Dabigatran affects thrombin-dependent platelet aggregation after a week-long therapy

Abstract

Objective. Dabigatran is a direct thrombin inhibitor. As the main adverse event is bleeding, it is relevant whether dabigatran has additional effects on platelet function. If so, it could affect the bleeding risk. We aimed to assess in vitro aggregation in patients with atrial fibrillation (AF) receiving dabigatran. Design. We evaluated 32 AF patients treated with dabigatran (study group) and 18 non-anticoagulated non-AF blood donors (control group). We assessed light transmittance platelet aggregation (LTA) with 100 nmol/L γ-thrombin in both groups. The LTA was performed at two time-points in our dabigatran group of patients. Results. The thrombin-induced platelet aggregation was significantly lower two hours after dabigatran was taken compared to baseline measurement (9% ± 6% vs. 29% ± 21%) in our study group. Moreover, we observed that the baseline value of platelet aggregation in patients on dabigatran treatment was significantly lower compared to healthy volunteers (29% ± 21% vs. 89 ± 8). However, one subanalysis showed that this significant reduction in platelet aggregation at baseline was only observed in patients who received dabigatran for over a week. Conclusion. The thrombin-induced platelet aggregation is reduced in non-valvular AF patients receiving dabigatran after a week-long therapy.

Keywords:

• atrial fibrillation
• dabigatran
• haemostasis
• platelets
• aggregation

Introduction

Dabigatran etexilate is a low-molecular-weight prodrug that exhibits no pharmacological activity. After oral administration, dabigatran etexilate is converted to its active form, dabigatran, a potent, competitive, and reversible direct inhibitor of thrombin [1–5]. Dabigatran binds to the active site of the thrombin, thereby inactivating both fibrin-bound and unbound (free) thrombin [6]. As dabigatran is a direct thrombin inhibitor, it has effect on the secondary haemostasis [7]. As the main adverse event of dabigatran treatment is bleeding [8], it is relevant whether dabigatran has additional effects on platelet function. If so, it could affect the bleeding risk and influence the choice of treatment based on the patient risk profile. The effect of dabigatran on platelet activation has been studied by Vinholt et al. [9]. In this study healthy donor blood was incubated with dabigatran 0, 50, 500 and 10,000 ng/mL. It was observed that the platelet activation was reduced after thrombin stimulation in samples with dabigatran levels ≥500 ng/mL. It could be an indirect effect due to the binding of dabigatran to generated thrombin which prevents thrombin from stimulating platelets. Platelets express two thrombin receptors, protease-activated receptor (PAR)-1 and PAR-4, which are both stimulated with thrombin [10]. The aim of the present study was to assess the effects of dabigatran on in vitro platelet aggregation in real patients receiving dabigatran.

Material and methods

The local Ethical Committee of the Jessenius Faculty of Medicine in Martin approved this study (EK 1702/2015). All study participants agreed to participate in the project and signed a written informed consent in accordance with the Declaration of Helsinki.

Dabigatran was administrated in patients with atrial fibrillation (AF) twice daily (at 7:00 PM and 7:00 AM). Blood samples were taken 12 hours after a previous drug dose administration (baseline, at 7:00 AM), followed by next blood sample after 2 hours (at 9:00 AM). To be sure that the drug was administered at the right and same time, we implemented the following measures. First, the drug was administered to the patients by a physician who was involved in this study. Second, treatment with dabigatran was monitored by Hemoclot Thrombin Inhibitor assay (Hyphen BioMed, Paris, France), according to the manufacturer’s instructions. This dilute thrombin time assay is certified in Europe and the United States for the quantitative determination of dabigatran plasma levels. All patients on dabigatran therapy included in our study where hospitalized on the 1^st Department of Internal Medicine during July and August 2017. We did not have any selection criteria. Concomitant treatment (e.g. beta blockers, proton-pump inhibitor…) was administered immediately after taking the morning dose of dabigatran.

We recruited 18 blood donors aged 19 to 61 years for the control group. These patients were healthy volunteers without any current drug history. Blood samples were collected from each donor and thrombin platelet aggregation was measured.

Light transmission aggregometry (LTA) was performed using the international protocol for the laboratory investigation of platelet function [11]. We want to emphasize that testing was performed on patients without any antiplatelet or non-steroidal anti-inflammatory drugs (10–14 days before measurement) and with normal platelet count (≥150 × 10⁹/L). The antecubital venous blood was collected into tubes containing 3.2% buffered sodium citrate (anticoagulant-blood ratio 1:9) to assess platelet aggregation. Platelet aggregability was tested with platelet-rich plasma (PRP) using platelet aggregometry (PACKS-4 aggregometer, Helena Laboratories, USA). The platelet count in PRP was between 400–450 × 10⁹/L. Blood samples were stimulated with human γ-thrombin in final concentration 100 nmol/L (Mybiosource Inc., San Diego, USA).

Data are presented as numbers with frequencies for categorical variables and means with standard deviations (± SD) for continuous variables. For comparison of the different groups, the closed-test-principle was used. An overall comparison was performed, followed by pairwise comparison if the results were significant. The p values less than 0.05 were considered statistically significant. Data were analysed with SPSS 21.0.0.0 (SPSS Inc, Chicago, Illinois, USA).

Results

Table 1 presents full clinical baseline characteristics of the patients. Thirty-two patients with non-valvular AF were enrolled (study group). The mean age was 72 ± 9 years (range 51-88 years), 14 patients were woman and the mean CHA₂DS₂VASc score was 3 ± 1. All patients began treatment with dabigatran as an initial anticoagulant treatment. Eight patients had an initial duration ≤7 days. Dabigatran doses were 110 mg (59%) or 150 mg (41%) twice daily.

Table 1. Clinical baseline characteristics of the patients.

Characteristics	Number of patients or value
Mean age, years ± SD (range)	72 ± 9 (51–88)
≤65 years	9
>65 years	23
Sex
Male	18
Female	14
Duration of dabigatran treatment
Mean (range) ± SD	616 ± 706 (0–2612) days
≤7 days	8
>7 days	24
Indication
Non-valvular atrial fibrillation	32
Paroxysmal	19
Persistent	7
Permanent	6
Dose
2 × 110 mg	19
2 × 150 mg	13
Risk factors
Diabetes mellitus	12
Arterial hypertension	32
Renal disease	6
Dialysis, transplant, creatinine >2.26 mg/dL	0
Glomerular filtration rate	56 ± 13 (28–78)
Liver disease	5
Cerebral stroke/transient ischemic attack history	7
Coronary artery disease
One vessel	4
Two vessels	3
History of pulmonary embolism	0
Myocardial infraction history	5
BMI (kg/m^2)
Normal weight (18.5–24.9)	11
Overweight (25.0–29.9)	13
Obese (30.0–39.9)	8
Ischemic heart disease – Classification according to New York Heart Association
I	3
II	25
III	4
CHA2DS2VASc∗ ± SD	3 ± 1
HAS-BLED^ score	2 ± 1
Platelets × 10^9/L (range × 10^9/L)	190 (135–314)
Creatinine (μmol/L ± SD)	105 ± 18
Male
Normal (55 – 100)	7
Pathological (>100)	11
Female
Normal (44 – 95)	8
Pathological (>95)	6
ALT (μkat/L ± SD) (ALT)	0.4 ± 0.2
Normal (0.1 – 0.6)	26
Pathological (>0.6)	6
AST (μkat/L ± SD) (AST)	0.4 ± 0.2
Normal (0.1 – 0.6)	28
Pathological (>0.6)	4
GMT (μkat/L ± SD)	1 ± 1
Normal (0.07 – 0.63)	14
Pathological (>0.63)	18
Drugs
Beta blockers	22
Calcium channel blockers	14
Angiotensin converting enzyme blockers	6
Angiotensin II receptor antagonists	7
Antiplatelet drugs	0
Proton-pump inhibitor	8
Statins	7

∗CHADS2-VASC score is presented as points on a scale of 1–9 (±SD)

^{^}HAS/BLED score is presented as points on scale of 0–9 (±SD)

ALT: alanine transaminase; AST: aspartate transaminase; GMT: gamma glutamyltransferase; SD: standard deviation.

In sum, data from 18 blood donors were included in the analyses (control group). The mean age was 35 ± 11 years (range 19-61 years), 7 blood donors were woman.

As shown in Figure 1, the thrombin-induced platelet aggregation was significantly lower two hours after dabigatran was taken compared to baseline measurement (9% ± 6% vs. 29% ± 21%; p < .0001) in our study group. Moreover, we observed that the baseline value of platelet aggregation in patients on dabigatran treatment was significantly lower compared to healthy volunteers (29% ± 21% vs. 89 ± 8; p < .0001).

Figure 1. Results of thrombin induced platelet aggregation between groups.

The mean dabigatran concentration at baseline was 104 ± 91 ng/mL and 190 ± 111 ng/mL two hours after dabigatran was taken, respectively. The dose-response curve for the plasma-diluted thrombin time assay with dabigatran had a correlation coefficient of r² = 0.13 for baseline and 0.02 for followed measurement (after two hours), see Figure 2.

Figure 2. The dose-response curve for the plasma-diluted thrombin time assay with dabigatran. Line represent the best-fit regressions for dabigatran.

We have done 17 subgroup analyses in order to determine the impact on aggregometer measurement, see Table 2. We did not find any significant difference between the groups, with one exception. Thrombin induced platelet aggregation was depended on treatment duration. In short, we did not observe significant difference between healthy volunteers and baseline aggregation values for patients receiving dabigatran <7 days (89 ± 8 vs. 73% ± 24%; p > .05). Moreover, we observed significant difference between patients receiving dabigatran <7 days vs. >7 days (baseline measurement: 73% ± 24% vs. 28% ± 20%, p = .02; measurement after two hours: 25% ± 7% vs. 8% ± 5%, p = .03).

Table 2. Subgroup analysis

Subgroup analysis	Thrombin induced platelet aggregation∗	p value
≤65 years old vs. >65 years old	baseline: 36% ± 28% vs. 27% ± 17%	p = .23
	after 2 hours: 7% ± 4% vs. 10% ± 6%	p = .27
Treatment duration: ≤7 days vs. >7 days	baseline: 73% ± 24% vs. 28% ± 20%	p = .02
	after 2 hours: 25% ± 7% vs. 8% ± 5%	p = .03
Diabetes mellitus vs. without diabetes mellitus	baseline: 26% ± 10% vs. 32% ± 25%	p = .43
	after 2 hours: 8% ± 6% vs. 9% ± 5%	p = .69
With a history of renal disease vs without a history of renal disease	baseline: 18% ± 8% vs. 32% ± 22%	p = .12
	after 2 hours: 7% ± 4% vs. 10% ± 5%	p = .09
With a history of liver disease vs without a history of liver disease	baseline: 22% ± 10% vs. 31% ± 22%	p = .37
	after 2 hours: 8% ± 4% vs. 9% ± 6%	p = .72
Dose: 2×110 mg vs. 2×150 mg	baseline: 31% ± 22% vs. 28% ± 19%	p = .67
	after 2 hours: 10% ± 5% vs. 8% ± 6%	p = .27
Normal weight vs. overweight and obese	baseline: 29% ± 23% vs. 30% ± 20%	p = .9
	after 2 hours: 9% ± 7% vs. 9% ± 5%	p = .91
Normal creatinine level vs. pathological creatinine level	baseline: 31% ± 18% vs. 28% ± 23%	p = .66
	after 2 hours: 9% ± 5% vs. 9% ± 6%	p = .72
Normal AST level vs. pathological AST level	baseline: 30% ± 22% vs. 23% ± 7%	p = .48
	after 2 hours: 9% ± 6% vs. 10% ± 2%	p = .59
Normal ALT level vs. pathological ALT level	baseline: 31% ± 22% vs. 22% ± 5%	p = .33
	after 2 hours: 9% ± 6% vs. 11% ± 6%	p = .42
Normal GMT level vs. pathological GMT level	baseline: 34% ± 23% vs. 26% ± 18%	p = .24
	after 2 hours: 9% ± 5% vs. 9% ± 6%	p = .79
Beta blockers vs. no beta blockers	baseline: 29% ± 20% vs. 30% ± 23%	p = .98
	after 2 hours: 9% ± 5% vs. 9% ± 8%	p = .71
Calcium channel blockers vs. no calcium channel blockers	baseline: 26% ± 9% vs. 32% ± 26%	p = .48
	after 2 hours: 9% ± 5% vs. 8 ± 6%	p = .65
Angiotensin converting enzyme blockers vs. no angiotensin converting enzyme blockers	baseline: 32% ± 30% vs. 29% ± 19%	p = .7
	after 2 hours: 8% ± 5% vs. 9% ± 6%	p = .68
Angiotensin II receptor antagonists vs. no angiotensin II receptor antagonists	baseline: 37% ± 29% vs. 25% ± 16%	p = .08
	after 2 hours: 7% ± 4% vs. 9% ± 6%	p = .45
Proton-pump inhibitor vs. no proton-pump inhibitor	baseline: 21% ± 9% vs. 32% ± 23%	p = .18
	after 2 hours: 8% ± 5% vs. 9% ± 6%	p = .73
Statins vs. no statins	baseline: 30% ± 23% vs. 29% ± 20%	p = .9
	after 2 hours: 7% ± 7% vs. 9% ± 5%	p = .32

∗values are mean (± SD) for continuous variables.

ALT: alanine transaminase; AST: aspartate transaminase; GMT: gamma glutamyltransferase; SD: standard deviation.

Discussion

Dabigatran is a competitive reversible non-peptide antagonist of thrombin. Thrombin is not only a key protein in the cascade of fibrin clot formation but also a potent inducer of platelet aggregation [12–17]. This is the first prospective comprehensive study to test whether the dabigatran affects thrombin induced platelet aggregation.

This case-control study quantifies platelet aggregation in patients treated with dabigatran by LTA. The thrombin-induced platelet aggregation was significantly lower two hours after dabigatran was taken compared to baseline measurement in our study group. Moreover, we observed that the baseline value of platelet aggregation in patients on dabigatran treatment was significantly lower compared to healthy volunteers. However, we found a significant difference between patients receiving dabigatran <7 days vs. >7 days. In addition, we did not observe significant difference between healthy volunteers and baseline aggregation values for patients receiving dabigatran <7 days. This indicates that the reduction of thrombin inducted platelet aggregation is not necessarily an immediate effect of dabigatran but may be explained by the duration of chronic dabigatran therapy. Similar results were observed by TRAP-induced platelet aggregation in patients on dabigatran or xabans treatment [18,19]. Thus, data from our study and study which dealt with TRAP-induced platelet aggregation may indicate that aggregation is enhanced by a chronic, direct and indirect inhibition of thrombin. This might be due to changes in expression of PAR-1 and PAR-4 receptor on platelets.

No significant differences were found in the patients with a history of renal or liver disease, diabetes, coronary artery disease, myocardial infarction, and so on (see Table 2).

Our findings could have some important clinical implications because platelet aggregation and coagulation cascade are affected at the same time. The situation could be more worse during concomitant administration of antiplatelet or anticoagulant agents.

There were several limitations in our study. The small number of participants may have limited the ability to detect small drug effects on platelet function. Second, this study was not powered for clinical outcome. Therefore, it cannot be concluded that, for example, combination of antiplatelet therapy and dabigatran is not safe. Third, platelet aggregability is greatly affected by preanalytical issues and therefore interpretation of platelet hyperaggregability is potentially accordingly adversely influenced.

Disclosure statement

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Additional information

Funding

The study was supported by grants VEGA 1/0187/17.