Journal of Thrombosis and Haemostasis, 5: 20552061
ORIGINAL ARTICLE
Citrate anticoagulation and the dynamics of thrombin
generation
K . G . M A N N , M . F . W H E L I H A N , S . B U T E N A S and T . O R F E O
Department of Biochemistry, University of Vermont, Colchester, VT, USA
To cite this article: Mann KG, Whelihan MF, Butenas S, Orfeo T. Citrate anticoagulation and the dynamics of thrombin generation. J Thromb
Haemost 2007; 5: 205561.
Summary. Background: Sodium citrate has been used as an
anticoagulant to stabilize blood and blood products for over
100 years, presumably by sequestering Ca++ ions in vitro.
Anticoagulation of blood without chelation can be achieved by
inhibition of the contact pathway by corn trypsin inhibitor
(CTI). Objective: To evaluate the inuence of citrate anticoag-
ulation on the performance of blood, platelet-rich and platelet-
poor plasma assays. Methods: Blood was anticoagulated in
three ways: by collection into citrate, CTI and citrate with CTI.
Plasma was prepared using each anticoagulation regimen.
Functional analyses included calibrated automated thrombog-
raphy, thromboelastography, plasma clotting, the synthetic
coagulation proteome and platelet aggregation. Coagulation
reactions were initiated with tissue factorphospholipid and
Ca++ (when indicated). Results: In all cases, citrate anticoag-
ulation resulted in reaction dynamics signicantly altered
relative to blood or plasma stabilized with CTI alone.
Subsequent experiments showed that calcium citrate itself
impairs coagulation dynamics. Conclusion: Coagulation anal-
yses using blood that has been exposed to citrate and recalcied
do not yield reliable depictions of the natural dynamics of blood
coagulation processes.
Keywords: anticoagulation, platelet aggregation, thrombin,
thrombograms, thromboelastography.
Introduction
The essential roles for calcium ion in blood clotting reactions
were demonstrated by Arthus between 1890 and 1896 [2].
Subsequently, Sabbatani [3,4] and Pekelharing [5,6] observed
that the Ca++ required for blood clotting could be neutralized
Correspondence: Kenneth G. Mann, Department of Biochemistry,
University of Vermont, 208 South Park Drive, Suite 2, Colchester, VT
05446, USA.
Tel.: +1 802 656 0335; fax: +1 802 656 2256; e-mail: kenneth.mann@
uvm.edu
A preliminary account of this work was presented at the December 2006
meeting of the American Society of Hematology [1].
Received 12 March 2007, accepted 18 July 2007
 2007 International Society on Thrombosis and Haemostasis
by the addition of Na citrate. The prothrombin time (PT) [7]
and the activated partial thromboplastin time [8] make use of
citrate plasma, a source of Ca++ and either tissue thrombo-
plastin or contact activation. Typically [9,10], blood is collected
into 3.2% or 3.8% sodium citrate and plasma is prepared by
differential centrifugation. Reactions are initiated by the
addition of Ca++ solutions sufcient to overcome the citrate.
Citrate anticoagulation has enabled tests of coagulant function
and has provided stable sources of both blood and plasma for
clinical use, protein isolation and biochemical studies.
Calcium ions are essential for the conformational transitions
involving Gla, kringle and EGF domains [11] of the vitamin
K-dependent (VKD) proteins, and for the protein and
membrane-binding [12,13] required for complex formation.
The subunit associations of factor (F) Va [14,15] and FVIIIa
[16,17] and FXIII activation are also Ca++ dependent. Platelet
activation involves Ca++ uxes [18].
Consequently, during the evolution of reactions initiated by
the addition of Ca++ into citrate-treated blood, platelet-rich
plasma (PRP) or platelet-poor plasma (PPP), many time-
dependent reactions associated with reconstitution to Ca++
homeostasis occur [1922]. Thus, the start of Ca++-initiated
tests corresponds to a vague zero time during which multiple
processes re-establishing Ca++ homeostasis occur.
The use of corn trypsin inhibitor (CTI) to block contact
pathway activation provides a non-chelating method for
stabilizing blood [23], permitting the examination of the effect
of citrate/recalcication on the reaction dynamics.
In the present study, we utilized CTI to evaluate the inuence
of citrate on the performance of reactions in plasma, PRP and
whole blood. Our results indicate that the procedure of citrate
anticoagulation/recalcication results in artifactual representa-
tions of the dynamics of the blood coagulation process.
Materials and methods
Reagents
Human VKD zymogens were prepared using the methods of
Bajaj et al. [24], and purged of traces of active enzymes [25]. FV
and antithrombin III were from plasma [26,27]. Recombinant
human FVIIa was from Dr U. Hedner (Novo Nordisk,
2056 K. G. Mann et al
Denmark). Tissue factor pathway inhibitor (TFPI) was a gift
from Dr K. Johnson (Chiron Corp., Emeryville, CA, USA).
Recombinant tissue factor (TF) (1-242) and FVIII were gifts
from Drs Liu and Lundblad (Hyland Division, Baxter
Healthcare Corp., Duarte, CA, USA). CTI was isolated in-
house [28,29]. Spectrozyme TH was from American Diagnos-
tica, Inc. (Greenwich, CT, USA). 1,2-Dioleolyl-sn-glycero-3-
phospho-L-serine (DOPS) and 1,2-dioleoyl-sn-glycero-3-phos-
phocholine (DOPC) were from Avanti Polar Lipids, Inc.
(Alabaster, AL, USA), EDTA was from Sigma (St. Louis,
MO, USA), and analytical-grade Na3 citrate2H2O was from
Fisher (Pittsburgh, PA, USA). Phospholipid vesicles (PCPS)
composed of 25% DOPS and 75% DOPC and the TfPCPS
reagent [(Tf): 3.5 nM Tf, 10 lM PCPS, 5 mM Ca++] were
prepared as described [29,30]. Monoclonal anti-TF (a-Tf-5)
and anti-FXIa (a-FXI-2) antibodies were prepared in-house.
The substrate benzyloxycarbonyl-Gly-Gly-Arg-7-amido-
4methylcoumarin HCl (Z-GGR-AMC) was from Bachem
(Torrance, CA, USA). Type 1 collagen, adenosine 5 diphos-
phate (ADP), 1-epinephrine bitartrate and ristocetin were from
Chronolog Co. (Havertown, PA, USA). The thromboelasto-
gram (TEG) analyzer was from Dr Eli Cohen, Haemoscope
Corporation (Niles, IL, USA). The Calibrated Automated
Thrombogram (CAT) was loaned by Dr Worfolk of
Diagnostica Stago (Parsippany, NJ, USA).
Blood and plasma preparation
Healthy donors were recruited and advised according to a
protocol approved by the University of Vermont Human
Studies Committee. All donors provided informed consent.
Whole blood drawn by venipuncture was collected in four
ways, as follows. (i) Citrate blood: 9 parts blood were mixed
with 1 part sodium citrate (3.2%; nal concentration 10.8 mM).
(ii) CTIcitrate blood: CTI (0.1 mg mL)1 CTI nal) was added
to citrate blood [1 part CTI stock (5 mg mL)1) to 50 parts
citrate whole blood, v/v]. (iii) CTI blood: 9 parts blood were
added to 1 part of a 1 mg mL)1 stock of CTI in 20 mM
HEPES, 0.15 M NaCl, pH 7.4 (HBS). (iv) a-FXI-2 blood: 9
parts blood were added to 1 part of a 1 mg mL)1 stock of a-
FXI-2 in HBS. PRP and PPP were prepared by centrifugation
at 190 g (PRP) and 2500 g (PPP) for 15 min.
Ca++ measurements
Free Ca++ was measured using a Ca++-selective electrode
(Orion 97-20, Thermo Electron Co., Beverly, MA, USA) in
5 mL samples in a stirred cell maintained at 37 C. A standard
curve was generated by titrating CaCl2 into a buffer containing
0.15 M NaCl, 15 mM Tris, 4% human albumin (Ca++ free) at
pH 7.4.
Thrombogram (CAT) studies
A 100 mM solution in DMSO of Z-GGR-AMC was prepared
and diluted to 2.5 mM in 40 mM HEPES, 6% bovine serum
albumin, pH 7.5, with 0.1 M CaCl2 (substrate 1) or without
CaCl2 (substrate 2). Experiments involving citrate and CTI
citrate plasma were performed by adding 20 lL of Tf (6
30 pM), 12 lM PCPS in HBS to an Immulon 96-well plate
(Thermo Electron Co., Waltham, MA, USA). 80 lL of plasma
was added, incubated at 37 C for 3 min, and 20 lL of
substrate 1 was injected. For preincubation of Ca++ in CTI
citrate plasmas, 80 lL of plasma was mixed with 20 lL of
substrate 1, incubated at 37 C for 3 min, and 20 lL of 6
30 pM Tf, 12 lM PCPS in HBS was added. In experiments with
CTI plasma, 20 lL of substrate 2 and 80 lL CTI plasma were
added, allowed to incubate for 3 min at 37 C, and the reaction
was initiated with 20 lL of Tf (630 pM), 12 lM PCPS in HBS.
Thrombin generation curves were generated using Thrombino-
scope software (Thrombinoscope BV, Maastricht, the Neth-
erlands) and calibrator provided.
Thromoboelastography (TEG)
TEG experiments were performed at 37 C using the Haemo-
scope TEG. A 350 lL volume of citrate, CTIcitrate or CTI
blood was added to the TEG cups preloaded with varying
concentrations of Tf (15 pM) at 37 C. For citrate blood the
cups contained 5.5 lL of 1 M CaCl2, yielding an excess Ca++
concentration of 15 mM. For Ca++ preincubation of CTI
citrate blood, Ca++ (to 15 mM) was mixed with blood for
3 min prior to Tf addition.
Plasma clotting assays
Assays were performed using the STart coagulation analyzer
(Diagnostica Stago, Asnie`res, France). CTI plasma was divided
into three sample sets: (i) CTI plasma + HBS (9:1 v/v); (ii) CTI
plasma + a preformed solution of 108 mM citrate with
150 mM CaCl2 (9:1 v/v); and (iii) CTI plasma + 3.2% citrate
(9:1 v/v). Sample sets 1 and 2 (280 lL) were incubated for
3 min at 37 C and then activated with 20 lL of a solution
containing 751500 pM Tf, 30 lM PCPS in HBS. Sample set 3
(280 lL) was either incubated with 15 mM CaCl2 for 3 min at
37 C and then initiated as above, or subjected to a 3-min
incubation at 37 C and initiated with simultaneous additions
of the Tf solution and CaCl2 (15 mM nal).
Synthetic coagulation proteome
A modication of the methods of Lawson et al. [31] and van t
Veer et al. was used [25]. Mixtures of FVIIa, FV, FVIII, VKD
zmogens, TFPI and AT-III were constructed in: (a) HBS,
2 mM CaCl2; (b) HBS, 17 mM CaCl2 and 10.8 mM Na citrate;
and (c) HBS, 2 mM CaCl2 and 10.8 mM Na citrate. Reaction
mixtures (a) and (b) were preincubated for 10 min at 37 C and
then activated with 5 pM Tf and 2 lM PCPS in HBS. Reaction
mixture (c) was treated in two ways: either incubated for 7 min
at 37 C, supplemented with CaCl2 (to 17 mM nal) [CaCl2
equivalent to 15 mM (nal) was added; this plus the 2 mM
Ca++ already present gives 17 mM] for an additional 3 min
 2007 International Society on Thrombosis and Haemostasis
 Citrate coagulation assays 2057

and activated as above; or incubated for 10 min at 37 C and
then activated by the simultaneous additions of Tf, PCPS and
CaCl2 (5 pM, 2 lM and 17 mM nal, respectively). Thrombin
generation was measured using 200 lM Spectrozyme TH and a
Molecular Devices THERMOmax microplate reader (Molecu-
lar Devices, Sunnyvale, CA, USA) .
Platelet aggregation
Platelet aggregation studies were conducted using an Aggre-
gometer 490-2A (Chrono-log Co., Havertown, PA, USA).
500 lL of citratePRP, CTIcitratePRP or CTIPRP and its
matching PPP (background control) were added to four
cuvettes in the aggregometer and allowed to stabilize at 37 C
for 5 min. Platelet aggregation was initiated by the addition of
one agonist to each cuvette in the sequence (staggered at 5 s
intervals): type 1 collagen, ADP, ristocetin and epinephrine.
of CaCl2 reached 1213 mM, and they were stable up to the
addition to 15 mM exogenous [Ca++]. In CTIcitrate plasma
made from CTI plasma, the transition to minimum clotting
time required 7 mM of added CaCl2, with subsequent
increases of CaCl2 up to 15 mM having little inuence
(10%) on clotting times. Therefore, our experiments were
standardized by CaCl2 addition to achieve 15 mM. This value is
within the range of other recalcication protocols [9,3739].
Segregation of coagulation pathways
Several reports have recognized the importance of contact
pathway suppression by CTI in the application of the
thrombogram and thromboelastograph techniques to citrate
plasma and blood [36,40,41].
In the thrombogram, when CaCl2 and PCPS are added to
citrate plasma in the presence of a-Tf-5 (Fig. 1A), signicant

Results A
100
Free Ca++ levels in blood and plasma: chelation and the
restoration of Ca++ levels 80

The free [Ca++] in CTI blood is 1.34 0.2 mM (n = 5). The

IIa (nM)
60
[Ca++] in PPP, PRP and serum prepared via Tf-initiated
clotting were not signicantly different than those measured in 40
the CTI blood from which they were prepared. Titration
(n = 3) of exogenous CaCl2 into an individuals CTI blood or 20
plasma yields increases in signal coincident with those observed
in the albumin containing reference buffer. Thus, Ca++ 0
additions above physiological translate equivalently to higher 0 10 20 30 40 50 60
free [Ca++]. The absence of any signicant additional Time (min)
buffering capacity for Ca++ in non-chelated blood or plasma
means that the variability in Ca++ reconstitution schemes for B
100
chelated blood and plasma results in different nal free [Ca++]
[3235].
80
For CTIcitrate blood, a free [Ca++] equivalent to that
measured in CTI blood was achieved when the added CaCl2
IIa (nM

60
reached 7 mM (n = 4), consistent with an earlier report [36].
For CTIcitrate plasma derived from CTIcitrate blood, the
40
establishment of free [Ca++] equivalent to that in CTI blood
required the addition of 12.5 mM CaCl2 (n = 4). In CTI
20
citrate plasma prepared from CTI plasma, the addition of
CaCl2 to 7.5 mM (n = 4) was required to re-establish the free
[Ca++]. These results are consistent with the expected parti- 0
0 10 20 30 40 50 60
tioning of citrate into the plasma during the preparation of Time (min)
citrate plasma from citrate blood, and with the expected
reduction of the titratable citrate concentration because of its Fig. 1. Segregation of pathways. (A) () Citrate plasma with 5 pM
recombinant tissue factor-PCPS reagent (Tf) and 2 lM PCPS triggered at
chelation of endogenous plasma Ca++ and Mg++. The effect 15 mM CaCl2 (exogenous); (h) citrate plasma + a-FXI-2 with 5 pM Tf,
of calcium citrate on CTI plasma pH was examined and no 2 lM PCPS triggered at 15 mM CaCl2 (exogenous); ( ) citrate plasma +
signicant change was observed. a-Tf-5, 2 lM PCPS triggered at 15 mM CaCl2 (exogenous); ( ) citrate
In order to assess functional consequences of the addition of plasma + a-FXI-2, 2 lM PCPS, and 15 mM CaCl2 (exogenous), no Tf. (B)
Ca++ in excess of that required to reproduce the pre-citrate (d) Corn trypsin inhibitor (CTI) or (h) a-FXI-2 citrate plasma was with
5 pM Tf, 2 lM PCPS and triggered at 15 mM CaCl2 (exogenous). (s) CTI
free [Ca++], a titration study was conducted using a modied citrate plasma with 2 lM PCPS and trig at 15 mM CaCl2 (exogenous).
PT assay. In CTIcitrate plasma derived from CTIcitrate Each thrombogram represents the average prole from a triplicate
blood, minimum clotting times were observed when additions determination.

 2007 International Society on Thrombosis and Haemostasis

2058 K. G. Mann et al

thrombin is generated after a prolonged initiation phase, 150

presumably by the contact pathway. Treatment of the same
plasma sample with a-FXI-2 antibody yields no thrombin for 125
60 min upon CaCl2, PCPS addition. With a-FXI-2, CaCl2,
100
PCPS and 5 pM Tf, thrombin is generated with a shorter

initiation phase, but at weaker intensity. The addition of CaCl2,
PCPS and 5 pM Tf to citrate plasma produced a similar
initiation phase and signicantly higher levels of thrombin
because of the contributions of both the extrinsic and intrinsic
pathways of thrombin generation.
Suppression of the contact pathway using either a-FXI-2 or
CTI in the absence of Tf is presented in Fig. 1B. In the presence
of 5 pM Tf, citrate plasma additionally anticoagulated with
either CTI or a-FXI-2 gave similar thrombograms. The small
suppression of peak activity observed with the FXIa inhibitor
was a consequence of defeat of thrombin feedback activation of
FXI [29,42]. With no Tf added, CTI plasma produced no
thrombin upon PCPS and Ca++. Tf titration suggests that the
magnitude of the contact pathway contribution is approxi-
mately equivalent to that provided by 1 pM Tf when the
contact pathway is suppressed.
Similar experiments using thromboelastography were per-
formed by subjecting citrate blood to TF in the presence or
absence of contact pathway inhibition. Reactions were initiated
by the addition of CaCl2 and Tf. Analyses of the behavior of
citrate blood and CTIcitrate blood in terms of the TEG
parameter R (the initial change in viscosity signaling the onset
of clot formation) yielded the following data (mean,
min SD). (i) Citrate blood: no Tf, R = 24 5.5
(n = 10); 1 pM Tf, R = 17 2.5 (n = 5); 5 pM Tf,
R = 10.1 2 (n = 13). (ii) CTIcitrate blood: no Tf,
R = 66 19 (n = 12); 1 pM Tf, 21.9 4.2 (n = 5); 5 pM
Tf, 9.1 1.5 (n = 10). These analyses also suggest that the
magnitude of the contact pathway contribution to the clotting
process is in the range of that provided by 1 pM Tf when the
contact pathway is suppressed.
Influence of chelation on thrombin generation
Figure 2 presents thrombograms contrasting thrombin gener-
ation in CTI plasma with the identical CTIcitrate plasma.
Reactions were initiated by the addition of TfPCPS, or a Tf
PCPS/CaCl2 solution. Thrombin generation with 15 pM Tf
was analyzed. Data for 5 pM Tf are presented in Fig. 2.
Signicantly larger amounts of thrombin were generated in
CTI plasma after a somewhat shorter lag period when
IIa (nM)
75
50
25
0
0 10 20 30 40 50 60
Time (min)
Fig. 2. Corn trypsin inhibitor (CTI) vs. CTIcitrate thrombograms. ( )
CTI plasma with the thrombin substrate, and the reaction triggered with
the addition of 5 pM recombinant tissue factor-PCPS reagent (Tf) and
2 lM PCPS (nal); () CTIcitrate plasma was premixed with 5 pM Tf and
2 lM PCPS (nal), and the reaction triggered with CaCl2 (15 mM exoge-
nous) and thrombin substrate. (4) CTIcitrate plasma preincubated with
15 mM CaCl2 and thrombin substrate (3 min) followed by 5 pM Tf, 2 lM
PCPS. Each thrombogram represents the average prole for three deter-
minations.
compared with CTIcitrate plasma. With CTIcitrate plasma
the maximum level of thrombin was attenuated, and the
initiation phase prior to robust thrombin generation was
extended from 5 min to 7 min. At lower Tf concentrations, the
effect on the duration of the lag phase remained in the range of
23 min. Similar results were obtained with plasma from four
donors.
In order to eliminate the contributions of recalcication
protein/complex dynamics on the thrombogram, CTIcitrate
plasma was preincubated for 3 min with CaCl2 prior to the
addition of TfPCPS. Surprisingly, while these data (Fig. 2)
revealed increased peak thrombin generation, a more pro-
longed lag was observed.
These data illustrate that in the absence of a contact pathway
contribution, studies of Tf-dependent reactions conducted
using citrate-chelated plasma and initiated by CaCl2 addition
are awed with respect to interpretations of the reaction
dynamics. The dynamic parameters are altered even when
plasma is re-equilibrated with Ca++, indicating that the citrate
plasma is not reversed by Ca++ addition.
The complexities observed with respect to the dynamics of
thrombin generation in recalcied citrate plasma were also
observed in whole blood. TEG experiments (Table 1)

Table 1 Citrate eects on Tf-TEG parameters

Anticoagulant R* (min) P K (min) P

CTI (n = 9) 7.1 0.9 3.3 0.5

a-FXI-2 (n = 3) 7.4 1.1 3.0 0.4
CTI-Citrate (n = 5) Ca++ Initiation 9.1 1.2 0.023 4.7 1.1 0.023
CTI-Citrate (n = 5) Ca++ Preincubation 7.4 1.6 0.774 7.5 0.6 <0.001
CTI (n = 5) 66.1 18.7 <0.001 NA
*
R the time to detectable clot formation. K the time after R to achieve a xed degree of viscoelasticity. No Tf. P value relative to CTI-blood.

 2007 International Society on Thrombosis and Haemostasis


300
250
200
IIa (nM)
150
100
50
0 relative to the control in the duration of the initiation phase and
0 3 6 9 12 15 18
Time (min)
Fig. 3. Anticoagulation and recombinant tissue factor (Tf)-initiated
thrombin generation in the synthetic coagulation proteome. All reactions
were initiated with 5 pM Tf-PCPS reagent (Tf) and 2 lM PCPS (nal). ( )
Control, proteins equilibrated in 2 mM CaCl2; () proteins in 2 mM CaCl2
+ 10.8 mM Na citrate, reaction initiation by simultaneous addition of Tf
PCPS with CaCl2 to 17 mM nal concentration; (4) proteins in 2 mM
CaCl2 equilibrated with 10.8 mM Na citrate and incubated for 3 min with
CaCl2 to 17 mM (nal) prior to initiation; ( ) proteins in 2 mM CaCl2
equilibrated with premixed 10.8 mM Na citrate + 15 mM CaCl2.
compared blood collected into CTI with blood from the same
draw collected into citrate and CTI, triggered by the addition of
5 pM Tf or 5 pM Tf and 15 mM CaCl2, respectively. CTI blood
with no Tf gave no signal over 60 min. In the absence of citrate,
a shorter R-time was observed for CTI and a-FXI-2 antico-
agulation. Preincubation with CaCl2 prior to Tf addition
delayed K-values for CTIcitrate whole blood.
In contrast to the observation of an extended lag time
following CaCl2 preincubation of CTIcitrate plasma (Fig. 2),
the R-values were restored by CaCl2 pre-equilibration while the
K-values were prolonged. The differences between thrombo-
gram and thromboelastogram results were probably conse-
quences of the fact that the latter depends upon platelet
activation and brin polymerization while the former reports
only thrombin generation per se.
The calcium citrate effect
The lack of restoration of normal coagulation dynamics in
citrate plasma and blood, even after Ca++ re-equilibration,
suggested that calcium citrate itself might alter the natural
dynamics of Tf-induced blood coagulation. This possibility was
explored by measuring clot time as a function of [Tf] for CTI
plasma, CTIcitrate plasma, with and without preincubation
with CaCl2, and CTI plasma to which a calcium citrate solution
(10.8 mM Na citrate + 15 mM CaCl2 nal) was added. For
CTIcitrate plasma, clotting initiated by the addition of CaCl2
was signicantly delayed relative to CTI plasma. While
preincubation with Ca++ partially alleviated this effect, cal-
cium citrate itself delayed Tf-induced clotting of CTI plasma.
The synthetic coagulation proteome provides a dened
system for testing the effect of citrate on reaction dynamics.
Figure 3 illustrates control data for proteome mixtures
 2007 International Society on Thrombosis and Haemostasis
Citrate coagulation assays 2059
equilibrated with 2 mM CaCl2 and subjected to a 5 pM Tf,
2 lM PCPS stimulus. For the 2 mM Ca++ proteome treated
with citrate and the reaction started by the simultaneous
addition of CaCl2, TfPCPS, the initiation phase was more
than doubled, and the peak thrombin concentration was
reduced (compare with Fig. 2). Preincubation (3 min) of the
citrate-treated proteome with CaCl2 (17 mM nal) prior to the
addition of TfPCPS shortened the initiation phase and
partially restored peak thrombin to that of the control. When
the Ca++ proteome was treated with premixed citrate and
CaCl2 (10.8 mM and 17 mM nal), the reaction was still altered
peak thrombin.
Platelet function
Figure 4 is representative of experiments (n = 4) comparing
the aggregation proles for PRPs prepared from CTI blood,
CTIcitrate blood and citrate blood. Signicant differences in
the aggregation curves between the three plasmas are observed
for each of the four platelet agonists tested. The magnitude of
the alterations is decreased in the order epinephrine>ristoce-
tin>ADP> collagen. The same pattern was observed in PRP
from four individuals. A complete lack of reactivity to
epinephrine in CTIPRP (Fig. 4A) was observed in three
individuals, with one individual showing a partial response. In
all four PRPs, the addition of collagen to the epinephrine-
stimulated CTIPRP resulted in a typical collagen response.
Chelator-induced alterations in platelet function have previ-
ously been reported by Phillips and coworkers [22].
Discussion
The data presented show that the collection of blood into
citrate, with or without subsequent cellular fractionation,
results in alterations that are not reversible by Ca++ replace-
ment. The data further show that calcium citrate, independent
of chelation, has a negative inuence on the dynamics of
thrombin generation following TfPCPS addition.
Blockade of the contact pathway with CTI or a-FXI-2
avoids the changes induced by Ca++ chelation while yielding
minimally altered whole blood and plasma that can be used in
both thrombogram and thromboelastogram methodologies to
study TF-initiated processes [23]. Changes in the biological
functions of plasma, PRP, and blood by citrate are most
evident when reactions are initiated by the addition of CaCl2,
but occur even when a regimen is followed that involves
preincubation with Ca++ at concentrations sufcient to
overcome chelation.
In thromboelastographic experiments, CTIcitrate blood,
even pre-equilibrated with Ca++ prior to Tf initiation,
consistently displays a greater K-value than that observed with
CTI blood from the same blood draw. Even in the absence of
chelation, calcium citrate prolongs the initiation phase of
thrombin generation whether the model evaluated is plasma or
a synthetic coagulation proteome. Overall, the data do not
2060 K. G. Mann et al

A 120 the intrinsic and extrinsic FXase complexes, but has only a
small impact on the prothrombinase complex. In the latter
100 instance, only an initial lag in thrombin generation is observed
for reactions initiated by Ca++ addition.
% Transmittance

80 Our observations in no way challenge the established clinical

utility of tests that assess function of plasma coagulation
60
factors, platelet aggregation, thrombin generation (CAT), and
generation of tensile strength in whole blood clot (TEG);
40
however, some comments are in order regarding the use of
20 citrated specimens for studies of the physiology of coagulation
with CAT and TEG assay systems. These techniques provide
0 signicant increases in the information collected relative to that
0 50 100 150 200 250 300 available with clotting tests for the evaluation of coagulation
Time (s) disorders. This added information may prove valuable in the
diagnosis and stratication of patients with hemorrhagic
B 120
disease [41,42]. However, as presently formulated with cit-
rate-anticoagulated blood and plasma these techniques are not
100
suitable for the interpretation of the quantitative dynamic
aspects of the native coagulation system induced by low
% Transmittance

80
concentrations of TF.
60
Author contributions
40
K. G. Mann participated in design of experiments and data
20 analyses, M. F. Whelihan performed CAT, TEG and platelet
experiments, S. Butenas performed synthetic proteome exper-
0 iments, and T. Orfeo performed Ca++ measurements and
0 50 100 150 200 250 300 participated in design and data analyses.
Time (s)

C 120 Acknowledgements

100 We thank S. L. Liu and R. Lundblad for recombinant TF, E.

Cohen of the Haemoscope Corp. (Niles, IL, USA) for the
TEG analyzer and L. Worfolk of Diagnostic Stago (Parsip-
% Transmittance

80
pany, NJ, USA) for the use of the Calibrated Automated
60 Thrombogram, U. Hedner, Novo Nordisk (Copenhagen,
Denmark) and K. Johnson, Chiron Corp. (Emoryville, CA,
40 USA).
This study was supported by grant P01 HL46703 from the
20 National Institutes of Health.

0
0 50 100 150 200 250 300
Time (s)
Fig. 4. Anticoagulants and platelet aggregation. Platelet aggregation in
response to collagen, 2 lg mL)1 (h); adenosine diphosphate (ADP), 2 lM
(); ristocetin 1 mg mL)1 ( ); epinephrine, 10 lM (s). (A) Corn trypsin
inhibitor (CTI) plasma; (B) CTIcitrate plasma; (C) citrate plasma.
Platelet aggregation was initiated with the addition at 5 s intervals of the
agonists in the sequence: type 1 collagen, ADP, ristocetin and epinephrine.
Aggregation data were digitally acquired and every fourth point is plotted.
support the conclusion that citrate chelation/recalcication is a
neutral event with respect to blood coagulation dynamics.
A systematic analysis of the effects of citrate on reaction
dynamics is outside the scope of this study. However,
preliminary studies indicate that calcium citrate inhibits both
Disclosure of Conflict of Interests
K.G. Mann holds US patents on the use of corn trypsin
inhibitor as an anticoagulant: 6 403 381; 6 566 140; 7 235 377.
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