through 30). This doublet is unexpected from studies in
purified systems and is observed to resolve as a single band
under reducing conditions (not shown). The heavy-chain
doublet is observed to be reduced in intensity from 10 to 60
minutes. The disappearance of the M, = 105,000 band precedes the M, = 103,000 band, suggesting the M, = 103,000
product is more resistant to degradation. Other immunoreactive bands are observed at M, = 70,000 (a, Fig lC, lanes 18
through 30), M, = 45,000 (b, Fig lC, lanes 22 through 30),
and M, = 30,000 (c, Fig lC, lanes 25 through 30). The M,
= 70,000 and M, = 30,000 bands migrate identically with
the previously described activated protein C degradation
products of factor Va heavy chain resulting from the inactivating cleavages at Arg,,, and Arg,,,, respectively."' The M,
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BLOOD CLOTTING IN MINIMALLY ALTERED WHOLE BLOOD
4
3437
Minutes
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 9 10 12 14 16 18 20 25 30 35 40 50 60
A
- Fgn
B
- FV
- FVa,
-a
C
-b
-C
D
Tla
E
TAT
II
.-.
TAT‘
-- Pr
II
TAT
-- dle
ATlll
F
*
G
----
H
1 2
3
4
5
6
--
PF,
7 8 9 10 11 12 1314 1516 1718 19 20 21 22 23 24 25 26 27 28 2930
Fig 1. Composite Western blots of fibrinogen, factor V, prothrombin, antithrombin 111 (AT-Ill), and platelet factor 4 in clotting blood.
Transverse sections of immunoblots from individual gels for various analytes separated on electrophoresis gels prepared with bloodlserum
samples, obtained from individual BT and subjected t o tissue factor-initiated coagulation in corn trypsin inhibitor-inhibked whole blood.
Gels on the same samples taken sequentially during the course of the coagulation process were run under both reducing and nonreducing
condkions, transferred, and probed with the appropriate monoclonal or polyclonal antibody. Fibrinogen, factor V and factor Va light and heavy
chains, and PF4 are analyzed under nonreducing conditions. Prothrombin and AT-Ill were analyzed under reducing conditions. At the top of
the figure are indicated the time intervals at which the sample was obtained, while the bottom legend indicates the lane number on the
electrophoresis gel. Clot time (4.5 minutes) is indicated by bold arrow. The letters A through H on the left margin correspond t o various
transverse gel sections corresponding t o different analytes (see text), while the legend on the right side of the gel corresponds t o the various
analytes. The analytes labeled a, b, c, Tla, (TAT’), II, and dle are not yet positively identified.
= 45,000 band most likely corresponds to the product of
initial APC cleavage at Arg,,, or might be the result of
factor Xa or plasmin cleavage at Arg,, and Arg,, in the
factor Va heavy chain. However, this fragment is not well
resolved because of the presence of plasma albumin, which
interferes in the molecular weight (M,) range between M,=
45,000 and 50,000 on nonreduced gels. Staining with antiHFV-9 shows the light chain migrating as the expected doublet at M, = 74,000 to 72,000 (FVaLc, Fig ID, lanes 8
through 30). This product is produced at a slower rate when
compared with the heavy chain.
A quantitative display of the appearance and disappearance of factor V and its reaction products obtained by densitometric analyses of the immunoblots of Fig 1B through D
is presented in Fig 3. Factor V ( 0 )is observed to disappear
within the first 5 minutes of the reaction with a concomitant
appearance of the heavy chain (0).At late time intervals
(beyond 8 minutes) a gradual decrease in factor Va heavychain concentration occurs with the appearance of the fragments “a,” “b,” and “c.” In contrast the light chain (X)
is produced at a much slower rate and becomes the limiting
factor with respect to complete activation of factor V. An
inspection of light- and heavy-chain concentrations at any
point in the reaction provides the absolute concentration of
factor Va available to form prorhrombinase. The factor Va
heavy chain reaches 65% of maximum at 4.5 minutes (clot
time) compared with only 10% of the maximum level
achieved for light chain (Fig 3). Therefore, at a normal factor
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3438
RAND ET AL
1
stable, high molecular weight components represent less than
2% of the thrombin B-chain density observed at 60 minutes;
however, their intensities may underrepresent the concentrations of these inhibited thrombin species because of nonidentical reactivities of the antibody with thrombin that is present
in these complexes.
The thrombin B-chain density was quantitated by comparison to a standard density curve derived from known amounts
of purified a-thrombin run on the same gel. From the blot
data the thrombin B-chain can first be detected at 2.5 minutes
and reaches a maximum of 360 n m o m at 60 minutes (Fig
4). The thrombin B-chain estimated at clot time (4.5 minutes)
is 15 nmoVL (inset, Fig 4). A curve representing the thrombin concentration calculated from the rates of FPA generation (Fig 2, see Materials and Methods) is also presented in
Fig 4 (inset) and gives 2.5 nmol/L thrombin at clot time.
Based on these analyses, only 15% of the thrombin produced
at clot time is acting on fibrinogen. By either determination,
less than 2% of the initial prothrombin (1.24 pmol/L, see
below) is converted to a-thrombin (or meizothrombins) at
clot time.
By densitometry of a less exposed film and comparison
to standards, prothrombin is estimated at 1.24 pmoVL at
time zero and is reduced to 0.3 pmoVL at 60 minutes (Fig
lE, lane 30) corresponding to 76% prothrombin cleavage
over the entire course of the reaction. The thrombin B-chain,
360 nmol/L at 60 minutes (Fig lF, lane 30), represents 29%
of the initial prothrombin concentration. The M, = 36,000
band, representing prethrombin 2 (Pre 2, Fig lF), reaches a
maximum density at 25 minutes, which does not decrease
over 60 minutes. Assuming equivalent reactivity of the antibody with prethrombin 2 and thrombin B-chain, 440 nmol/
L prethrombin 2 is formed at 60 minutes, which accounts
0.8
-
f
0.6
E.
5
0.4
%
G
0.2
0
0
:
1
2
3
4
5
6
7
I
*
8
9
0
10
the (minutes)
Fig 2. Generation of fibrinopeptide A and depletion of fibrinogen.
Fibrinogen cleavage was monitored by fibrinopeptide A (FPA) levels
(0)
and fibrinogen levels (0)in the solution phase of clotting blood.
FibrinopeptideA was determined by ELSA assay and fibrinogen was
determined by densitometry as described in Materials and Methods.
At clot time, indicated by the bold arrow, FPA is 9.5 pmol/L, representing 45% of meximum (21 pmol/L). At this time, fibrinogen is
approximately 80% depleted from solution.
V concentration (20 nmomM)factor Va is 2 n m o m at clot
time.
Prothrombin activation and thrombin generation. A
polyclonal antiserum raised against human a-thrombin was
employed to detect prothrombin activation products. Prothrombin, prethrombin 1, prethrombin 2, a-thrombin, and
thrombin-inhibitor complexes are recognized by this antibody. Fig 1E and F show the migration of anti-a-thrombin
immunoreactive products under reducing conditions. Plasma
prothrombin is observed at M, = 72,000 and migrates identically with purified prothrombin (11, Fig lE, lanes 1 through
30). Activation products can be seen at 3.5 minutes (Fig lF,
lane 8) and consist of a band at M, = 36,000 and a band at
M, = 30,000. The M, = 30,000 band migrates identically
with a-thrombin B-chain (IIaB, Fig 3, lanes 8 through 30).
The M, = 36,000 band is deduced to be prethrombin 2 (Pre
2, residues 274 to 579 in prothrombin) because it displays
epitopes for the a-thrombin antibody, migrates identically
with prethrombin 2 and is M, = 6,000 larger than the Bchain, a difference accounted for by the size of the A-chain
of a-thrombin and previously shown to be prethrombin 2.
Other reactive bands of lesser intensity are seen at M, =
97,000 and M, = 93,000 (bands TAT, Fig lE, lanes 19
through 30) and a band at M, = 135,000 (band TIa, Fig lE,
lanes 17 through 30). The M, = 93,000 band (TAT’) migrates
identically with purified covalent thrombin-AT-I11complex.
The M, = 97,000 band appears at approximately the same
time and with the same intensity as the M, = 93,000 band.
This product has similar mobility to that reported for the
thrombin-heparin cofactor I1 ~ o m p l e xThe
. ~ band accumulating at M, = 135,000 (TIa) remains unidentified but is, most
likely, an SDS-stable thrombin-inhibitor complex. A reactive
band (TAT’) is observed at M, = 69,000, which appears at
12 minutes and remains up to 60 minutes. This component
represents an SDS-stable thrombin-AT-I11 complex fragment
(Fig IE, lanes 20 through 30).65 Collectively these SDS-
1
08
0.6
a
0
0.4
02
0
I
time (minutes)
Fig 3. Quantitation of factor V, factor Va heavy and light chains,
and platelet factor 4. Factor V (01,factor Va heavy chain (0)were
measured by densitometry of the blot in Fig 5B. Light chain ( x was
measured from the blot in Fig 5C. Values are expressed as percent
maximum density for each chain observed over the @minute reaction time course. At clot time (bold arrow, 4.5 minutes) the light
chain is approximately 10% of maximum, whereas the heavy chain
is 65% of maximum. The decrease in the heavy-chaind d t y is seen
to plateau at approximately 50% of maximum after Bo minutes. Platelet factor 4 ( + ) was determined by densitometry of Fig 1H. At clot
time (bold arrow, 4.5 minutes) PF4 reaches approximately 50% of the
maximum.
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3439
BLOOD CLOTTING IN MINIMALLY ALTERED WHOLE BLOOD
400
Fig 4. Concentration of thrombin B-chain in the fluid phase of
clotting blood. Thrombin Bchain was quantitated by densitometry as described in Materials and Methods and is seen t o
appear rapidly after an initial lag
phase. The B-chain concentration (A)reaches a maximum of
360 nmol/L at 60 minutes. The
inset shows the relative concentration of B-chain calculated
from rates of FPA generation
and by densitometry (m) over
the first 350 seconds. Clot time
(4.5 minutes) is indicated by the
bold arrow.
A
t
O
20
lo
-1
700
600
500
F 400
300
200
100
0
0
5
10
15
20
25
30
35
minutes
Fig 5. Quantitative analyses of the generation of prothrombinrelated products by immunoblot and commercial ELISA assays. The
molar concentrations of fragments v time are plotted. 0,ELISA values of fragment 1.2; V,ELISA values for TAT; 0, immunoblot analysis
of prethrombin 2;
concentration of thrombin B-chain at any point
in time; A, the sum of the immunoblot data for prethrombin 2 plus
thrombin B-chain.
+,
40
50
60
time (minutes)
for 36% of the initial prothrombin. All together, the thrombin
B-chain (a-thrombin and meizothrombin) and prethrombin
2 in the fluid phase account for 65% of the initial prothrombin. Thus, of the 76% of initial prothrombin cleaved, 11%
is not accounted for by these identifiable products and is
likely dispersed among the uncharacterized components and
SDS-stable inhibitor complexes. In addition, some reaction
products may be obscured by serum proteins.
Figure 5 displays the progress curves for formation of
800
30
thrombin B-chain and prethrombin 2 deduced from densitometry of Western blots and the progress curves determined
by the ELISA for TAT and F1.2 (corrected, see Materials
and Methods) determined on nondenatured samples. TAT
concentration levels are slightly lower than thrombin B-chain
levels, indicating that most of the thrombin represented by
the immunoblot corresponds to inhibited thrombin present
as SDS-unstable complex(es) with AT-111. The sum of the
profiles of prethrombin 2 and B-chain concentrations (Fig
5, A) is initially similar to the F1.2 levels determined by
ELISA; these diverge from approximately 8 to 20 minutes
and then become almost equivalent at longer time intervals.
Immunoblots conducted with a monoclonal anti-prothrombin
fragment 2 antibody are largely consistent with these corrected F1.2 ELISA data (data not shown). In general F1.2
levels are similar to the sum of thrombin B-chain plus prethrombin 2 concentrations over most of the course of the
reaction. The observations with respect to F1.2, however,
must be tempered by the fact that a large correction was
required for the commercial assay and the kit standard
“F1.2” is electrophoretically quite different from genuine
F1.2 (see Materials and Methods). Collectively, these data
suggest that the excess prothrombin products relative to the
prothrombin F1.2 produced during the early phase of the
reaction are accounted for as other prothrombin fragments
(see for example II(x) in Fig LF) and stable inhibitor complexes other than unmodified AT-111. Other SDS-stable covalent thrombin-inhibitor complex products are identified by
the products TIa, TAT’, and possibly II(x) (Fig 1E).
AT-III complex formation. On reduced gels, AT-I11 is
seen to migrate at M, = 58,000 (ATIII, Fig 1G). Upon extended exposure of the blot, an M, = 93,000 band is first
seen at 5.5 minutes; this band reaches a plateau of density
at time points between 30 and 60 minutes (TAT, Fig lE,
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3440
lanes 26 through 30). This band migrates identically to the
thrombin-AT-I11complex (TAT) and with the M, = 93,000
band seen with anti-a-thrombin staining (Fig 1E) and is
thus deduced to be TAT.h' This band represents less than
0.1% of the density of the initial AT-111, indicating less than
5 nmoln of SDS-stable TAT complex is formed. Far less
SDS-stable TAT is formed than had been anticipated, especially when considering approximately 360 nmol/L thrombin
B-chain has been generated. TAT complexes formed in vitro
using purified AT-I11 and thrombin are stable in the analytical gel system used unless an agent such as hydroxylamine
is used to hydrolyze the acylenzyme-inhibitor bond. Thus,
the observation that significant amounts of thrombin B-chain
are liberated upon denaturation with SDS leads unalterably
to the conclusion that the complex relevant to the blood
clotting process is not stable under the denaturing conditions
used: 9 5 T , 1% SDS, (see Materials and Methods). In contrast to the limited amounts of SDS-stable covalent thrombin-AT-111 complexes observed on the immunoblots, it is
apparent that SDS-dissociable TAT complexes are formed
at levels almost equivalent to the thrombin B-chain formed
(see Fig 5).
Other stable AT-I11 immunoreactive complexes are observed at M, = 76,000 (d, Fig 1G) and 74,000 (e, Fig 1G)
and represent either cleaved forms of thrombin-AT-I11 or
AT-111 complexes with other enzymes. It should again be
mentioned that the concentrations of these complexes may
be underestimated because of nonequivalent reactivity of the
antibody with the complexed-AT-111products; however, this
conclusion is less likely with polyclonal antibodies, which
are reactive with multiple epitopes.
Platelet factor 4 retease. The a-granule protein, PF4, is
a soluble component readily detectable in the fluid phase of
clotting blood and is used to serve as an indicator of platelet
a c t i v a t i ~ n . ~ Fig
~ . ~ ' IH shows the appearance of an M, =
8,300 band over time that migrates identically with purified
PF4. Quantitation by densitometry demonstrates a rapid increase of PF4, which reaches a maximum density at 12 minutes (Fig 3, +). Comparison with standard amounts of PF4
gives approximately 4.4 ,ug/mL in the fluid phase of blood
at the maximum, a value consistent with quantitative release
of PF4 and, hence, complete platelet activation. At clot time
(4.5 minutes) approximately 50% of the maximum PF4 is
released. This is consistent with either complete activation
of 50% of the platelet population or the release of 50% of
the PF4 from the whole platelet population, which has been
partially activated. The time-dependent release of PF4 shares
a profile similar to that observed for FPA (Fig 2, 0) but is
displaced in time. In the interval of 12 to 20 minutes PF4
is observed to decrease, reaching a final value of 70% of
maximum, which persists through 60 minutes. Previous studies with purified intact platelets give estimates of 2,700 prothrombinase sites per intact activated platelet'; thus the potential concentration of platelet sites available to support
prothrombinase can be estimated. Subject BT with 1.9 X
lo8 platelets/mL of blood, or approximately 3.2 X 10* platelets/mL plasma, has a minimum of 4.3 X 10" prothrombinase siteslmL (or -700 pmol/L) (assuming 50% platelet site
activation, Fig 3).
RAND ET AL
18
14.4
-3
loa
7.2
5
3.6
0
2
4
6
8
1
0
1
2
1
4
0
time (minutes)
zok
0-
time (minutes)
Fig 6. Prothrombinase expression. (A) The molar concentration
of platelet prothrombinase sites (0) (left axis) anticipated to be
formed as a consequence of platelet activation is plotted v time.
The molar concentration of factor Va interpreted from the limiting
concentrations of intact heavy chain and light chain (Wis plotted v
time. Clot time is at 4.5 minutes. (B) The molar concentration of
prothrombinase inferred from the generation of thrombin B-chain
(illustrated in the fitted curve of Fig 4) is plotted vtime. The kinetics
used for calculation are appropriate for platelet bound prothrombinase (see Materials and Methods) at 22°C. On synthetic phospholipids,,k increases approximately 2.78-fold in going from 25" to 37°C.
Thus, the prothrombinase concentrations are overestimates.
Prothrombinase activity. Estimates of prothrombinase
concentration were calculated from the rate of thrombin Bchain generation using the Michaelis-Menten expression described in the Materials and Methods section. These data are
plotted in Fig 6 together with data on the concentration of
fully activated factor Va, deduced from the limiting (heavy
chain or light chain) component, and the molar concentration
of prothrombinase sites available on intact platelets, based
upon the extent of platelet activation (PF4 release, see Fig
3). Prothrombinase reaches approximately 7 pmoVL at clot
time (4.5 minutes) and reaches a maximum of 155 pmoV
L at 7 minutes. Prothrombinase activity is then quenched,
becoming approximately 10 pmoYL by 12 minutes. This loss
of activity is not due to depletion of substrate, which is still
at almost 300 nmoVL after 60 minutes (see Fig 4) nor is it
a consequence of APC inactivation of factor Va, which is
present at 17 nmol/L (see Fig 3), nor is it due to unavailability of prothrombinase sites on activated platelets,
1.4
nmol/L. Because neither platelet prothrombinase sites nor
-
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BLOOD CLOTTING IN MINIMALLY ALTERED WHOLE BLOOD
Table 2.
Concentration
at Clot Time
Component
(4.5 min)
Factor Va
Platelet prothrombinase sites
Prothrombinase
Thrombin
Thrombin-AT-Ill§
2
700
7
15
nmol/L*
pmol/Lt
pmol/L
nmol/L
ND
Maximum
Observed
16 nmol/L*
1,400 pmol/Lt
155 pmol/L
360 nmol/L*
<5 nmol/L
Abbreviations: ND, not detectable; SDS-PAGE, sodium dodecyl SUIfate-polyacrylamide gel electrophoresis.
*Assuming M, = 74/72,000, light chain as the limiting component
and a normal (20 nmol/L) factor V concentration (50).
t Assuming 2,700 sites/platelet (45).
Based on thrombin B-chain concentration.
§ Covalent complex on SDS-PAGE.
*
factor Va is limiting, the prothrombinase concentration must
be limited by the active factor Xa concentration. These data
suggest that the termination of prothrombinase activity is
primarily a consequence of the inhibition of factor Xa. In
addition, the data are consistent with the conclusion that
factor Xa production by factor VIIIa factor IXa and factor
VIIa tissue factor have already been terminated at this point
in the reaction. This determination suggests that, assuming
a normal plasma concentration of factor X (170 nmol/L),68
the active factor Xa participating in prothrombinase at clot
time represents less than 0.004% of the zymogen and does
not exceed 0.09% at the maximum. As the reaction progresses between 30 and 60 minutes, the rate of thrombin Bchain production diminishes to levels below that observed
at clot time. This reduced rate occurs despite substantial
remaining prothrombin substrate (11, Fig 3, lanes 26 through
30) and is therefore consistent with the almost complete
downregulation of prothrombinase activity. This downregulation most likely occurs through direct inhibition of prothrombinase as well as the intrinsic and extrinsic tenase
complexes.
DISCUSSION
In the model presented the coagulation process is studied
in a system approaching native blood (in vitro) and thus,
permits the investigation of reactions under near physiological conditions. Because all blood components are present in
the system, the observations made with this model may be
compared with those made using purified in vitro systems to
define areas of consistency and incongruency and, therefore,
identify molecules or mechanisms in need of further evaluation. The model has the following advantages: (1) the influence of contact activation is eliminated by specific inhibition
of factor XIIa, ( 2 ) the reaction mixture is prealiquoted for
consistent sampling independent of physical state, and (3)
analyses of the reactants and products by quantitative Westem blotting and ELISA provide the physical state and quantitation of the proteins during various stages of the coagulation
process.
Clot time serves as a relevant point of reference and is
commonly referred to in the following discussion. A quantitative comparison of the degrees of coagulation factor activation at the time of clot formation is summarized in Table 2.
3441
The evaluation of the interval after clot formation is useful
in defining ongoing events and identifying stable end products of the reaction. The results are best used in comparative
evaluations of the temporal relationships among reactants
(Fig 1) when blood clots.
Concurrent analyses of the cleavage of fibrinogen, factor
V, prothrombin, platelet activation, and formation of thrombin-inhibitor complexes lead to the following conclusions:
1. Significant amounts of fibrinogen are incorporated into
the insoluble clot initially formed at clot time.
2. Most thrombin is formed subsequent to clot time and
25% of prothrombin remains uncleaved. Nearly equal
amounts of prethrombin 2 and thrombin are ultimately
formed.
3. The thrombin produced is nearly accounted for by the
formation of TAT complex as measured by native
ELISA. However, most thrombin is not found in SDSstable covalent complexes with AT-I11 or other inhibitors.
4. Factor Va heavy chain is quantitatively generated by
clot time, whereas factor Va light chain is generated
at a slower rate than the heavy chain with only 10%
of the possible light chain accounted for at clot time.
5. At clot time approximately 50% of the platelet a-granule stores have been released.
6 . Prothrombinase is approximately 7 pmoVL at clot time
and reaches approximately 155 pmoVL at maximum.
Prothrombinase activity is eventually inhibited after
cleavage of about 75% of the prothrombin.
7. The clot occurs after generation of approximately 15
nmoVL thrombin B-chain or about 1% to 2% of prothrombin activation. However, only 2.5 nmoVL of the
thrombin generated in blood at clot time is acting on
fibrinogen, suggesting that the major fraction of thrombin activity is occupied by other substrates and inhibitors present in plasma.
8. Significant amounts of prethrombin 2 are produced as
a stable product either by cleavage of prothrombin at
Arg2,, by factor Xa or at Arg,,, by thrombin or by
leading to the formation of significantly more
prothrombin fragment 1.2 than thrombin or TAT complex.
9. The degradation of the factor Va heavy chain gives
rise to activated protein C-like digestion products suggesting either the presence of this enzyme, either because of its constitutive presence or its activation in
the time subsequent to clot f~rmation.~"
We have previously reported the activations of the procoagulation factors presumed essential in a mixture composed
of purified components at plasma concentrations system.38
This system used purified human prothrombin, factors X,
IX, and V, recombinant human factor VIII, and PCPS vesicles. The final addition of factor VIIa and relipidated recombinant human tissue factor to the mixture served to initiate
the reaction. A comparative analysis of this previously described system and the current whole blood model is instructive in defining the mechanisms of the numerous reactions
involved.
As in the present whole blood system, the purified system
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3442
RAND
displays a factor VIIa-tissue factor concentration dependent
lag in thrombin generation. Subsequently, prothrombin is
observed to be consumed with the ultimate rate of thrombin
propagation varying only fivefold over three orders of magnitude of factor VIIa-tissue factor used.3s In the present
whole blood experiments, performed over a fivefold range
in TF concentration (60 to 300 pmol/L), the only observed
difference in the generation of thrombin was a difference in
lag time, whereas the maximal rates of B-chain production
observed were virtually identical. Two recent studies from
our laboratory have compared the influence of AT-111, TFPI,
protein C, and thrombomodulin added to the synthetic mixture of procoagulants when the latter are at normal physiological con~entration.~’,~
These studies display many but not
all of the qualities present in the whole blood system. In
particular these studies emphasize how the factor VIIa
tissue factor pathway and TFPI influence the lag time of the
reaction. In contrast, the influence of the activated protein
C system is principally on the rate of the propagation phase
whereas in the purified system AT-I11 appears to provide
mainly a scavenger function, suppressing enzyme products
subsequent to the coagulation related events.
The available sites for prothrombinase formation in both
purified and whole blood systems are in excess (see Table
2 and Fig 6) and thus, not likely to be limiting. It appears
that the concentration of fully activated factor Va (M, =
105,000 heavy chain, M, = 74,000 light chain) is well in
excess of the prothrombinase formed. Thus, it follows that
factor Xa formation is the limiting component in prothrombinase generation in the whole blood system. Yet, as mentioned above, the thrombin generation rates observed during
the propagation phase vary little in response to a broad range
of factor VIIa tissue factor in the purified system, suggesting
that the extrinsic tenase, factor VIIa tissue factor does not
significantly influence the ultimate level of factor X activation. This implies that the intrinsic tenase (factor IXa * factor
VIIIa) is the major contributor of factor Xa in both systems.
It should be appreciated from the present system that
picomolar concentrations (or less) of factor IXa factor
VIIIa complex are all that is required for the factor Xa generation observed to be associated with clot formation.
By Western blotting it was shown previously that activation of the various zymogens and cofactors produced no
unexpected major products in the purified systems3*In contrast, as discussed above, prethrombin 2 represents a major
prothrombin cleavage product not predicted from purified in
vitro analyses. Prethrombin 2-like product(s) are formed by
a single cleavage of prothrombin at Arg,,, by factor Xa or
at Argzg, by thrombin or both. It follows that prethrombin 2
could arise in whole blood by three possible mechanisms.
Free factor Xa acting in solution could cleave at Arg,,,. This
reaction is unlikely since factor Va and platelet prorhrombinase sites are in excess of prothrombinase; hence, nearly all
the factor Xa would be in this complex. Alternatively, the
relative rates of cleavage at Arg,,, and Arg,,,, in prothrombin
by prothrombinase could be altered in the whole blood environment. A precedent for this exists in the observations of
Kung et a17’ in which PCPS vesicles containing saturated
acyl side chains gave rise to near equal proportions of pre-
-
ET AL
thrombin 2 and a-thrombin. Thus, the composition of the
natural cell membranes present in whole blood may support
a similar change in specificity of prothrombinase. Third, athrombin or meizothrombin produced during the reaction
could perform feedback cleavage at kg284 in prothrombin.
Although initial cleavage at Arg,,, by a-thrombin is preferential in vitro,69the whole blood conditions may favorably
influence the susceptibility of the ArgZg4thrombin cleavage
site in prothrombin. Although these three mechanisms remain a possibility, further experimentation in this system and
in purified systems is warranted to resolve the mechanisms
directing these observed cleavage products of prothrombin.
In either case, prethrombin 2 has no known enzymatic activity or biological activation, other than as a potential intermediate in a-thrombin formation. These tentative conclusions
are supported by previously reported indirect evidence for
prethrombin 2 formation in blood and plasma. Rabiet et
a1” reported generation of “prothrombin fragment 1.2.3’’
(residues 1 to 284) in clotting plasma that would potentially
generate prethrombin 2 as the other product. In addition,
Teitel et a173reported that the rate and end point level of FI .2
formation exceeds that of TAT formation in spontaneously
coagulating blood. These authors conclude that F1.2 is produced without corresponding thrombin activity and therefore, a significant amount of stable prethrombin 2 must be
formed in clotting blood. In addition, the present study demonstrates that SDS-stable TAT complexes represent only a
small fraction of the thrombin B-chain that is ultimately
generated. As a whole, the above conclusions and the present
observations should caution the researcher in using the F1.2
and TAT determinations as a direct index of thrombin activity.
Other prothrombin cleavage products not yet identified
have been observed in the present studies. These include
a potential thrombin-heparin cofactor I1 complex, which is
formed almost equivalently with the TAT SDS-stable complex. Other products requiring analysis include the product
II(x), which either represents an additional inhibitor-thrombin product or an additional cleavage product of prothrombin, and the high molecular weight inhibitor complex TIa.
We had anticipated that the covalent thrombin-AT-I11
(TAT) or modified complex (TAT‘) would be major products
based on previous work in plasma and purified systems in
vitr0.7~As seen in Fig 5 the ELISA for TAT suggests that
nearly all thrombin B-chain is accounted for by complex
with AT-111; however, SDS-stable TAT complexes account
for less than 2% of the thrombin B-chain and 0.1% of the
AT-111, suggesting that thrombin activity is not accounted
for by SDS-stable covalent complex formation with AT-111.
It is unlikely the complex is disrupted during analysis because it has been previously established to be stable to the
SDS gel reducing conditions used. Furthermore, the noncovalent thrombin-AT-I11complex has too weak an association
(& = 1.4 mmol/L) to account for thrombin inhibition in the
current system. The stable form of TAT complex observed
on an SDS gel is most likely the acylenzyme, which can be
dissociated into the products a-thrombin and modified AT111 by hydroxylamine.’* This complex must be preceded by
a tetrahedryl adduct between the enzyme and inhibitor,
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3443
BLOOD CLOTTING IN MINIMALLY ALTERED WHOLE BLOOD
which may not be stable in the SDS gel reducing conditions
used. It is therefore possible that the TAT tetrahedryl complex was generated and subsequently dissociated by the stopping reagents before the formation of the more stable inhibitor-acylenzyme complex. It is also important to consider that
the characterization of thrombin inhibition by AT-IT1 has
been conducted with a-thrombin almost exclusively. This is
despite evidence that a-thrombin and F1.2 associate tightly
(I(d = lo-’’) and are in complex when thrombin is generated
in situ.76Furthermore, fragment 2 has been reported to reduce the rate of TAT complex formation threef0ld.7~Therefore, the lack of SDS-stable TAT complex formation in
whole blood may be a result of a contribution of F1.2 to the
inhibition process. A reevaluation of AT-I11 interactions with
the various forms of thrombin in purified systems and in this
system is critical to resolving the role of AT-111 in attenuating
thrombin activity in clotting blood.
Factor V activation clearly plays a major role in controlling the coagulation process. An additional complexity not
previously recognized is the identification of a new heavy
chain of 103,000 molecular weight. The previously unidentified M, = 103,000 factor Va heavy-chain species appears to
show greater resistance to degradation, suggesting it is a
poorer substrate for APC than the larger product. The difference of the M, = 105,000 and M, = 103,000 may be explained by differences in posttranslational modification.
However, if the difference lies in the polypeptide, it is most
likely in the COOH terminus because multiple NH,-terminal
amino acid sequence determination of the heavy chain has
not resulted in two sequences.
The factor Va heavy-chain products a, b, and c appear to
be APC associated. The activation of protein C by thrombin
is quite slow in the absence of thrombomodulin.8 Although
the major fraction of thrombomodulin is contributed by the
endothelium, platelet- and plasma-derived thrombomodulin
have been d e ~ c r i b e d ~and
~ . ’ ~most likely contribute to the
apparent thrombin activity in the present system. Expansions
of the present system to include various endothelial cells
as an additional source of thrombomodulin will clarify the
contributions of various sources of this thrombin cofactor.
Our studies must be compared with two recent investigations that have employed clotting of minimally altered blood.
Thrombin generation in whole blood has recently been described by Kessels et
In this system, blood clotting was
initiated with human brain thromboplastin as a source of
tissue factor. The blood was maintained in an unstirred condition at 37°C and thrombin was monitored by chromogenic
substrate activity in dilute, fluid phase samples. These investigators showed that thrombin activity reaches a maximum
of 163 nmol/L and is followed by a decrease to a plateau of
50 nmol/L. In addition, these authors reported that the cleavage of prothrombin is quantitative during the clotting of
whole blood. However, this analysis is limited in that the
thrombin concentration measured is based on activity toward
a small chromogenic substrate. The various thrombin activities are only selective toward physiological macromolecular
substrates. The present observations indicate that thrombin
activity is almost quantitatively inhibited based on the native
TAT ELISA and incomplete cleavage of prothrombin. The
discrepancy between Kessels et a1 and the current observations may be attributed to inhibitors of thrombin that do not
occupy the active site, such as cr2-macroglobulin, as was
shown by Tans et a17*in a plasma system in which thrombin
generation was initiated by thromboplastin. In addition, the
protocol of Kessels et a143calls for removal of the clot from
the reaction mixture at the point it obstructs sampling. This
is despite evidence from their group and others that clotassociated thrombin influences thrombin generation and fibrinogen ~leavage.~’
Bierdermann et ala’ have described a model of spontaneously clotting of contact-activated whole blood for the purpose of investigating the effects of endothelial cells on prothrombin activation. In this system, in the absence of
endothelial cells, FPA generation proceeds to 14.5 pmol/L,
prothrombin cleavage, judged by F1.2 assay, goes to 68%
(950 nmol/L) after 60 minutes of clotting, and TAT reaches
383 nmoVL. These data are consistent with those of the
present model TAT = 317 nmol/L, F1.2 = 1014 nmoVL,
and FPA = 15.2 pmoVL at 60 minutes.
In summary, the model presented here examines extrinsic
pathway initiated clotting of whole blood in a dynamic system at physiological temperature. This model, in conjunction
with previous purified model systems, permits insight into
the relevant proteolytic mechanisms of in vivo coagulation
events. This model also provides opportunities for precise,
holistic examinations of the fitness of blood from patients
displaying thrombotic and hemorrhagic tendencies. Finally,
evaluation of natural and synthetic modulators of coagulation
in a relatively inexpensive, noninvasive para-vivo setting can
be conducted in this system.
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1996 88: 3432-3445
Blood clotting in minimally altered whole blood
MD Rand, JB Lock, C van't Veer, DP Gaffney and KG Mann
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