Keywords
thrombin generation - hemophilia - bleeding - FXIa - FVIII
Introduction
Hemophilia A (HA) is a congenital X-chromosomal bleeding disorder characterized by
a deficiency of coagulation factor VIII (FVIII). Its clinical phenotype is classified
according to the remaining FVIII activity: less than 1% being severe, 1 to 5% being
moderate to severe, and more than 5% regarded as mild.[1] Despite this classification, the severity of the bleeding phenotype within these
three groups varies, suggesting that not only the level of FVIII determines the coagulation
potential in HA patients.
Global coagulation assays such as the thrombin generation assay (TGA) by calibrated
automated thrombography has been mainly used in research settings for the assessment
of hypercoagulability.[2] Over the past few years, TGA has seen an increase in applications for hypocoagulability
disorders such as HA.[3]
[4]
[5] Although TGA offers an attractive option to measure global coagulation potential
in HA populations, (pre)analytical standardization is often lacking. Although FVIII
measurements are able to precisely measure FVIII levels as low as approximately 1%.
By solely focussing on FVIII quantity, both the one-stage and the chromogenic variants
are unable to correlate their FVIII measurements with clinical bleeding phenotype.
TGA, which is a global coagulation assay, could better reflect the coagulation potential
of HA patients as FVIII is measured in its plasma context. However, current TGA assay
protocols have a limited sensitivity to low FVIII concentrations. Increasing the assay
sensitivity in these lower FVIII ranges, typically at trough levels, might allow for
better characterization of individual patients with severe HA and aid in predicting
their clinical bleeding tendency.
In this article, we report on optimization of TGA for measurements in low FVIII ranges
in HA patients. Based on our results, criteria for optimized TGA are proposed, which
might lead to a more precise and accurate estimation of the coagulation potential
in individual patients with hemophilia. The optimized hemophilia TGA will aid in analyzing
therapeutic effects of different FVIII-based treatments and emerging non-FVIII-based
therapeutics.[6]
[7]
[8]
Materials and Methods
Blood Withdrawal and Plasma Preparation
Blood was drawn using vacutainer 21-gauge needles (Becton Dickinson, Plymouth, United
Kingdom). Blood was collected in 3.2% citrated vacutainer tubes containing 25 µg/mL
thermostable inhibitor of contact activation (TICA) as the contact activation inhibitor.[9] Withdrawal was performed after collection of one small vacutainer clot activator
tube, which was discarded. Platelet-poor plasma was made by double centrifugation:
whole blood was centrifuged at 2,500 × g for 15 minutes, after which plasma was removed
for a secondary centrifugation at 2,500 × g for 15 minutes. Both centrifugation steps
were performed at room temperature without centrifugal break. Centrifugation leads
to concentration of TICA, resulting in a final concentration of around 50 µg/mL depending
on patient sample hematocrit. The resulting plasma was transferred into a new tube
and snap-frozen at −180°C and stored at −80°C until use.
Hemophilia A Plasma Pool
Blood of 10 severe HA patients at trough levels were combined into a hemophilia plasma
pool. All patients were on FVIII-replacement therapy. Trough levels were confirmed
to be between <1 and 3% with an average of 1.8%. Samples were handled as described
above, stored at −80°C and were thawed only once. Patients provided informed consent.
The study was conducted in accordance with the ethical standards and principles as
agreed upon in the Declaration of Helsinki.
Thrombin Generation
TGA was performed using calibrated automated thrombography (Thrombinoscope B.V., Maastricht,
The Netherlands).[2] TGA was activated by tissue factor (TF; Innovin, Baxter Diagnostics Ltd.) in absence
or presence of activated factor XI (FXIa; Innovative Research, Michigan, United States).
Recombinant FVIII spiking was performed with octocog alfa (Advate, Takeda Netherlands).
The assay was performed in presence of 30 µM phospholipids (PLs) 20/60/20 PS/PC/PE
at 37°C. TGA was assessed by addition of the Z-Gly-Gly-Arg 7-amino-4-methylcoumarin
low-affinity fluorescent substrate. TF and PLs were incubated for 1 hour in 25 mM
Hepes pH 7.4, 150 mM NaCl, 5% BSA (HNBSA 5% buffer). FXIa, when used, was co-incubated
with the TF/PL mix for 1 hour. FVIII was incubated with the plasma for 5 minutes before
addition of the TF/PL/(FXIa)-mix. Each measurement well consisted of 80 µL plasma,
25 µL CaCl2-containing coagulation trigger (with TF, FXIa, or combinations thereof), and 20 µL
of fluorescent substrate. In experiments including FVIII titration, the well setup
was altered to 80 µL plasma, 10 µL FVIII in HNBSA 5%, 15 µL coagulation trigger, and
20 µL of fluorescent substrate, totaling 125 µL. The Fluoroskan Ascent Reader (Thermo
labsystems, Helsinki, Finland) utilizing a 390/460 nm filter was used in conjunction
with the Thrombinoscope software to measure and calculate thrombin generation (TG).
All TG measurements included normal pool (NP) samples on the 96-well plates to monitor
intra- and inter-assay reproducibility.
Results
The contribution of contact activation during blood drawing and assay procedure was
investigated. A FVIII titration in a TF-activated (1 pM) setup was performed in presence
and absence of the contact activation inhibitor TICA ([Fig. 1]) in the blood drawing tube. TF-activated TG in severe HA patient plasma was significantly
influenced by contact activation. Peak height and endogenous thrombin potential (ETP)
were reduced by 45 to 63% and 23 to 46%, respectively, over the range of added FVIII
in presence of a contact activation inhibitor ([Fig. 1]). As a result, it was decided to add TICA to blood-drawing tubes during collection
for our severe HA plasma pool.
Fig. 1 Effect of contact activation on thrombin generation in severe hemophilia plasma.
Thrombin generation assays were performed in 2pM TF-initiated severe hemophilia plasma
in absence (A) and presence (B) of contact activation inhibitor. The plasma was spiked with 0% (blue), 2% (red),
5% (green), 10% (purple), 20% (orange), 50% (black), or 100% (brown) FVIII. Averages
of duplicate experiments are shown. FVIII, factor VIII; TF, tissue factor.
Next, a TF titration between 1 to 5 pM was performed in NP and HA pool plasma (HAPP).
NP plasma showed a gradual increase of both ETP and peak height with increasing concentrations
of TF ([Fig. 2A]). All curves in NP plasma showed a representative curve shape reflecting initiation,
propagation, and inhibition of TG. TF titration in HAPP, as expected, showed a significantly
decreased ETP and peak height compared to NP plasma ([Fig. 2B]). Moreover, TG at both 1 pM and 2 pM TF showed aberrant curve shapes due to lack
of a clear peak and due to prolonged tailing. As overstimulation of the extrinsic
pathway by an excess of TF might reduce the sensitivity toward the FXI–FVIII propagation
loop, the lowest TF concentration with a representative TG curve (3 pM TF) was chosen
for further experiments ([Fig. 2B]).
Fig. 2 Tissue factor (TF) titration with thrombin generation in normal plasma compared to
severe hemophilia plasma. TF titrations (1–5 pM TF) were performed in normal pool
plasma (A) and severe hemophilia A pool plasma (B). TF was added in concentrations of 1 pM (blue), 2 pM (red), 3 pM (green), 4 pM (purple),
and 5 pM (orange). Averages of duplicate experiments are shown.
A FVIII titration TGA was performed in pooled hemophilia plasma, initiated by 3 pM
of TF ([Fig. 3A]). FVIII activities between 0 and 5% failed to show a dose-dependent increase of
ETP or peak height. Moreover, the shallow curve shape with a flattened peak and prolonged
tail did not allow accurate analysis of ETP. In contrast, measurements of TGA in pooled
hemophilia plasma with added FVIII levels between 10 and 100% did show a dose-dependent
increase of TG in both ETP and peak height with adequate curve shapes. Thus, the sensitivity
towards added FVIII levels became apparent only at levels of FVIII of >10% and was
less pronounced in the lower, more clinically relevant FVIII ranges of <10% ([Fig. 3B]).
Fig. 3 FVIII titration in severe hemophilia plasma activated by 3 pM of tissue factor. Thrombin
generation in pooled severe hemophilia plasma was initiated by 3 pM of tissue factor.
FVIII titrations of 0% (blue), 2% (red), 5% (green), 10% (purple), 20% (orange), 50%
(black), and 100% (brown) were performed (A). Panel (B) shows a logarithmic extrapolation of peak heights from the data from panel (A).
Averages of duplicate experiments are shown. FVIII, factor VIII.
To increase sensitivity of TGA for lower levels (<10%) of FVIII, alternative conditions
for initiation of coagulation were investigated. Activation of TGA by addition of
only FXIa was measured in both NP plasma and severe hemophilia plasma. NP plasma showed
a dose-dependent increase in TG; however, addition of FXIa to severe hemophilia plasma
did not result in measurable TGA curves (data not shown). Only in presence of low
amounts of TF (1 pM), FXIa-dependent TG was observed ([Fig. 4A, B]). This observation was most likely explained by activation of low FVIII levels by
thrombin generated through extrinsic activation. In contrast to NP plasma, higher
concentrations of FXIa were needed to generate baseline TG curves in pooled HA plasma
([Fig. 4B, C]). Based on the results, 100 pM of FXIa, in combination with 1 pM TF, was chosen
as the optimal condition, as reliable TG was obtained with room for upward and downward
modulation. We next assessed possible replacement of FXIa with FIXa. In a similar
setup as with TF/FXIa, TF/FIXa was unable to generate sufficient TG in severe HA plasma
([Fig. 4D]). Alternatively, the replacement of the accessory component TF was investigated
by exchanging it for either FXa or thrombin ([Fig. 5]). TF/FXIa and Xa/FXIa showed TG in severe HA plasma, which was enhanced by the addition
of 5% FVIII in a comparable way. In contrast, thrombin/FXIa showed comparable results
to TF/FXIa and Xa/FXIa in the presence of 5% FVIII, but not in absence ([Fig. 5]). In order to obtain the most sensitive TG conditions, TF was chosen over FXa.
Fig. 4 Factor FXIa titration in pooled severe hemophilia A plasma and normal pooled plasma.
Thrombin generation in normal pooled plasma and severe hemophilia A plasma was measured.
(A) Normal plasma was titrated with FXIa. (B) Severe hemophilia A plasma was titrated with FXIa in presence of 1 pM of TF. (C) Normal plasma was titrated with FXIa in presence of 1 pM of TF. FXIa concentrations:
0 pM (blue), 25 pM (red), 50 pM (green), 75 pM (purple), 100 pM (orange), 150 pM (black),
250 pM (brown), and 500 pM (navy blue). (D) Severe hemophilia A plasma was titrated with FIXa in presence of 1 pM of TF. FIXa
concentration (panel D): 0 pM (blue), 50 pM (red), 100 pM (green), 250 pM (purple),
500 pM (orange), and 1 nM (black). Averages of duplicate experiments are shown. FXIa,
activated factor XI; TF, tissue factor.
Fig. 5 Thrombin generation in pooled severe hemophilia A plasma activated by FXIa in combination
with either TF, FXa, or thrombin. For each trigger combination, thrombin generation
was measured in presence (A) and absence (B) of 5% added FVIII. Triggers: 100 pM FXIa/1 pM TF (blue); 100 pM FXIa/5 pM thrombin
(red); 100 pM FXIa/12.5 pM FXa (green). Averages of duplicate experiments are shown.
FXIa, activated factor XI; TF, tissue factor.
In order to assess the optimal concentration of TF, a titration was performed in the
presence of the chosen fixed FXIa concentration (100 pM) in severe HA plasma ([Fig. 6]). As only an increase in TG is expected as a result of higher FVIII levels, the
lowest amount of TF (1 pM TF) was chosen for further experiments.
Fig. 6 Tissue factor titration in severe hemophilia plasma in presence of FXIa. Thrombin
generation in pooled severe hemophilia A plasma was initiated by addition of 100 pM
FXIa and various concentrations of tissue factor. Tissue factor was added in concentrations
of 1 pM (blue), 2 pM (red), 3 pM (green), 4 pM (purple), and 5 pM (orange). Averages
of duplicate experiments are shown. FXIa, activated factor XI.
To investigate the effect of inhibition of contact activation on the TF/FXIa dual
activated TGA, FVIII titration in the TF/FXIa-activated (1 pM/100 pM) TGA was performed
in presence and absence of a contact activation inhibitor ([Supplementary Fig. S1], available in the online version). No obvious differences were seen between absence
and presence of contact activation inhibitor, in contrast to results shown in absence
of FXIa ([Fig. 1]). The higher baseline TG in absence of added FVIII is explained by traces of FVIII
replacement therapy resulting in FVIII of 2% rather than ideal trough levels of <1%.
Although there were no differences in TG as a result of TICA addition in TGA measurements
in this experiment, differences in contact activation between several blood withdrawals
from individual patient with variable work-up time might still lead to variable levels
of contact activation and resulting FXIa levels. Thus, it was decided to maintain
TICA in the blood-drawing tubes.
Using the described method with the addition of both FXIa and low TF initiation (the
dual-activated TGA), we performed a FVIII titration in our pooled hemophilia plasma
([Fig. 7A]). Compared to the TF-only TGA setup ([Fig. 3]), the sensitivity for FVIII was significantly increased. The increase in sensitivity
to FVIII especially applied to lower, clinically relevant FVIII levels (<10%). Within
the titration range, both peak height and ETP increased in a dose-dependent manner,
while lag times decreased with increasing amounts of FVIII. To further explore the
benefit for dual TGA activation, current conditions were compared to FVIII titrations
in absence of FXIa ([Fig. 7B]). In the presence of 1 and 3 pM TF and in the absence of FXIa, the sensitivity for
FVIIIa in the lower range (<10%) was almost completely lost ([Fig. 7B]). The decrease in both peak height and ETP in absence of exogenous FXIa emphasized
the critical priming role of FXIa in this assay.
Fig. 7 FVIII titration in TF/FXIa-activated thrombin generation. Thrombin generation in
pooled severe hemophilia plasma was initiated by 100 pM of FXIa and 1 pM of TF. Addition
of FVIII was performed in increments of 0% (blue), 2% (red), 5% (green), 10% (purple),
20% (orange), 50% (black), and 100% (brown), respectively (A). Averages of duplicates are shown. Panel (B) shows FVIII sensitivity shown as peak height for 1 pM TF (red), 3 pM TF (green),
and 100 pM FXIa/1 pM TF (blue)-activated thrombin generation. Averages of duplicate
experiments are shown. FXIa, activated factor XI; TF, tissue factor.
To investigate application of the assay to individual patients rather than pooled
plasma, FVIII titrations in individual patients were performed ([Supplementary Fig. S2], available in the online version). The 10 individual severe hemophilia patients
that constitute the HAPP with trough levels between <1 and 3% were measured individually
as a function of added FVIII. All 10 measurements of patient's plasma showed reliable
baseline curves. Subsequent FVIII titration showed an increase in TGA in a similar
pattern compared to the average pool measurements. In all individual patient samples,
TGA showed similar sensitivity to the addition of FVIII in the lower ranges. All curves
leveled off around 20% FVIII after which a gradual shallow, more linear increase in
TG was seen up to the final spiked concentration of 100% FVIII.
Discussion
Measurement of TG in HA plasma can be a challenging effort. Due to low amounts of
FVIII present in HA plasma, it is tempting to overstimulate plasma samples with high
concentrations of TF to obtain measurable and reproducible TG curves. However, overstimulation
with TF will irrevocably lead to direct TG through extrinsic prothrombinase and thereby
reduce the sensitivity for the intrinsic pathway. When in turn TF concentrations are
reduced in severe hemophilia plasma, TGA curves will become indiscriminate or, in
some situations, even will become absent.[10]
[11] One could argue that these issues might be only relevant in a small subset of the
hemophilia patient samples, mostly at trough level situations of severe hemophilia
patients. However, this is exactly the population where improved sensitivity of coagulation
measurements is needed to aid clinical decision making and could provide the most
clinically relevant benefit.
The goal of this study was to optimize the use of TGA in the HA population by increasing
the sensitivity for the impaired intrinsic pathway. The optimized TGA should be able
to assess coagulation potential in the lower FVIII levels, mimicking trough levels
in patients. For reference purposes, a pool plasma consisting of 10 different severe
HA patients was created. The hemophilia pool plasma offered a better reference for
hemophilia patients as a group rather than individual patients.
During the setup of the assay, we specifically looked at the quality of the resulting
TGA curves in each of the development stages. We consider the shape of TGA curves
to be essential for correct data interpretation, although this is a vastly overlooked
aspect of the assay.[12] Due to low amounts of thrombin formed in HA plasma, especially when using regular
low TF-only activated TGA, a proper coagulation pattern consisting of initiation,
propagation, and inhibition might not occur, which is reflected in an aberrant curve
shape. However, the Thrombinoscope will still provide its calculated parameters like
peak height and ETP. To better understand the quality of the data, it is essential
to either show or describe the quality of curves in TGA for diagnostic research, especially
in hypocoagulant states such as hemophilia.
The benefit of contact activation inhibitors in standardizing the assay was subsequently
investigated. Standardizing and controlling analytical and preanalytical variables
in TGA have been shown to drastically improve assay variability.[6]
[13] One of the most significant and uncontrolled variables in TGA, especially in hemophilia,
is contact activation. As such, the use of contact activation inhibitors has already
been recommended for TGA within the HA patient population.[8] The extent of contact activation can vary greatly. It is influenced by a multitude
of factors including blood withdrawal, transport of samples, sample handling (time),
and materials used. The level of contact activation directly impacts the amount of
intrinsic activation of the sample. Our data showed that TGA is significantly influenced
by the amount of contact activation, especially in the currently used TF-only assay.
Intrinsic activation of the coagulation pathway increases sensitivity for FVIII, as
does contact activation. However, the amount of contact activation per sample is variable
and difficult to quantify. Addition of a contact activation inhibitor is therefore
crucial in controlling the amount of intrinsic activation and thereby increasing reproducibility.
In addition, adding an amount of 100 pM FXIa as an intrinsic activator leads to controlled
and reproducible TG in the plasma of hemophilia patients.
The focus on TGA initiation through the intrinsic pathway was chosen to make use of
levels of FVIII as the rate-limiting step of HA plasma to provide a new functional
HA assay within its specific plasma environment. This is especially relevant in the
context of new and emerging treatments for HA, which are mostly non-FVIII therapies.
Interestingly, both TF/FXIa- and FXa/FXIa-initiated TG gave similar TG curves, strengthening
the hypothesis that both intrinsic and extrinsic activators are required for increased
assay sensitivity in the lower FVIII ranges. Increasing the sensitivity for the intrinsic
pathway in the optimized dual TF/FXIa-activated TGA in HA has several advantages.
The most clinically relevant changes in coagulation potential are seen in severe hemophilia
patients. Small increases of coagulation potential, for example by medication, can
drastically improve the bleeding phenotype, whereas a similar increase in coagulation
potential in mild hemophilia patients will most often not modify the bleeding phenotype
at all. With the current TGA setup, we are now able to better measure TGA on these
clinically FVIII ranges.
Although TF-only activated TGA in severe HA plasma does not seem to possess the required
sensitivity to show small changes in coagulation potential, it might still be useful
in some settings. Several studies showed its use in patient monitoring,[3]
[4]
[14]
[15]
[16]
[17] bypassing agent dosing[18]
[19]
[20]
[21]
[22] and evaluation of novel treatments.[23]
[24]
[25]
[26]
[27] Due to the low sensitivity of TF-activated TG for low amounts of FVIII, currently
only studies are performed that either measure large changes in coagulation potential
by adding larger amounts of FVIII or measure averages in large population-based studies.
Implementing the optimized dual TF/FXIa-activated TGA will aid current and future
uses of TG in predicting clinical efficacy of treatment in the individual HA patient
by providing a baseline measurement and additional sensitivity for small increases
in coagulation potential by factor replacement or alternative treatment strategies
even at low FVIII ranges.
Conclusion
We propose optimization of the TGA in severe HA by dual-activation of the assay by
TF/FXIa leading to increased sensitivity toward added FVIII compared to activation
by TF only, especially in lower FVIII ranges. The proposed setup allows for more sensitive
and reproducible measurements at low, clinically relevant, FVIII levels than the currently
used TF-only activated assay. With many new therapeutic options for hemophilia being
introduced recently, it has become increasingly important to have a robust and sensitive
assay to measure the coagulation potential of HA patients. The increased sensitivity
of the optimized dual TF/FXIa-activated TGA might allow for better individual characterization
at baseline, interventions, and follow-up regardless of treatment strategies.
What is known about this topic?
-
Adequate assessment of thrombin generation at trough levels in severe hemophiliacs
is hampered by current protocols.
-
Thrombin generation is able to differentiate between plasmas from severe, moderate,
and mild hemophiliacs.
-
Contact activation inhibition is a relevant determinant of thrombin generation in
severe hemophilia A.
What does this paper add?
-
Optimized protocols for thrombin generation allow for measurement of trough levels.
-
Dual TF/FXIa activation of thrombin generation enables high sensitivity assessment
of plasma from individuals within severe hemophilia A populations.
-
The variable unknown amount of contact activation in plasma can be blocked by contact
activation inhibitors, while maintaining a controlled amount of intrinsic activation
by addition of FXIa.
