Thrombin generation assays: accruing clinical relevance.
ABSTRACT 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.
- SourceAvailable from: Yvonne P J Bosch[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: In this study the value of thrombin generation parameters measured by the Calibrated Automated Thrombography for prediction of blood loss after cardiac surgery with cardiopulmonary bypass was investigated. METHODS: Thirty male patients undergoing first-time coronary artery bypass grafting were enrolled. Blood samples were taken pre-bypass before heparinisation (T1) and 5 min after protamine administration (T2). Thrombin generation was measured both in platelet-rich plasma and in platelet-poor plasma. Besides thrombin generation measurements, activated clotting time, haematocrit, haemoglobin, platelet number, fibrinogen, antithrombin, D-dimers, prothrombin time and activated partial thromboplastin time were determined. Blood loss was measured and the amount of transfusion products was recorded postoperatively until 20 hours after surgery. Patients were divided into two groups based on the median volume of postoperative blood loss (group 1: patients with median blood loss <930 ml; group 2: patients with median blood loss >=930 ml). RESULTS: On T1, patients of group 2 had a significantly lower endogenous thrombin potential and peak thrombin (p<0.001 and p=0.004 respectively) in platelet-rich plasma, a significantly lower endogenous thrombin potential (p=0.004) and peak thrombin (p=0.014) in platelet-poor plasma, and a lower platelet count (p=0.002). On T2 both endogenous thrombin potential and peak thrombin remain significantly lower (p=0.011 and p=0.010) in group 2, measured in platelet-rich plasma but not in platelet-poor plasma. In addition, platelet number remains lower in group 2 after protamine administration (p=0.002). CONCLUSIONS: The key finding is that the Calibrated Automated Thrombography assay, performed preoperatively, provides information predictive for blood loss after cardiac surgery.Journal of Cardiothoracic Surgery 06/2013; 8(1):154. · 0.90 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In women who suffer venous thrombosis (VT) during oral contraceptive (OC) use, a transient risk factor (OC) is removed during the acute event, while most co-existing forms of thrombophilia persist and presumably continue to maintain hypercoagulability. The aim of this study was to establish if hypercoagulability persists long after OC-related VT and if it could be attributed to thrombophilia. 60 women (age 33.0±8.5years) were investigated 5 - 64 (median 33) months after OC-related VT (patients) and compared to 63 apparently healthy women (controls). All women were tested for thrombophilia, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, P-selectin and C-reactive protein. Thrombin generation was measured by Technothrombin® TGA assay. Overall haemostasis potential (OHP) assay with overall coagulation potential (OCP) and overall fibrinolytic potential (OFP) as supplementary parameters were measured by repeated fibrin formation and degradation registration. In patients increased endogenous thrombin potential (4205±440nM x min vs 4015±421nM x min, p=0.017), increased OCP (22.6±4.6 Abs-sum vs 20.8±4.1 Abs-sum, p=0.025), shorter APTT (30.9±3.8s vs 33.4±3.6s, p<0.001) and lower antithrombin activity (99, 93-105% vs 104, 100-109%, p<0.05) were observed. Thrombophilia was observed in 22/60 (36%) patients and in 5/63 (7.9%, p<0.001) controls. The only significant difference between thrombophilic and non-thrombophilic patients was higher soluble P-selectin in the former subgroup (22, 20-33μg/L vs 17, 12-22μg/L, p=0.012). In women with a history of OC-related VT persistent hypercoagulability was observed, which, however was not augmented by the presence of thrombophilia.Thrombosis Research 09/2013; · 3.13 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Introduction Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are susceptible to haemostatic disturbances. Monitoring the haemostatic capacity by conventional clotting tests is challenging. Materials and Methods Thrombin generation (TG) by Calibrated Automated Thrombography, clotting tests and tissue factor pathway inhibitor (TFPI) measurements were performed to describe the relationship between haemostatic changes and alterations in these tests. Blood samples were collected before, during and after CPB. Furthermore, it was investigated whether TG measured intraoperatively, is associated with increased risk of bleeding postoperatively. Results TG diminished significantly (p < 0.01) after heparinization in the presence and absence of platelets (37% and 50%) compared to baseline. After the start of CPB, TG elevated and persisted till the end of surgery but remained lower than preoperatively. Activated clotting time increased after heparinization and after the start of bypass compared to baseline (400% and 500%). Anti-FXa activity reduced on the start of CPB compared to the level after heparinization, to almost the baseline value following protamine reversal of heparin. The plasma levels of total and free TFPI elevated 9 and 14 fold during bypass and remained after protamine administration higher than preoperatively. Plasma D-dimer levels reduced (p < 0.01) when bypass started. However, a marked elevation was observed in the following time points. TG in platelet-rich plasma measured after heparinization and after the start of CPB associated (p < 0.05) with postoperative blood loss. Conclusions TG can be determined during CPB despite the high heparinization level, it reflects the haemostatic capacity better than clotting-based assays and might better predict bleeding when performed intraoperatively.Thrombosis Research 01/2013; · 3.13 Impact Factor
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.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• Of special interest
••Of outstanding interest
Hayem: Du Sang et de Ses Altérations Anatomiques. G. Masson Editeur;
Kyrle PA, Mannhalter C, Béguin S, et al.: Clinical studies and thrombin gen-
the prothrombin gene. Arterioscler Thromb Vasc Biol 1998, 18:1287–1291.
Siegemund T, Petros S, Siegemund A, et al.: Thrombin generation in severe
haemophilia A and B: the endogenous thrombin potential in platelet-rich
plasma. Thromb Haemost 2003, 90:781–786.
Thrombin generation was measured in PRP from hemophilia A and hemophilia B
patients. Platelets increase ETP in the hemophilias. There was an almost linear
limit at 1011platelets/L. The influence of platelets diminishes with increasing con-
centration of either FVIII or FIX.
Kakkar VV, Hoppenstead DA, Fareed J, et al.: Randomized trial of different
regimens of heparins and in vivo thrombin generation in acute deep vein
thrombosis. Blood 2002, 99:1965–1970.
Hemker HC, Béguin S: Phenotyping the clotting system. Thromb Haemost
Mann KG, Brummel K, Butenas S: What is all that thrombin for? J Thromb
Haemost 2003, 1:1504–1514.
Mattsson C, Bjorkman JA, Abrahamsson T, et al.: Local proCPU (TAFI) acti-
vation during thrombolytic treatment in a dog model of coronary artery throm-
bosis can be inhibited with a direct, small molecule thrombin inhibitor (mela-
gatran). Thromb Haemost 2002, 87:557–562.
Verstraete M: Clinical application of inhibitors of fibrinolysis. Drugs 1985,
Ruf W, Dorfleutner A, Riewald M: Specificity of coagulation factor signaling.
J Thromb Haemost 2003, 1:1495–1503.
in atherosclerosis and thrombosis: lessons from thrombin receptor knockout
mice. Arterioscler Thromb Vasc Biol 2003, 23:931–939.
Jensen AH, Béguin S, Josso F: Factor V and VIII activation “in vivo” during
bleeding: evidence of thrombin formation at the early stage of hemostasis.
Pathol Biol (Paris) 1976, 24(suppl):6–10.
Undas A, Brummel K, Musial J, et al.: Blood coagulation at the site of micro-
vascular injury: effects of low-dose aspirin. Blood 2001, 98:2423–2431.
Kessels H, Béguin S, Andree H, Hemker HC: Measurement of thrombin gen-
eration in whole blood: the effect of heparin and aspirin. Thromb Haemost
Thrombin generation assays Hemker et al. 173
Brummel KE, Paradis SG, Butenas S, Mann KG: Thrombin functions during
tissue factor-induced blood coagulation. Blood 2002, 100:148–152.
Monroe DM, Hoffman M, Roberts HR: Platelets and thrombin generation.
Arterioscler Thromb Vasc Biol 2002, 22:1381–1389.
This review provides a summary of the evolution of knowledge with respect to
present-day concepts of TG via the TF pathway and its regulation, seen from a
Mann KG: Thrombin formation. Chest 2003, 124:4S–10S.
most 2001, 85:958–965.
Hemker HC: Thrombin generation in a reconstituted system: a comment.
Thromb Haemost 2002, 87:551–554.
Béguin S, Kumar R, Keularts I, et al.: Fibrin-dependent platelet procoagulant
activity requires GPIb receptors and von Willebrand factor. Blood 1999,
Béguin S, Lindhout T, Hemker HC: The effect of trace amounts of tissue
factor on thrombin generation in platelet rich plasma: its inhibition by heparin.
Thromb Haemost 1989, 61:25–29.
endogenous thrombin potential. Thromb Haemost 1995, 74:134–138. Pub-
lished erratum appears in Thromb Haemost 1995, 74:1388.
Hemker HC, Béguin S: The love of the artist for his model: of thrombin gen-
eration. J Thromb Haemost 2004, 2:400–401.
clotting factors and TG in a system consisting of platelets and TF-bearing mono-
cytes and purified clotting factors. TG increases linearly with prothrombin concen-
tration but reaches an upper limit at minimal concentrations of the other clotting
factors. The authors explain this in terms of enzyme kinetics of the clotting process.
We consider it to demonstrate diffusion limitation of cell-bound TG.
Allen GA, Hoffman M, Roberts HR, Monroe DM 3rd: 2004.
Heemskerk JW, Bevers EM, Lindhout T: Platelet activation and blood coagu-
lation. Thromb Haemost 2002, 88:186–193.
Reverter JC: Fondaparinux sodium. Drugs Today (Barc) 2002, 38:185–194.
Béguin S, Kumar R, Keularts I, et al.: Fibrin-dependent platelet procoagulant
activity requires GPIb receptors and von Willebrand factor. Blood 1999,
bin generation in platelet-rich plasma in a VWF-GPIb-dependent process,
defective in Bernard-Soulier syndrome. J Thromb Haemost 2004, 2:170–
Induction of fibrin polymerization during the lag phase of TG by a snake venom
enzyme induces an immediate burst of TG that is inhibited by a monoclonal anti-
body against GPIb. Inhibition of polymerization decreases TG. So polymerizing
fibrin interacts with VWF so as to activate GPIb and produce platelet procoagulant
Hemker HC, Willems GM, Béguin S: A computer assisted method to obtain
the prothrombin activation velocity in whole plasma independent of thrombin
decay processes. Thromb Haemost 1986, 56:9–17.
Hemker HC, Wielders S, Kessels H, Béguin S: Continuous registration of
thrombin generation in plasma, its use for the determination of the thrombin
potential. Thromb Haemost 1993, 70:617–624.
Thrombin substrate binding to fibrinogen is mediated through exosite 1. Nonsub-
strate binding of thrombin to fibrin occurs at low affinity in the fibrin E domain and
at high affinity to a variant gamma chain, found in ∼15[ref] of the fibrinogen mol-
ecules (fibrinogen 2). Fibrin formation (antithrombin I) thus inhibits the appearance
of thrombin in the fluid phase and “high-affinity” thrombin-binding plays a dominant
role in this process.
Mosesson MW: Antithrombin I: inhibition of thrombin generation in plasma by
fibrin formation. Thromb Haemost 2003, 89:9–12.
Kumar R, Béguin S, Hemker HC: The influence of fibrinogen and fibrin on
thrombin generation: evidence for feedback activation of the clotting system
by clot bound thrombin. Thromb Haemost 1994, 72:713–721.
de Bosch NB, Mosesson MW, Ruiz-Saez A, et al.: Inhibition of thrombin gen-
eration in plasma by fibrin formation (Antithrombin I). Thromb Haemost 2002,
Kumar R, Béguin S, Hemker HC: The effect of fibrin clots and clot-bound
thrombin on the development of platelet procoagulant activity. Thromb Hae-
most 1995, 74:962–968.
Rijkers DT, Hemker HC, Tesser GI: Synthesis of peptide p-nitroanilides mim-
icking fibrinogen- and hirudin-binding to thrombin: design of slow reacting
thrombin substrates. Int J Pept Protein Res 1996, 48:182–193.
Hemker HC, Giesen P, Al Dieri R, et al.: Calibrated automated thrombin gen-
eration measurement in clotting plasma. Pathophysiol Haemost Thromb
By using a “slow” fluorogenic thrombin substrate and continuous comparison to a
simultaneously run calibrator, TG can be monitored automatically, on line, in clot-
ting PPP or PRP. The resulting thrombogram in PPP measures hypocoagulability
(hemophilias, oral anticoagulants, heparins, and heparin-likes), direct inhibitors)
and hypercoagulabilities (AT deficiency, prothrombin hyperexpression, protein C
and S deficiency, factor V Leiden, oral contraceptives). In PRP it is diminished in
thrombopathies, in von Willebrand disease, by antibodies blocking GPIIb-IIIa or
GPIb, or by antiplatelet drugs like aspirin and clopidogrel.
endogenous thrombin potential. Thromb Haemost 1995, 74:134–138.
Rosing J, Tans G, Nicolaes GA, et al.: Oral contraceptives and venous throm-
bosis: different sensitivities to activated protein C in women using second-
and third-generation oral contraceptives. Br J Haematol 1997, 97:233–238.
Sorensen B, Johansen P, Christiansen K, et al.: Whole blood coagulation
thrombelastographic profiles employing minimal tissue factor activation.
J Thromb Haemost 2003, 1:551–558.
Whole blood thrombelastography was used according to the traditional technique
curves appeared to be dependent on the nature and severity of the hemostatic
deficit in hemophiliacs and could be normalized with recombinant factor VIIa.
Carr ME, Martin EJ, Kuhn JG, Spiess BD: Onset of force development as a
marker of thrombin generation in whole blood: the thrombin generation time
(TGT). J Thromb Haemost 2003, 1:1977–1983.
Reports an assay that measures platelet contractile force as a surrogate marker of
Shima M: Understanding the hemostatic effects of recombinant factor VIIa by
clot wave form analysis. Semin Hematol 2004, 41:125–131.
Yamamoto J, Yamashita T, Ikarugi H, et al.: Gorog Thrombosis Test: a global
in-vitro test of platelet function and thrombolysis. Blood Coagul Fibrinolysis
Reports a technique for testing ex vivo blood flow. Aggregation and the explosive
TG result in occlusion. Occlusion time was dose-dependently inhibited by mono-
clonal antibody against GPIb, aurin tricarboxylic acid, monoclonal antibody against
GPIIb/IIIa, a GPIIb/IIIa antagonist, argatroban, and anti-vWF, but not by antifibrino-
gen. The test also measures thrombolysis.
Hemker HC, Lindhout T: Interaction of platelet activation and coagulation.
Fuster et al. 2004;to be published
Duchemin J, Pittet JL, Tartary M, et al.: A new assay based on thrombin gen-
eration inhibition to detect both protein C and protein S deficiencies in
plasma. Thromb Haemost 1994, 71:331–338.
risk factors of venous thrombosis on a thrombin generation-based APC re-
sistance test. Thromb Haemost 2002, 88:5–11.
Regnault V, Béguin S, Wahl D, et al.: Thrombinography shows acquired re-
sistance to activated protein C in patients with lupus anticoagulants. Thromb
Haemost 2003, 89:208–212.
Using thrombinography, APC resistance can be demonstrated in patients with lu-
pus anticoagulants. A long time lag is observed before the thrombin burst (lupus
anticoagulant effect) together with a marked inability of APC to diminish the throm-
bin activity. The effects were more outspoken in the presence of phospholipids
from patients’ platelets than with added phospholipids.
Al Dieri R, Peyvandi F, Santagostino E, et al.: The thrombogram in rare inher-
ited coagulation disorders: its relation to clinical bleeding. Thromb Haemost
The relation between clotting factor concentration, the ETP, and the severity of
bleeding was investigated in patients with congenital deficiency of factors II, V, VII,
X XI, and XII. In all the patients with severe bleeding, the ETP was less than 20[ref]
of normal. Bleeding tendency was absent or mild in patients with an ETP of 30[ref]
factor-induced thrombin generation by the mouse/human chimeric 7E3 anti-
body: potential implications for the effect of c7E3 Fab treatment on acute
thrombosis and “clinical restenosis.” J Clin Invest 1996, 98:863–874.
Keularts IM, Hamulyak K, Hemker HC, Béguin S: The effect of DDAVP infu-
sion on thrombin generation in platelet-rich plasma of von Willebrand type 1
and in mild haemophilia A patients. Thromb Haemost 2000, 84:638–642.
Turecek PL, Varadi K, Keil B, et al.: Factor VIII inhibitor-bypassing agents act
by inducing thrombin generation and can be monitored by a thrombin gen-
eration assay. Pathophysiol Haemost Thromb 2003, 33:16–22.
A TG assay seems suitable for monitoring the pharmacokinetics of inhibitor by-
passing agents during treatment and possibly for predicting responses to treat-
174 Hemostasis and thrombosis
A TG assay enables the pharmacodynamic and pharmacokinetic properties of fac-
Azar AJ, Cannegieter SC, Deckers JW, et al.: Optimal intensity of oral antico-
agulant therapy after myocardial infarction. J Am Coll Cardiol 1996,
Al Dieri R, Wagenvoord R, van Dedem GW, et al.: The inhibition of blood
coagulation by heparins of different molecular weight is caused by a common
functional motif: the C-domain. J Thromb Haemost 2003, 1:907–914.
For any type of heparin, the capacity to inhibit the coagulation process in plasma is
primarily determined by the concentration of the AT-binding pentasaccharide with
12 or more sugar units at its nonreducing end, ie, the structure that induces anti-
thrombin activity. Antifactor Xa activity hardly influences either ETP or APTT.
Varadi K, Negrier C, Berntorp E, et al.: Monitoring the bioavailability of FEIBA
with a thrombin generation assay. J Thromb Haemost 2003, 1:2374–2380.
Al Dieri R, Alban S, Béguin S, Hemker H. Thrombin generation for the control
of heparin treatment: comparison to the activated partial thromboplastin time.
J Thromb Haemost 2003;2:yy.
In 12 volunteers, 9000 IU of four heparins of different molecular weight distribution
was injected. The aPTT showed the effect of heparin in 34[ref] of the samples; the
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.
in platelet-rich plasma in the rat. Thromb Haemost 1999, 81:957–960.
Kessels H, Béguin S, Andree H, Hemker HC: Measurement of thrombin gen-
eration in whole blood: the effect of heparin and aspirin. Thromb Haemost
Thrombin generation assays Hemker et al. 175