Thrombin generation assays: accruing clinical relevance
H. Coenraad Hemker, Raed Al Dieri and Suzette Béguin
Purpose of review
After decades of near oblivion, thrombin generation is being
revived as an overall function test of the plasmatic coagulation
system in platelet-poor plasma (PPP). In platelet-rich plasma
(PRP) it assesses platelet procoagulant functions as well.
The recently developed use of special fluorogenic thrombin
substrates allows monitoring of thrombin concentration in
clotting PPP and PRP on line in up to 24 parallel samples.
Studies in model systems stress the importance of cell-bound
thrombin generation such as measured in PRP.
The method can be profitably applied to various hitherto
unyielding problems such as the control of
(low-molecular-weight) heparin therapy, the detection of lupus
anticoagulant, and various forms of thrombomodulin and
activated protein C resistance (including the use of oral
contraceptives) as well as monitoring the treatment of
hemophiliacs by factor VIII bypassing therapy. In PRP it
reflects the abnormalities encountered in von Willebrand
disease and Glanzmann and Bernard-Soulier thrombopathy as
well as the action of antiplatelet drugs.
thrombin generation, platelet-poor and platelet-rich plasma,
monitoring antithrombotics, lupus anticoagulant, hemophilia
and von Willebrand disease
Curr Opin Hematol 11:170–175. © 2004 Lippincott Williams & Wilkins.
Because of its numerous positive and negative feedback
controls, the hemostatic-thrombotic system is so compli-
cated that it is practically impossible to judge the overall
hemostatic function of the blood from the concentration
or structure of its components. Information on details of
the system therefore is not an alternative for an overall
function test. Clotting times (prothrombin time, acti-
vated partial prothrombin time [aPTT], activated whole
blood clotting time) do not indicate hypercoagulability
and are insensitive to mild bleeding disorders. For over a
century (eg, Hayem ), the generation of thrombin in
clotting blood or plasma has been used to assess the
coagulation system, but only recently have technical de-
velopments brought it into reach of the nonspecialized
Ex vivo thrombin generation (TG) should be distin-
guished from in vivo TG, revealed by products of an
ongoing clotting process in the body (prothrombin frag-
ment 1-2, D-dimer etc.). Increased in vivo TG indicates
an ongoing pathologic process. Increased or decreased ex
vivo TG means that the function of the coagulation pro-
cess is abnormal (eg, hyperprothrombinemia , hemo-
philia [3••], use of anticoagulants ). It does not nec-
essarily mean ongoing pathology but indicates an
increased risk of thrombosis or bleeding . In vivo TG
is a smoke detector signaling ongoing evil; ex vivo TG is
like the smell of gasoline indicating an increased risk.
The first law of hemostasis and thrombosis
“The more thrombin the less bleeding but the more
thrombosis, the less thrombin the less thrombosis but
the more bleeding” may be called the “first law of he-
mostasis and thrombosis.” The converse is not true. The
cause of thrombosis or bleeding can be in the vessel wall,
with the hemostatic function of the blood being perfectly
normal. The circumstantial evidence for the first law is
overwhelming; we know of no refutation. Bleeding may
be caused by a lack of any known clotting factor or by an
excess of antithrombin activity (antithrombin Baltimore,
heparin); thrombosis by an excess of clotting factor (eg,
prothrombin); or a shortage of coagulation delimiters (an-
tithrombin, proteins C and S).
The time course of TG (the “thrombogram”) is shown in
Figure 1. After a lag time, a burst of thrombin is ob-
served. Clotting occurs at the end of the lag time, when
more than 95% of all thrombin is still to be formed. We
Synapse BV, Cardiovascular Research Institute, Maastricht, The Netherlands
Correspondence to H. C. Hemker, Synapse BV, Cardiovascular Research Institute,
PO Box 616, 6200MD Maastricht, The Netherlands
Current Opinion in Hematology 2004, 11:170–175
activated protein C
activated partial prothrombin time
calibrated automated thrombogram
endogenous thrombin potential
protease activatable receptor
von Willebrand factor
©2004Lippincott Williams & Wilkins
may well ask what the purpose is of all the thrombin that
is formed after clotting . We see two main functions:
In vivo thrombin diffuses out, around the primary focus
of its formation (eg, wound, ruptured plaque). Above a
certain threshold concentration, it will autocatalytically
promote more thrombin formation and thus thrombus
growth. Under that threshold it will be washed away or
neutralized. The amount of thrombin formed in a focus
will thus determine the extent of a thrombus/hemostatic
plug. Thrombin in a clot also prevents subsequent lysis
via the activation of thrombin-activatable fibrinolysis in-
hibitor [7,8]. This explains why hemophiliac bleeding
often develops after a bleeding-free interval, as if a
formed hemostatic plug is precociously dissolved (eg,
Verstraete ). Thrombin also acts on a number of dif-
ferent cells in the neighborhood of a focus and has a
function in tissue repair and proliferation of surrounding
Models of ex vivo thrombin generation
The course of thrombin concentration in a hemostatic
plug or thrombus is technically impossible to measure.
Measuring thrombin (-products) in samples from the
blood in a wound comes close [12,13], as does subsam-
pling from clotting blood [14,15]. Both require heavy
experimentation. Two essential different types of model
are of more practical use: reconstituted systems and
Reconstituted systems (for reviews see Monroe et al. 
and Mann [17•]) use purified clotting factors to represent
the physiologic situation. Reaction conditions are under
tight control and can be varied at will. To investigate TG
at cell surfaces, notably platelets with or without cells
bearing tissue factor (TF) or thrombomodulin (TM)
(monocytes, endothelial cells) they proved very useful
. However, purified factors will not necessarily retain
their native activity (eg, Hemker ). Reconstituted sys-
tems are as realistic as our insight into the clotting
mechanism allows; extrapolation to physiology should
therefore be regarded with due suspicion. Minor players
(eg, ?1glycoprotein Ib, ?2macroglobulin [?2M]) and a for-
tiori unrecognized proteins/functions escape notice; fi-
brin(-ogen) and von Willebrand factor (vWF) are often
absent but do play a role in rendering platelets proco-
agulant ; see below).
Thrombin generation in plasma (in platelet-poor or
platelet-rich plasma, PPP or PRP [21,22]) represents the
function of a relevant slice of the in vivo system with all
the plasma proteins present, unmodified, near their
physiologic concentrations and independent of a priori
hypotheses. It represents a function test of the “isolated
organ” PPP or PRP. The vessel wall is lacking, however.
To simulate its presence, the two most important known
elements, TF and TM, may be added to the plasma
Cell-bound thrombin generation: the role
The arm-to-tongue circulation time of the blood (∼30
seconds) is short compared with a whole blood clotting
time, so thrombin formed in flowing blood in vivo is
rapidly diluted and inactivated before clotting can occur.
Thrombin will only build up in unstirred boundary layers
at cell surfaces and in the unstirred plasma caught in a
clot or an aggregate. Transport by diffusion will therefore
tend to govern reaction rates. According to our interpre-
tation  diffusion limitation, for instance, explains the
kinetics observed in a cell-bound model of TG by Allen
et al. [24•].
Cell-bound TG is dependent on TF-bearing cells
(monocytes, perivascular cells) and platelets [18,16]. The
role of the platelet in physiologic thrombin generation is
twofold. By adhesion and aggregation it forms a maze in
which plasma can clot without the thrombin being
washed away; conversely, activated platelets provide the
surface on which TG can take place. Upon activation of
the platelet , procoagulant phospholipids appear at
its outside. Thrombin (PAR 1) and collagen (GPVI)
bring about this process, especially in combination.
GPIIb/IIIa plays a role as well and GPIIb/IIIa antago-
nists inhibit . vWF adsorbs onto polymerizing fibrin
and this probably brings about a molecular change (like
ristocetin), which makes it interact with GPIb-V. This
enhances the formation of a procoagulant surface
Techniques of thrombin
The thrombogram can be obtained through subsampling
 or through monitoring the conversion of a suitable
substrate directly added to the clotting plasma . The
former method is straightforward and time consuming;
Figure 1. Thrombin generation curves as obtained with the
calibrated automated thrombogram (CAT) technique
Platelet-poor plasma (PPP) was triggered with low (5 pM) tissue factor (TF);
platelet-rich plasma (PRP) with traces (0.5 pM) of TF to mask variations in
endogenous TF. Bold lines: no addition; dotted lines: 10 nM thrombomodulin
(TM) added; thin lines: 6 nM APC added (lower curve: PPP; upper curve PRP).
Thrombin generation assays Hemker et al. 171
the latter allows automatic continuous measurement of
many samples in parallel. Via subsampling, the thrombin
(∼30% of total) that adsorbs onto the formed clot
[31•,32,33] escapes notice. Such thrombin can activate
factors V, VIII, and XI or platelets and thus probably is
essential in thrombin growth .
Added thrombin substrate occupies part of the thrombin
formed. Enough free thrombin should remain to allow
for natural feedback reactions and for adequate removal
of thrombin by antithrombins; therefore binding should
be relatively loose (low Km). Suitable substrates should
also be converted slowly (low kcat) so as not to be con-
sumed during the experiment .
The fluorescent signal has the drawback of not being
linear with product concentration. To compensate for
this and for the effects of substrate consumption, the
calibrated automated thrombogram (CAT) method has
been developed that continuously compares the signal
from the experimental sample to that of a fixed known
thrombin activity [36•]. This method allows visualizing
the thrombin concentration in clotting PPP or PRP in 24
Typical thrombograms as obtained with the CAT are
shown in Figure 1. The three most important parameters
are the lag time, the peak value, and the area under the
curve or endogenous thrombin potential (ETP), which
quantifies the enzymatic “work” that thrombin can do
during its lifetime (“person-hours” of thrombin) .
Plasma clots at the end of the lag phase so the clotting
time can be read from the thrombogram. During the lag
phase the reaction mechanisms are essentially different
from those during the thrombin burst ); this is one of
the reasons that the clotting time does not represent TG.
The normal values and coefficients of variation as ob-
tained with the CAT-method are given in Table 1.
Both the lag time and the ETP can be obtained by al-
ternative techniques without monitoring the complete
thrombogram. The clotting time represents the lag time.
The ETP can be assessed by measuring the product
from any natural or added substrate that is not exhausted
during the clotting process. One natural substrate is ?2M,
which, in defibrinated plasma, will bind ∼30% of the
thrombin formed (∼5% with fibrinogen). The final con-
centration of the (amidolytically active) ?2M-thrombin
complex is proportional to the ETP . Rosing et al.
 used this approach to demonstrate acquired acti-
vated protein C (APC) resistance through the use of oral
contraceptives. The ETP can also be assessed by mea-
suring the end level of conversion of a slow-reacting ar-
tificial substrate, provided that it does not react with
Surrogate techniques of thrombin generation
Several techniques have been published that depend on
fibrinogen polymerization. Apart from the clotting time,
they give little information on thrombin generation be-
cause fibrinogen is exhausted before 5% of all thrombin
is formed. In so far as the properties of the clot are de-
termined by the velocity or the amount of thrombin
formed, some information can be retained, however. In-
deed tensile strength [39•], clot retraction [40•], turbid-
ity , and fibrinolysis [42•] are derived variables that
to some extent are determined by the amount or velocity
of thrombin formation. They recently have been (re-)used
for the assessment of overall hemostatic function. Some
of these methods have the advantage of using full blood.
A disadvantage is that the indicating substance (fibrino-
gen) may increase when TG decreases, eg, in active
thrombosis under anticoagulant treatment. It may be ex-
tremely confusing that curves are obtained that resemble
real TG curves but are not.
Applications of thrombin
Thrombin generation measurement has been shown to
be a useful tool in several different domains.
Thrombin generation has been instrumental in unveiling
the role of platelet receptors in the production of a pro-
coagulant surface by platelets. In short, a role of
GPIIb/IIIa, of GPIb/V, and vWF [28•] and of GPI (col-
lagen), as well as of the PAR receptors, has been dem-
onstrated (see Hemker and Lindhout  for a review).
Detection and quantification of thrombotic tendency
Deficiencies of proteins S or C are readily recognized
when TM is added to the plasma, factor VLeidenas well
[44,45]. APC resistance, either acquired (oral contracep-
Table 1. Normal values and variability
nAverage (pop.)StDev (pop.)CV (pop.) CV (ind.)CV (exp.)
PPP ETP (nM.min)
CV,; ETP, endogenous thrombin potential; PPP, platelet-poor plasma; PRP, platelet-rich plasma.
172 Hemostasis and thrombosis
tive treatment) or congenital, can also be detected with a
TG-based endpoint technique .
Thrombin generation also solves the long-standing
enigma of the prothrombotic anticoagulant in lupus ery-
thematodes. It has been shown that this antibody pro-
longs the lag phase (ie, is anticoagulant in clotting tests)
but induces TM and APC resistance [46••].
Detection and quantification of bleeding tendency
In deficiencies of factors II, V, VII, VIII, IX, X, and XI,
it has been demonstrated that TG is diminished in PPP
and that clinical bleeding is observed at ETP values less
than 30% [3••,47].
The thrombasthenias of Glanzmann  and Bernard-
Soulier [28•], as well as severe thrombopenia, show a
moderately diminished TG in PRP. Von Willebrand dis-
ease, unless accompanied by severe factor VIII defi-
ciency, shows normal TG in PPP but decreased TG in
Control of procoagulant therapy
As expected, restoration of the factor VIII level of he-
mophiliac plasma restores TG, as does DDAVP treat-
ment in mild hemophilia and vWD . More interest-
ing: inhibitor bypassing therapy with either factor VIIa,
or this factor in combination with other factors (Feiba),
can be monitored with TG [50•,51••].
Control of antithrombotic therapy
Under oral anticoagulation the incidence of bleeding in-
creases as soon as the international normalized ratio
drops below 3 , which is equivalent to ETP = 20%
[36•]. Heparins, including the low-molecular-weight
types, inhibit TG primarily by increasing thrombin
breakdown [53•]. Twofold prolongation of the aPTT
corresponds to ∼80% inhibition of the ETP [54•]. TG is
the only available method to quantify the combined ef-
fect of heparin and vitamin K antagonists or other anti-
Platelet “aggregation” inhibitors in general such as ab-
ciximab , clopidogrel , and aspirin  also in-
hibit TG to a certain degree. This is not to say that
inhibition of aggregation as such would not have an—or
even be the main—antithrombotic action. It is an inter-
esting possibility that through decreasing the size of the
platelet aggregate, the volume in which thrombin can
form undisturbed by flow is also diminished.
As yet we have not encountered any antithrombotic, ei-
ther anticoagulant or antiaggregant, that did not inhibit
TG . We can assume that any drug that inhibits TG to
∼50% of normal will show an antithrombotic effect at an
acceptable bleeding risk. By introducing TG as an inter-
mediate step between the biochemical experiments and
thrombosis models in experimental animals, we can sig-
nificantly diminish the latter, especially in dose-finding
experiments. The test can also be used to assess the
effects of a candidate molecule in volunteers.
To understand thrombin generation, we have to measure
thrombin generation, the whole thrombin generation,
and nothing but thrombin generation—under conditions
as close as possible to those in vivo. This offers a wealth
of information that is not otherwise available. Measure-
ment in an undisturbed fibrin clot in which activated
platelets are fixed, as is possible with fluorogenic sub-
strates, probably resembles the situation in a hemostatic
plug or thrombus more closely than stirred systems do.
The calibrated automated technique makes it possible to
obtain a graph of thrombin concentration against time in
real time in up to 24 parallel experiments. Whole blood
measurement is as yet technically impossible.
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174 Hemostasis and thrombosis
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recognized by the aPTT in 55[ref] of the cases and by the ETP in 98[ref]. There
were no large differences between the different types of heparin.
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