Keywords
thrombin - dabigatran - microscale thermophoresis - platelets
Introduction
Thrombin is not only the prima ballerina in the coagulation cascade but also a strong
platelet activator binding to the protease activated receptors (PARs) 1 and 4.[1] The catalytic activity and specificity of thrombin are highly dependent on two intramolecular
recognition sites located distant from the active site. These domains, designated
as fibrinogen recognition site and heparin binding site, or exosite I and II, respectively,
facilitate proteolysis by interacting with anionic surfaces on various substrates
([Fig. 1]). Exosite II binds to heparin,[2] factors V and VIII,[3] PAR4,[4] and may also bind to glycoprotein Ib (GPIb)[4] on platelets. Exosite I is the binding epitope for bivalirudin, fibrinogen, thrombomodulin,
hirudin, and PAR1 and contributes to binding to GPIb.[5] The exosites I and II on thrombin may be blocked by the DNA oligomers HD1 and HD22,[6] respectively. The binding of thrombin exosite I to PAR1 is mediated by a hirudin-like
sequence immediately downstream of the tethered ligand domain in PAR1.[7] This sequence does not exist on PAR4. Instead, thrombin has been reported to have
low affinity for the thrombin cleavage site of PAR4.[8]
Fig. 1 Important structural components of human α-thrombin. Human α-thrombin bound to dabigatran
in the active site pocket has been shown. The secondary structural components of thrombin
are represented as cartoons and dabigatran as sticks. The structural units of thrombin
like exosite 1 (red), exosite 2 (blue), active site (orange), and the 60 loop (green),
which are considered important for binding, catalytic activity, and substrate specificity,
are shown as spheres.
Binding of thrombin to GPIb via exosite II facilitates the binding to PAR1,[4]
[9] whereas exosite I binds directly to the hirudin-like domain of PAR1.[10] Thrombin's strong affinity for PAR1 allows transient PAR1 signaling sufficient to
elicit significant α-granule release and platelet aggregation in presence of therapeutic
heparin concentration at physiological antithrombin levels. In contrast, inhibition
of thrombin-induced PAR4-mediated platelet activation was complete and sustained.[11]
Oral anticoagulants are used worldwide to prevent thrombosis, for example, in patients
with atrial fibrillation, venous thrombosis, and prosthetic heart valves. In recent
years oral reversible direct thrombin and factor Xa inhibitors have substituted vitamin
K antagonists to a large extent for most indications, with the exceptions of prosthetic
heart valves and patients with thromboembolism and being triple positive for lupus
anticoagulant. The first drug of this class in clinical use at present, approved in
2010, was dabigatran etexilate from Boehringer-Ingelheim which is a prodrug metabolized
to its active form dabigatran by endogenous esterases.[12]
[13] Dabigatran is a direct, reversible thrombin inhibitor with half-maximal inhibitory
concentration (IC50) of 9.3 nmol/L,[14] Ki 4.5 nmol/L.[15] Dabigatran inhibits both free and fibrin-bound thrombin and has no effect on thrombin
binding to a glycoprotein Ibα peptide.[16] In this study we report that along with blocking the active site, dabigatran also
inhibits binding of thrombin to platelets.
Materials and Methods
Reagents and Antibodies
H-D-Phe-Pro-Arg-chloromethylketone (PPACK) dihydrochloride, apyrase, PGI2, 14-azido-3,6,9,12-tetraoxatetradecan-1-amine and disposable PD 10 desalting columns
were from Merck, Darmstadt, Germany. Human α-thrombin was purchased from Haematologic
Technologies, Vermont, USA. The DNA aptamers HD1 and HD22 and dibensocyclooctyne (DBCO)
functionalized clickable aptamers were from Biomers.net (Ulm, Germany). Dabigatran
was purchased from Selleckchem (Munich, Germany). Argatroban (Novastan) was purchased
from FrostPharma, Danderyd, Sweden. Melagatran was purchased from MedChemTronica,
Sollentuna, Sweden. Blood collection tubes were from Greiner Bio-One GmbH, Frickenhausen,
Germany. Amine coupling kit, HBS-EP buffer, and acetate buffer were from Cytiva Europe
GmbH, Uppsala, Sweden. Antibodies, anti-CD62P (P-selectin)-phycoerythrin (PE) or allophycocyanin
(APC) with corresponding isotype antibodies, were from BD Biosciences, thrombin monoclonal
antibody (MA1–43019) and Alexa Fluor™ 555 Antibody labeling kit were from Life Technologies
Europe BV, and fluorescein isothiocyanate (FITC)-conjugated chicken antibodies directed
toward human fibrinogen was provided by Diapensia AB (Linköping, Sweden, www.diapensia.se). Protein labeling kit RED-NHS 2nd generation and Monolith Premium Capillaries were
from NanoTemper Technologies, Munich, Germany.
Blood Collection and Sample Preparation
Blood from healthy human volunteers was collected, after informed consent, in Vacuette®, 9 mL ACD-A tubes to prepare platelets rich plasma (PRP) and washed as reported previously.[17] Briefly, the blood was centrifuged at 150 × g for 15 minutes at room temperature
(RT) to get PRP and then again at 480 × g after addition of 1 U/mL apyrase and 100 nM
PGI2 to pellet the platelets. The plasma phase was carefully replaced by Krebs Ringer
glucose (KRG; 20 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, 10 mM glucose, pH 7.3) buffer supplemented with apyrase and PGI2 and platelet count
was adjusted after recalcification. The platelet samples were incubated for 30 minutes
at RT after isolation before usage in any experiments. The collection of blood samples
from healthy donors was approved by the local ethics committee in Linköping, Sweden,
decision No. 2012/382–31.
Chromogenic Thrombin Substrate Assay
Thrombin activity was measured using the Chromogenix S-2238™ substrate (DiaPharma,
West Chester, USA), according to the manufacturer's instructions using Enspire multimode
plate reader (Perkin Elmer, Sollentuna, Sweden) to evaluate and compare the effect
of labeling of thrombin and the treatment of PPACK (covalent inhibitor of active site).
Thrombin was labeled using protein labeling kit RED-NHS 2nd generation according to
the manufacturer's instructions. For PPACK treatment, thrombin was first incubated
with double molar concentration of PPACK dihydrochloride for 30 minutes and unbound
PPACK was removed using PD10 chromatography column. Briefly, unlabeled, labeled, and
PPACK thrombin were diluted in 50 µL buffer (50 mM Tris, with 0.2% bovine serum albumin
(BSA) pH 8.3) and incubated at 37°C for 4 minutes. The reaction was started by mixing
50 µL of S-2238 substrate and incubated at 37°C for 3 minutes. The final concentration
of thrombin and PPACK in the reaction mixture was 0.16 µM. The reaction was stopped
with the addition of 25 µL of 20% acetic acid, and the absorbance was measured at
405 nm. The data was calculated using one-way ANOVA followed by Bartlett's test using
GraphPad Prism version 10.0.2 for Windows, GraphPad Software, Boston, Massachusetts,
USA, www.graphpad.com.
Flow Cytometry
Binding of thrombin to platelets was analyzed by flow cytometry using washed platelets
(300 × 109/L). RED-NHS-labeled thrombin (0.5 µM) was used to determine the binding of thrombin
to platelets in the presence or absence of various inhibitors. To study the contribution
of the thrombin exosites, DNA aptamers HD1 (exosite 1 inhibitor) or HD22 (exosite
2 inhibitor) was used at 1 µM concentration as previously reported.[4] To study the role and contribution of the thrombin active site, four different active
site inhibitors dabigatran (318 nM), argatroban (318 nM), melagatran (1 µM) or PPACK
was used. For experiments with PPACK, it was prepared as described in above section
and 0.5 µM PPACK thrombin labeled with RED-NHS 2nd generation was compared with RED-NHS
2nd generation labeled thrombin. HD1 and HD22 (1 µM) were also used with PPACK thrombin.
Platelet activation was assessed by using anti-human-CD62P (P-selectin, PE or APC
labeled and 1.25 µg/mL) and FITC-conjugated anti-human fibrinogen chicken antibody
(1 µg/mL). Corresponding isotype antibodies were used as a control. Briefly, washed
platelets were incubated with various combinations of thrombin, antibodies, and inhibitors
in HEPES buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM glucose, 1 mg/mL bovine serum albumin, and 20 mM HEPES, pH 7.40) and dimethyl
sulfoxide (DMSO) (final concentration 0.5% v/v) in the dark at RT for 10 minutes.
Samples were then diluted 1:10 with HEPES buffer and analyzed by flow cytometry on
a Gallios™ flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA). The median fluorescence
intensity (MFI) was used for analysis. MFI of thrombin without any inhibitors was
set to 100% and other results were normalized against it and presented as relative
fluorescence intensity (RFI). These experiments were also performed with unlabeled
thrombin using and Alexa Fluor™ 555 labeled thrombin monoclonal antibody which resulted
in similar outcome. The data was calculated using one-way ANOVA followed by Brown-Forsythe
test using GraphPad Prism. For IC50 analysis, washed platelets were “spiked” with
dabigatran to the final concentrations of 2 to 600 nM and 0.5% (v/v) DMSO in all samples
in HEPES and analyzed as described above. The logarithm of the dabigatran concentration
was plotted against the response (expressed in MFI) and the IC50 values were calculated
using the GraphPad Prism's built-in equation “Dose-Response - Inhibition” and “log(inhibitor)
vs. response - Variable slope (four parameters).” The curves are fit using nonlinear
regression with the drug concentrations transformed to a logarithmic scale.
Microscale Thermophoresis
Microscale thermophoresis (MST) was used to analyze the interaction of thrombin with
platelets. Thrombin was labeled using NHS-Red 2nd generation and the concentration
of thrombin to be used was determined by the degree of fluorescence using pretest
analysis of the Monolith NT.115 i software (NanoTemper) and the average concentration
used was 55 nM. The platelet concentration used was approximately 1,800 × 109 /mL and 1,276 PAR1,[18] 539 PAR4,[19] and 25,000 GPIb[20] receptors per platelet were used for the calculation of receptor concentration.
Thus, the highest ligand concentration of platelets in the MST experiment was 80 nM
which was diluted 16 times serially and evaluated for binding to constant thrombin
concentration to get the binding curve. Briefly, washed human platelets (highest concentration
1,800 × 109 /mL) were diluted serially 16 times using PBS + 0.005% NP40 buffer, pH 7.4 and mixed
with equal amount (v/v) of RED-NHS-labeled thrombin (55 nM) diluted in the same buffer
in the presence or absence of various inhibitors like HD1 (1 µM), HD22 (1 µM), dabigatran
(318 nM), or argatroban (318 nM) and transferred to the Monolith Premium Capillaries.
The capillaries were placed in the Monolith instrument and thermophoresis was measured
at RT with medium MST power and 80% LED intensity. The data was analyzed using Monolith
NT.115 i software. PPACK thrombin was used instead of regular thrombin for the experiments
of PPACK. To investigate the effect of dabigatran (318 nM) on thrombin exosites, binding
of DNA aptamers HD1 (highest concentration 1 µM) or HD22 (highest concentration 1
µM) or heparin (highest concentration 1 U/mL) to thrombin in a buffer without platelets
was evaluated. As described above the ligand (HD1/HD22/heparin) was diluted 16 times
serially and mixed with equal amount (v/v) of RED-NHS-labeled thrombin (55 nM) in
the presence or absence of dabigatran 318 nM or 3,180 nM and binding was evaluated
using MST. Data analyses were performed using the MO. Affinity analysis software (NanoTemper)
and normalized display of fraction bound versus ligand concentration, or ΔFnorm (%)
versus ligand concentration were used. For plots with fraction bound, ΔFnorm values
of the curve are divided by curve amplitude resulting in range of values between 0
and 1. To compute change in normalized fluorescence ΔFnorm (%) the baseline Fnorm
values were subtracted from all data points. For the statistical calculations shown
in [Fig. 2], the binding curves from the MST experiments are fitted using the one phase decay
equation in the exponential model using GraphPad Prism.
Fig. 2 Inhibition of thrombin binding to platelets as measured by microscale thermophoresis
(MST). Binding of thrombin to the platelets and the effect of inhibitors were evaluated
using MST. In this normalized display, the change in normalized fluorescence (ΔFnorm)
of thrombin was plotted against the concentration (conc) of the thrombin receptors
on platelets in log scale. Binding curve of thrombin to platelets without any inhibitors
(control) is compared with the binding of thrombin (55 nM) to platelets in the presence
of (A) thrombin exosite specific inhibitors HD1 (1 µM) or HD22 (1 µM) or both and (B) thrombin active site inhibitors dabigatran (318 nM), argatroban (318 nM), or PPACK
(55 nM). The data was calculated using the one phase decay equation in the exponential
model using GraphPad Prism. The results shown are mean with standard deviation (SD)
represented as shadow outlines around the binding curves, where number of blood donors
(n) ≥5.
Surface Plasmon Resonance
Surface plasmon resonance (SPR) was used to evaluate the effect of dabigatran on the
binding of thrombin to DNA aptamers HD1 and HD22 using a Biacore 3000 system. HD1
and HD22 were immobilized in multiple steps in separate channels on a CM5 sensor chip
(Biacore, Uppsala, Sweden). Briefly, 75 µL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) and N-hydroxysuccinimid (NHS) were injected at a flowrate of 5 µL/min; then
90 µL 10 mM 14-azido-3,6,9,12-tetraoxatetradecan-1-amine in pH 11 potassium dihydrogen
phosphate/NaOH buffer was injected at a flowrate of 1 µL/min. This was followed by
an injection of 60 µL ethanol amine at 10 µL/min. Finally, 90 µL DBCO functionalized
aptamer at a concentration of 42 µg/mL in pH 4 acetate buffer was injected at 1 µL/min.
The DBCO moiety on the aptamer will bind to the azide via click chemistry. Thrombin
was used at 100 nM as maximum response was obtained at this concentration. It was
mixed with three molar equivalents of dabigatran dissolved in DMSO or the corresponding
volume of DMSO and injections of 30 µL were performed at 10 µL/min, repeated three
times. The data was analyzed using MATLAB (R2023b). The data was filtered for noise
using the smooth function with a 5-point moving average over areas where the needle
movement of the Biacore induced additional noise. Spike artifacts induced by the large
jump in refractive index upon start and finish of injection were removed using the
Hampel function. Baseline correction was done using a straight line and an asymmetric
truncated quadratic function using script by Mazet.[21] The used MATLAB scripts and raw data are available upon request.
Results
Binding of Thrombin to Platelets and Effect of Inhibitors
The binding of thrombin to platelets was determined using fluorescently labeled thrombin
(0.5 µM) and washed platelets, measured by flow cytometry. The labeling of thrombin
had no significant effect on the enzymatic activity as confirmed by the chromogenic
substrate assay. Thus, compared with unlabeled thrombin (0.16 µM), RED-NHS-labeled
thrombin (0.16 µM) retained 94% activity whereas PPACK-treated thrombin (0.16 µM)
showed 0.5% activity ([Supplementary Fig. S1], available in the online version). When considering total thrombin binding to platelets
in the absence of any inhibitors as 100%, HD1 (1 µM) and HD22 (1 µM) inhibited thrombin
binding to platelets by 43 and 32%, respectively ([Fig. 3]). The combination of both aptamers inhibited binding completely, as expected. In
these experiments, dabigatran (318 nM), a thrombin active site inhibitor, was used
as a positive control that inhibits enzymatic activity. Surprisingly, along with inhibiting
the enzymatic activity, dabigatran also blocked thrombin binding to the platelets.
To confirm the efficacy of the inhibitors and the viability or reactivity of the platelets
used for the experiments, platelet activation/function markers like P-selectin exposure
and fibrinogen binding to platelet were also measured, which showed similar trends,
complementing the thrombin binding results ([Fig. 3]). The result on binding indicated that the blockage of the active site also blocks
the binding of thrombin exosites I and II. Thus, the active site, along with the exosites,
plays an important role in binding thrombin to platelets. To confirm this hypothesis,
we used argatroban (318 nM) and melagatran (1 µM) inhibitors, similar in size, nature,
and function to thrombin, which showed similar characteristics, i.e., inhibition of
thrombin binding to platelets along with blocking the active site ([Fig. 3] and [Supplementary Fig. S2], available in the online version). PPACK, another active site inhibitor, also blocked
thrombin binding to platelets, albeit not completely like dabigatran ([Supplementary Fig. S3], available in the online version). PPACK, however, is structurally different from
dabigatran and is a covalent inhibitor of the active site. To rule out any interference
of fluorescence labeling on thrombin binding to platelets, the results obtained with
labeled thrombin were confirmed by unlabeled thrombin and an antithrombin monoclonal
antibody ([Supplementary Fig. S4], available in the online version), which showed a similar trend and strengthened
our findings. Similarly, the effect of pre-incubation of dabigatran to thrombin was
also evaluated, which showed that dabigatran-inhibited thrombin binds to the platelets
irrespective of pre-incubation status ([Supplementary Fig. S5], available in the online version).
Fig. 3 Inhibition of thrombin binding to platelets and effects on platelet activation by
flow cytometry. Thrombin binding to platelets and its effect on platelet activation
have been shown in the presence and absence of various inhibitors of the thrombin
exosites and the active site. Platelet activation markers P-selectin exposure and
fibrinogen binding were measured to confirm inhibitors' efficacy and platelet reactivity.
Negative control test without thrombin has been compared and shown here as control.
Median fluorescence intensity of thrombin without any inhibitor has been considered
as 100% and the relative fluorescence intensity (%) to thrombin without any inhibitor
has been shown as bar graph. The data was calculated using a one-way ANOVA test and
results shown are the mean with standard error of mean (SEM) where number of blood
donors (n) ≥5. RFI, relative fluorescence intensity.
Fig. 4 Effect of dabigatran on thrombin binding to exosite ligands by microscale thermophoresis
(MST). The effect of dabigatran on exosite ligands binding to thrombin without platelets
was analyzed by MST. Binding of NHS-labeled thrombin (55 nM) to exosite 1 ligand HD1
(A; highest concentration 1 µM), exosite 2 ligand HD22 (B; highest concentration 1 µM), or another exosite 2 ligand heparin (C; highest concentration 1 U/mL) in the presence (+) or absence (−) of dabigatran (318 nM)
was measured by MST. In this normalized display, the change in normalized fluorescence
(fraction bound) is plotted against the concentration of the ligand in log scale.
All the results shown are mean with standard deviation (SD) where n ≥ 3.
Fig. 5 Surface plasmon resonance (SPR) sensogram of thrombin injected onto an SPR chip with
a carboxylated dextran with immobilized aptamers. The interactions between thrombin
exosites and DNA aptamers were analyzed. The aptamers HD1 and HD22 are immobilized
in separate flow channels to an increase in resonance units (RU) of 971 and 1,395,
respectively. Thrombin was injected at a concentration of 100 nM in the presence and
absence of 300 nM dabigatran. (A, B) Sensograms of thrombin in the presence and absence of dabigatran injected over immobilized
HD1 and HD22, respectively. To further evaluate the interaction between thrombin and
HD22 the injection volume was doubled to 60 uL (C). Overall, no significant differences between thrombin in the presence and absence
of dabigatran can be observed. Although response curves are of different shapes for
the two aptamers, the addition of dabigatran does not alter the response.
Binding Analysis Using Microscale Thermophoresis
We used MST to validate the dabigatran effect on thrombin binding to platelets. Complementing
the findings observed with flow cytometry, HD1 (1 µM) and HD22 (1 µM) inhibited thrombin
(55 nM) binding to platelets, but not completely ([Fig. 2A]). However, dabigatran (318 nM) showed complete inhibition of binding using MST ([Fig. 2B]). To further confirm the effect of blockage of active site on overall binding, we
used argatroban (318 nM) as well as the covalent inhibitor of active site, PPACK (55 nM),
both of which inhibited binding of thrombin to the platelets.
Effect of Dabigatran on Thrombin Exosites
To further investigate the mechanism by which dabigatran block thrombin binding to
platelets, we assessed the effect of dabigatran binding to thrombin active site on
the binding of HD1 and HD22 to the exosites. This was achieved by evaluating the binding
of thrombin to HD1 and HD22 in the presence and absence of dabigatran in a pure system
without platelets using two biophysical methods, microscale thermophoresis[22]
[23] and SPR.[24]
MST did not show any effect of the presence or absence of the dabigatran (318 nM)
on thrombin binding to HD1 (1 µM; [Fig. 4A]) or HD22 (1 µM; [Fig. 4B]) or heparin (1 U/mL) ([Fig. 4C]). Additionally, increasing the concentration of the dabigatran 10 times (3,180 nM)
also had no effect on exosite ligands binding ([Supplementary Fig. S6], available in the online version).
Fig. 6 IC50 of dabigatran for thrombin binding to platelets by flow cytometry. The inhibition
potential of dabigatran for thrombin binding to platelets and platelet activation
was assessed using flow cytometry. Increasing concentration of the dabigatran (2–600 nM)
as log transformation of concentrations to base 10 is plotted against the median fluorescence
intensity (MFI) of thrombin binding, P-selectin exposure, or fibrinogen binding and
IC50 values were calculated as described in the methods. The results shown are the
mean with standard error of mean (SEM) where number of blood donors, n ≥ 5.
SPR also did not indicate any effect of dabigatran binding to thrombin on interaction
with either HD1 ([Fig. 5A]) or HD22 ([Fig. 5B]). The shape and intensities (response signal) of the sensograms with and without
dabigatran were very similar. HD22 initially showed a minor trend where the thrombin–dabigatran
complex might have a slightly higher affinity to HD22; however, increased injection
volume (60 μL) showed no differences in the sensograms ([Fig. 5C]). The signal intensity of the SPR experiments were on a relatively low level, which
is consistent with other published data on thrombin in SPR.[25]
[26] To allow closer inspection of the obtained sensograms individual plots can be found
in [Supplementary Fig. S7] (available in the online version).
Dabigatran-Mediated Inhibition of Binding is Dose Dependent
To find out whether the dabigatran effect on thrombin binding is dose dependent or
not, IC50 analysis was performed. Dabigatran concentrations from 4 to 600 nM were
used for these experiments and their effect on thrombin binding and its functional
activity indicators P-selectin exposure and fibrinogen binding was evaluated using
flow cytometry and fluorescently labeled thrombin and the respective antibodies. We
found the effect of dabigatran to be dose dependent and that the IC50 for inhibition
of binding was 118 nM. The IC50 for inhibition of P-selectin exposure and fibrinogen
binding was 126 and 185 nM, respectively ([Fig. 6]).
Discussion
In this study we have shown that the thrombin active site inhibitor dabigatran effectively
inhibits thrombin binding to all platelet thrombin receptors. The binding was analyzed
using flow cytometry and further characterized by microscale thermophoresis and surface
plasmon resonance.
Most studies analyzing affinity or binding of thrombin to platelets used radiolabeled
thrombin and were performed more than 30 years ago; at that time the platelet thrombin
receptors PAR1 and 4 were not yet identified.[27]
[28] Although the use of radiolabeled proteins to analyze the interaction between two
biomolecules has been a classic method, it is not practical due to obvious health
hazards and strict regulations. Thus, to confirm our findings, we used two modern
biophysical techniques specialized on the measurement of interactions between biomolecules,
MST and SPR. Both the techniques are safe and have been successfully applied to measure
the affinity between biomolecules. MST can analyze biomolecular interactions in an
immobilization-free system where one of the binding partners is fluorescently labeled
and its motion in a micro temperature gradient is measured. This technique is particularly
useful for this study as live platelets can be used as one of the partners to analyze
the binding characteristics. On the other hand, SPR is a standard and valuable method
specialized in analyzing interactions between molecules with respect to both affinity
and kinetics.[29]
[30]
It is a possibility that the labeling of thrombin with a fluorescent dye might affect
its ability to bind or function properly; however, labeled thrombin's unaffected ability
to cleave S-2238 chromogenic substrate ([Supplementary Fig. S1], available in the online version), eliciting P-selectin exposure, fibrinogen binding,
and similar results obtained when using antithrombin antibody ([Supplementary Fig. S4], available in the online version) rule out this suspicion. Further, dabigatran quenching
the fluorescence signal or affecting exosite affinity can also be excluded by unaffected
binding of exosite ligands to the thrombin in the presence of dabigatran which is
demonstrated with both MST and SPR ([Figs. 4] and [5]). Although, PPACK is a potent inhibitor of the active site, it showed only 50% reduction
in the binding when evaluated by flow cytometry ([Supplementary Fig. S3], available in the online version), which has also been observed before.[31] Further, the remaining approximately 50% binding was not only due to the exosites,
as blockage of the exosites by aptamers could not abolish the binding to platelets
completely ([Supplementary Fig. S3], available in the online version). However, when analyzed by MST, no binding could
be detected to platelets which points toward the differences in techniques but strengthened
the finding of this study as dabigatran showed inhibition irrespectively. The inhibition
of thrombin by dabigatran has been shown to be the active site blockage with a reported
Kd of 10 nM[15] for thrombin-induced aggregation of gel-filtered platelets; however, to the best
of our knowledge, the effect of dabigatran on binding of thrombin exosites has not
been reported. The IC50 values were obtained using flow cytometry and washed platelets.
IC50 for platelet activation markers P-selectin exposure (122 nM) and fibrinogen binding
(185 nM) was much higher than the reported 10 nM[15] using aggregometry; however, the same study has reported the IC50 of 560 nM when
a thrombin generation assay was used. Similarly, another study[29] has reported the IC50 of 2 µM using thrombin generation method indicating a large
variation in the IC50 values obtained, probably dependent on the system and methodology
used. However, the IC50 values we obtained were well within the range of reported
drug concentrations in patients where the peak concentration in elderly healthy subjects
was approximately 540 nM.[13]
[32]
Our results demonstrate that the small molecules dabigatran, argatroban, and PPACK
binding to the active site of thrombin strongly attenuate the binding of thrombin
to the surface of platelets, in addition to the well-known inhibition of the enzymatic
activity. Our findings are in good agreement with an earlier study where numerous
important interactions between PAR1 N-terminus uncleaved fragment and thrombin active
site was shown by crystallography[10] and, thus, any disturbance in these interactions can lead to destabilization of
binding. Further, thrombin active site occupancy by a ligand has been reported to
decrease solvent accessibility of the regions in vicinity of the active site, induce
backbone dynamics changes, and inhibit exosite interaction to its ligands.[16]
[33]
[34] Among these, the most relevant study supporting our observations has shown that
dabigatran attenuates the binding of various substrates to exosite 1 or 2 of thrombin;
however, surprisingly in their study argatroban enhanced the exosite binding of its
substrates.[16] Contrary to these, it has been shown that dabigatran does not affect thrombin interaction
with fibrinogen, which indicates that dabigatran-bound catalytically inactive thrombin
may still be able to interact with its substrates via exosite 1. This observation
has also been reported earlier.[35]
[36] However, in these studies cells other than platelets were used and the effect that
they have measured, i.e., PAR1 expression on cell surface after prolonged incubation
or destabilization of the endothelial barrier, is not relevant in case of the platelets.
We also investigated the effect of dabigatran on binding of DNA aptamers to thrombin
exosites by MST and SPR, which showed that in the absence of platelets, dabigatran
does not inhibit ligand binding to thrombin exosites. Although the shape of the interaction
curve for thrombin and HD1 is not a typical curve obtained in SPR measurements it
is in good agreement with results published in this field. Yeh et al studied the binding
of thrombin and fibrinogen.[16] Wang et al[37] studied the interaction between thrombin and berberin. The resulting curves in both
publications were of similar shapes as in this study. The reason for the rapid binding
and releasing observed in these curves would be interesting to investigate but is
outside of the scope of this study. As SPR is only measured at one thrombin concentration
it is not possible to infer detailed conclusions on the possible effect on kinetics
that dabigatran might exert. The shapes and intensities of the curves in both presence
and absence are very similar and therefor the binding of exosites I and II is not
altered to a degree where it may explain the results where dabigatran greatly decreases
the binding of thrombin to platelets.
Studies using isolated systems of thrombin with its natural or synthetic substrates
do not have the similar outcome when cells or platelets have been involved. Thus,
our results indicate that dabigatran binding does not affect exosite structure per
se to a major extent, allowing small free molecules like peptides and DNA aptamers
or even larger molecules like fibrinogen to bind. Dabigatran still induces enough
dynamic change to affect its binding to the complex receptor system like platelets.
Structural, biochemical, and mutagenesis analysis suggests that in most cases, both
exosites are involved in binding and/or catalytic functions, either directly via the
substrate interaction or indirectly by facilitating the binding by interacting with
the cofactor.[38] PARs are known to have a crosstalk between same or other types of receptors and
show diversified interactions and functions. Dimerization is, therefore, a common
phenomenon in PAR activation.[39]
[40] GPIb acts as a cofactor for thrombin to bind to PARs on the platelets[41] and, similarly, PAR1 acts as a facilitator for PAR4 cleavage by thrombin.[42] The observation of various homo- or hetero-dimerization of receptors on platelet
surface like PAR1/PAR1, PAR4/PAR4,[43]
[44] PAR1/PAR4,[43] and PAR4/P2Y12[45] supports the hypothesis that thrombin binding to platelets is complex, possibly
requiring all three structural components together, which may explain the dabigatran
effect observed in this study. One of the limitations of this study is the lack of
knowledge on how dabigatran inhibited thrombin would react with ligands other than
platelets under physiological conditions. The other limitation is that the exact mechanism
behind inhibition elicited by dabigatran on the binding of thrombin to platelets is
unclear at present. Two insertion loops near the active site cleft, known as the 60-loop
and the γ loop ([Fig. 1]), have been reported to play key roles in determining the substrate and inhibitor
specificity as well as catalytic activity of thrombin[46]
[47]
[48] and might have a role in this mechanism. However, it will have to be evaluated structurally
which is beyond the scope of the current study.
The inhibition by dabigatran of thrombin binding to platelets may contribute to its
antithrombotic effect. So far dabigatran is the only direct oral anticoagulant drug
reported to be associated with less ischemic stroke compared with warfarin,[12] although there are no randomized clinical trials comparing DOACs head-to-head. However,
the dual mechanism inhibiting thrombin activation of platelets may increase bleeding
risk; possibly the effect would be most important at lower concentrations of dabigatran.
Interestingly, in a large Swedish registry study, patients with non-valvular atrial
fibrillation on reduced dose of apixaban had lower risk for major bleeding hazard
ratio (HR) of 0.62 (95% CI 0.44–0.88) in comparison with patients on a reduced dose
dabigatran. There was no such difference for standard doses.[49]
Furthermore, thrombin is probably the most versatile and broad-spectrum enzyme in
the cardiovascular milieu, and it has functions that spans from hemostasis, coagulation,
inflammation, to immunity, and for all these functions its ability to bind to its
substrates is a primary requirement. Thus, the inhibitory effect by dabigatran on
thrombin binding to PARs may have wider implications.
What is known about this topic?
-
Dabigatran is a small molecule chemical inhibitor of thrombin and binds to the active
site to block its function.
-
Thrombin binds to the protease-activated receptors on platelet membrane via its exosites
and then the active site cleaves the N-terminal to activate the receptor.
-
PPACK is a small peptide inhibitor of the thrombin and binds to the active site.
What does this paper add?
-
Dabigatran blocks the binding of thrombin to the platelets completely.
-
Along with the function, thrombin active site also plays major role in the binding.
