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The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression
Thrombosis Research 126 (2010) e327–e333
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression Christina Christersson a,b,⁎, Matilda Johnell b, Agneta Siegbahn b,⁎ a b
Department of Medical Sciences, Cardiology, Uppsala University, Sweden Department of Medical Sciences, Clinical Chemistry, Uppsala University, Sweden
a r t i c l e
i n f o
Article history: Received 16 December 2009 Received in revised form 9 March 2010 Accepted 30 March 2010 Available online 7 May 2010 Keywords: Direct thrombin inhibitors Myocardial infarction Platelet-monocyte aggregates Tissue factor
a b s t r a c t Introduction: High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs). Material and Methods: DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI. Results: In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p = 0.02) and to granulocytes (p = 0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p = 0.02-0.002). In the MI population, the DTI reduced the TF activity (p b 0.001). Conclusion: DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production. © 2010 Elsevier Ltd. All rights reserved.
Introduction Activated platelets form aggregates with leukocytes and increased levels of these aggregates, in particular platelet-monocyte aggregates (PMAs), have been found in patients with stable angina and early in acute myocardial infarction (MI) [1–3]. P-selectin binding to the counter receptor P-selectin glycoprotein ligand (PSGL)-1 on leukocytes is important for the stability of the formed aggregates [4]. Cellular signalling is also induced in monocytes leading to production of tissue factor (TF) and several proinflammatory cytokines with an enhancement of the inflammatory response [5–8]. Agonists such as thrombin and ADP both induce platelet activity generating thrombus formation. Several interactions between these agonists have been described, among others the ADP receptor (P2Y12), inducing activa-
⁎ Corresponding authors. Christersson is to be contacted at Department of Cardiology, Uppsala University Hospital, S-751 85 Uppsala, Sweden. Tel.: +46 18 611 90 68; fax: +46 18 50 66 38. Siegbahn, Department of Clinical Chemistry, Uppsala University Hospital, S-751 85 Uppsala, Sweden. E-mail addresses: [email protected] (C. Christersson), [email protected] (A. Siegbahn). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.03.019
tion of the thrombin receptor (PAR-1) [9,10]. TF, the main initiator of coagulation, is immediately presented to the blood stream upon activation through cell interactions involving monocytes and platelets without any increase of TF mRNA [11]. However, the source of the bloodborne TF is debated. During activation, platelets and monocytes expose encrypted TF, and activated platelets also induce formation of procoagulant microparticles (MPs) with TF on their surfaces [11–14]. Besides monocytes platelets activated by thrombin or ADP have been found to induce TF production [15,16]. Patients with MI have increased TF mRNA in platelets compared to controls [17]. However, the platelet contribution to TF-expression is contradictory with other groups that could not confirm the TF production or activity in platelets [18,19]. Oral direct thrombin inhibitors (DTIs) have been investigated in clinical trials as additional treatment after MI, and lead to long-term reduction of thrombin generation and fibrin turnover [20]. Oral DTIs also reduce the platelet P-selectin expression in atrial fibrillation and in experimental studies [21,22]. The aim of this study was to investigate whether DTIs could modulate the formation of plateletleukocyte aggregates, MPs and TF expression.
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Materials and Methods Reagents and antibodies The fibrin polymerization inhibitor Pephabloc FG® was purchased from Pentapharm (Basel, Switzerland). The commercial direct thrombin inhibitor melagatran® (Astrazeneca, Mölndal, Sweden) and dabigatran (a kind gift from Boehringer Ingelheim, Biberach, Germany) were used in the experiments with whole blood from healthy volunteers. Human α-thrombin and ADP were purchased from Enzyme Research Laboratories (South Bend, IN) and Chromolog Corporation (Havertown, PA), respectively. Anti-CD45-antibodies (Ab) conjugated with phycoerythrin (PE) or fluorescein isothiocyanate (FITC), anti-CD14PEAb, anti-CD16PEAb and negative control mouse IgG were from DakoCytomation (Glostrup, Denmark). AntiCD42aPEAb, anti-CD154PE Ab and Annexin VPE/FITC were purchased from BD Pharmingen (San José, CA). Anti-CD62PFITCAb and antiCD142FITCAb (4508CJ) were from Serotec ltd (Oxford, UK) and American Diagnostics (Greenwich, CA), respectively. Size Standard beads 1.97 μm, 1.48 μm, 1.30 μm and 1.01 μm were from Bangs Laboratories (Fishers, IN). MP-reagent, Thrombin Calibrator and FluCa-kit were from Thrombinoscope (Maastricht, the Netherlands). Anti-TF Ab, TF8-5G9, was a kind gift from Dr. J.H. Morrissey (UrbanaChampaign, IL). Whole blood collection and activation Blood was drawn from healthy volunteers with a 21G needle at no stasis into BD Vacutainer® tubes containing 3.8% citrate. To minimize platelet aggregation we used polypropylene tubes. 1-1.5 mg/mL Pephabloc FG®, a fibrin polymerization inhibitor, was added to all samples before stimulation in order to avoid clotting and platelet aggregation upon activation. Whole blood was stimulated with 5 nM thrombin or 20 μM ADP. The concentrations of agonists were chosen to reach maximal platelet expression of P-selectin. Whole blood was stimulated up to 1 hour at 37 °C, gently rotated after 30 minutes of incubation both with and without 30 minutes preincubation with 1 µM melagatran or 300 ng/mL dabigatran before stimulation. The concentrations of the direct thrombin inhibitors were chosen from the plasma concentrations found in clinical trials [20,23]. The study was approved by the local ethics committee at Uppsala University and followed the regulations of the Helsinki declaration. Platelet activity, formation of platelet-leukocyte aggregates and analysis of microparticles
granulocytes was verified by their CD14 and CD16 expression, respectively. The amount of platelets per leukocyte was detected by the mean fluorescence intensity (MFI) of CD42a-positive cells in the respective population. The amount of platelet-leukocyte aggregates positively stained for P-selectin, CD40L or TF and the MFI for these antigens was also analysed. MPs were defined as particles b1 μm, using size standard beads as reference limit, and positively stained for annexin V. The number of MPs was calculated as the number of MPs/10000 platelets in the same sample. RNA preparation, cDNA synthesis and mRNA quantification The total RNA from 2 mL of whole blood stimulated as described above was prepared by using QIamp®RNA Blood Mini Kits (QIAGEN, KJ Venlo, the Netherlands). Reverse transcription using oligoDT (Invitrogen Corporation, Carlsbad, CA) was used for cDNA synthesis and real time quantitative polymerase chain reaction (PCR) was carried out with the TaqMan real-time PCR assay (ABI PRISM™ 7000, Applied Biosystems, Foster City, CA). TF mRNA was analysed as previously described and ß 2 microglobulin was used as a housekeeping gene [25]. Fluorogenic analysis of tissue factor activity Platelet poor plasma (PPP) was prepared from citrated whole blood pre-incubated both with and without the DTIs before stimulation with thrombin or ADP for 15 minutes. Thrombin generation assay was performed using the Calibrated Automated Thrombogram (Thrombinoscope) measured in a 96-well plate fluorometer (Fluoroskan Ascent®, ThermoScientific, Waltham, MA) as previously described [26]. Briefly, 80 µL plasma was mixed with 20 µL of standardized reagent with 4 µM phospholipids final concentration to determine the activity of endogenous TF. PPP prepared from whole blood without direct thrombin inhibitors from the same donor was used for the Thrombin Calibrator. The fluorometric measurements were performed after automated addition of 20 µL of a fluorescent substrate (Z-Gly-Gly-Arg-AMC) and calcium chloride. The thrombin generation process was followed for 60 minutes. The initial phase of thrombin generation, the lag time, as a measurement for TF activity, was determined for each condition. In control experiments the contribution of TF to thrombin generation was assessed by preincubation of PPP at room temperature for 20 min with 10 µg/mL of the neutralising anti-TF Ab, TF8-5G9. Laboratory methods in the patient population
Flow cytometry was performed using an Epics XL-MCL (Beckman Coulter, Fullerton, CA). P-selectin and CD40 ligand (CD40L) were detected on platelets in whole blood labelled with anti-CD62PFITC and anti-CD154FITC Ab. Irrelevant FITC-conjugated isotype Ab was used as control. Stimulated or control whole blood was incubated with Ab 20 minutes in the dark at room temperature and fixed with cold 0.2% para-formaldehyde /hepes saline. No washing steps were used. The samples were diluted 1:20 in PBS before analysis. Single platelets were identified by their forward and side scatter characteristics, and the percentage of positive cells was determined. Platelet-leukocyte aggregate formation was determined in stimulated or control whole blood incubated with anti-CD45FITC or PE combined with anti-CD42aPE, anti-CD62PFITC, anti-CD154PE, antiCD142FITC or an irrelevant isotype AbFITC/PE. The samples were incubated for 20 minutes in the dark at room temperature and fixed in cold 0.5% formaldehyde in 0.9% NaCl [24]. For analysis, at least 5000 leukocytes were evaluated and CD45-positive cells were gated by the fluorescentdetection channel versus side scatter. The amount of monocytes, granulocytes and lymphocytes with conjugated platelets was determined. In separate experiments, the identity of monocytes and
In order to evaluate the in vitro results, we analysed plasma from 24 patients in the ESTEEM-trial. The ESTEEM-trial was a phase II study for safety and efficacy of the oral direct thrombin inhibitor ximelagatran [27]. Patients were randomized to one of four doses of ximelagatran together with aspirin or aspirin alone as antithrombotic treatment. Soluble (s)P-selectin and sCD40L were analysed by immunoassay (R&D Systems, Minneapolis, MN and Bender MedSystems GmbH, Vienna, Austria for sP-selectin and CD40L respectively) in plasma collected at randomization before the start of study treatment and after 1 week of treatment. The procoagulant TF activity after 1 week of treatment was calculated as described above, and PPP from the randomization visit, before the start of the study drug was used for calibration of each patient. Statistical analysis In the in vitro whole blood experiments results were described by mean or mean ± SEM if not stated otherwise. The MFI in each population was calculated as the MFI in the positive population/MFI of
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the isotype-Ab in the same sample. The student's t-test for dependent samples was performed to determine statistical significance between different data sets. The number of experiments in each analysis was 38. The results from the patient population were described by mean ± SEM. The Wilcoxon signed ranks test and Mann-Whitney U test were used for within and between group comparisons. Results Platelet activation and formation of platelet-leukocyte aggregates in vitro In order to investigate the formation and function of plateletleukocyte aggregates, we used an experimental model where whole blood was stimulated with thrombin or ADP for 15 and 60 minutes. Incubated whole blood without stimulation was used as control at the different time periods. In whole blood stimulated with thrombin for 15 minutes, 57.0 ± 6.0% of the single platelets expressed P-selectin, which was 7.9 ± 1.3 times more than in control whole blood (p = 0.003) where 8.1 ± 0.8% expressed P-selectin.. The amount of single platelets expressing CD40L also increased by 2.5 ± 0.4 times (p = 0.01) from 1.8 ± 0.1% to 4.8 ± 0.6%. Upon thrombin stimulation, 18.3 ± 3.1% of the monocytes formed aggregates with platelets compared to 10.5 ± 2.1% in control blood (p = 0.002,) and the amount of adhered platelets to each monocyte reflected by MFI for CD42a increased 2.4 ± 0.2 times (p = 0.002). Thrombin also induced an elevation of the amount of platelet-granulocyte aggregates (PGA) from 38.5 ± 4.1% to 85.0 ± 2.6% (p b 0.001) together with a 3.7 ± 0.5 times increase of MFI for CD42a on each granulocyte (p = 0.002). Moreover, thrombin increased the amount of formed plateletlymphocyte aggregates (PLAs) from 3.2 ± 0.4% in control blood to 5.1 ± 0.8% (p = 0.01), and the MFI for CD42a on each lymphocyte increased by 1.6 ± 0.1 times compared to control (p = 0.07). In ADP-stimulated whole blood, 21.7 ± 3.0% of the single platelets expressed P-selectin, an increase by 2.6 ± 0.3 times compared to control blood (p = 0.004) where 8.1 ± 0.8% expressed P-selectin. ADPstimulation also tended to increase the expression of CD40L on free platelets by 1.3 ± 0.1 times compared to control (p = 0.06). The amount of formed PMAs, PGAs or PLAs did not increase significantly upon ADP-stimulation. However, the amount of platelets bound to leukocytes analysed as MFI for CD42a increased for monocytes by 1.3 ± 0.1 (p = 0.06) times to 4.16 ± 0.87, for granulocytes 2.7 ± 0.6 (p = 0.03) times to 11.30 ± 2.70 and for lymphocytes 1.2 ± 0.1 times to 2.90 ± 0.37, compared to control whole blood. Melagatran and dabigatran diminish the formation of platelet-leukocyte aggregates in vitro Whole blood was pre-incubated with melagatran or dabigatran before stimulation with thrombin for 15 minutes. Melagatran and dabigatran inhibited induction of P-selectin expression in single platelets induced by thrombin stimulation by 86 ± 3% and 81 ± 3%, respectively (p b 0.001). The DTIs also decreased the CD40L expression in single platelets upon thrombin stimulation by 44 ± 9% and 46 ± 7% for melagatran and dabigatran, respectively (p = 0.001). DTIs added to unstimulated whole blood had no effect on the amount of formed PMAs. However, preincubation with both melagatran and dabigatran significantly reduced the number of adhered platelets to each monocyte upon thrombin stimulation (Fig. 1A). Melagatran also inhibited the amount of PMA expressing P-selectin to 18 ± 4% compared to 28 ± 4% in stimulated whole blood without the DTI, a reduction by 44±11% (p = 0.008). However, the DTIs had no significant effect on the CD40L expression within the PMAs induced by thrombin-stimulation (p = 0.6 and p = 0.1 for melagatran and dabigatran respectively). The amount of PGAs was significantly reduced by 50 ± 5% and 36 ± 6% by preincubation with melagatran
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and dabigatran, respectively, before thrombin stimulation (p = 0.001), and the amount of platelets bound per granulocyte also decreased significantly (Fig. 1B). Furthermore, the amount of PGAs expressing P-selectin was reduced to 24 ± 4% by melagatran compared to 91 ± 3% in thrombin stimulated whole blood (p b 0.001). The PGAs expressing CD40L also decreased from 44 ± 3% to 11 ± 3% by melagatran treatment (p b 0.001). Dabigatran also reduced the amount of PGAs expressing P-selectin and CD40L similar to melagatran (p = 0.001 and p = 0.004, respectively). In whole blood pre-incubated with melagatran or dabigatran before ADP-stimulation, the number of platelets adhered to monocytes tended to increase (p = 0.06) and was significantly elevated on granulocytes in comparison to ADP-stimulated blood without the DTIs (Fig. 1D-E). No effect was recorded of either the Pselectin or the CD40L expression within PGAs by preincubation with the DTIs upon ADP-stimulation (p = 0.4). Neither the number of formed PLAs nor the amount of platelets bound per lymphocyte at thrombin- or ADP- stimulation was affected by preincubation with DTIs (Fig. 1C-F). Tissue factor mRNA in whole blood and the effect of direct thrombin inhibitors In this experimental model, TF mRNA was determined in unstimulated lysed control blood after 1 hour or in blood stimulated by thrombin or ADP for 1 hour. In the control blood a low concentration of TF mRNA was found, 0.2 ± 0.08 arb units. Both agonists increased the TF mRNA concentration 2.4 ± 0.8 times upon thrombin stimulation and 2.6 ± 0.7 times upon ADP stimulation (p = 0.02 and p = 0.03, respectively). TF mRNA in thrombinstimulated whole blood preincubated with melagatran was reduced by 44 ± 8% (p = 0.002) to 0.25 ± 0.09 arb units, and by dabigatran 24 ± 8% (p = 0.04) to 0.34 ± 0.12 arb units compared to thrombin stimulation alone (Fig. 2). In contrast, neither melagatran nor dabigatran had any inhibitory effect regarding ADP-induced TF production. Direct thrombin inhibitors reduce TF expression in whole blood MPs, platelets and monocytes have been described as the sources of bloodborne TF [12,28,29]. We investigated the possible effects of DTIs regarding this type of TF, rapidly expressed upon activation without increase of mRNA levels. In control whole blood, there were 191 ± 33 circulating MPs/10000 single platelets, all expressing TF. In thrombin-stimulated whole blood, the amount of free MPs increased to 444 ± 96 MPs/10000 platelets (Fig. 3). Stimulation with 20 μM ADP increased the amount of MPs to 449 ± 88/10000 platelets. Preincubation with both melagatran and dabigatran, before thrombin-stimulation, decreased the amount of TF presenting MPs by 42 ± 12% (p = 0.02) to 204 ± 17/10000 platelets and 36 ± 12% (p = 0.03) to 231 ± 13/10000 platelets respectively. The MPs formed at ADP stimulation were also inhibited by melagatran with 21 ± 7% (p = 0.03) to 359 ± 72/10000 platelets, and by dabigatran with 39 ± 6% (p = 0.002) to 275 ± 46/10000 platelets (Fig. 3). The TF expression within the formed PMAs after15 minutes of thrombin or ADP stimulation was not affected by preincubation with the DTIs. However, TF expression measured within the PGAs after thrombin and ADP stimulation was lower in whole blood preincubated with melagatran (p = 0.005 and p = 0.007, respectively) than without the thrombin inhibitor (data not shown). Effect of direct thrombin inhibitors on TF activity The TF activity, determined as part of the lag time to start of thrombin generation, after addition of a standardised concentration of
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Fig. 1. The effect of DTIs on the formation of platelet-monocyte aggregates (A, D), platelet-granulocyte aggregates (B, E) and platelet-lymphocyte aggregates (C, F) in stimulated whole blood (n = 6). ***p-value ≤ 0.001 ,**p-value ≤ 0.005 and *p-value b 0.05.
phospholipids and calcium chloride in the CAT assay was 13.6 ± 1.6 minutes in plasma from control whole blood. In control experiments addition of the neutralizing anti-TF Ab prolonged the lag time by 4.53 ± 1.56 minutes (p = 0.001). The lag time in plasma from thrombin and ADP-stimulated whole blood was 5.4 ± 0.2 and 12.7 ± 0.3 minutes, respectively. Pre-treatment with melagatran before stimulation prolonged the lag time for thrombin stimulation to 24.7 ± 3.2 minutes (p = 0.02) and ADP stimulation to 53.4 ± 6.6 minutes (p = 0.03). Similar results were found after pre-treatment with dabigatran (Fig. 4).
The effect of an oral direct thrombin inhibitor after a myocardial infarction To evaluate the effect of oral direct thrombin inhibition in vivo, we analysed samples from 24 patients with recent MI randomised to 160 mg aspirin (n= 12) or aspirin in combination with 60 mg of the DTI, ximelagatran (n= 12) as antithrombotic treatment in the ESTEEM-trial [27]. Samples were collected at randomisation within 14 days after the acute MI, before the start of study treatment, and after 1 week of treatment, in the morning before intake of the study drug. SP-selectin
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Fig. 2. The effect of DTIs on TF production in stimulated whole blood. Results are presented relative to the TF mRNA concentrations of the thrombin- or ADP-stimulated samples in the same experiment (n = 6). **p-value ≤ 0.005 and *p-value b 0.05.
was significantly increased in the total population cohort after 1 week (p= 0.005). There was an elevation of sP-selectin by 29% (p= 0.02), from 29 ± 4 ng/mL to 36 ± 3 ng/mL within the aspirin group. A diminished, non significant, increase of sP-selectin by 14% (p= 0.05) was recorded within the aspirin + ximelagatran group, from 30± 3 ng/ mL to 33 ± 3 ng/mL. The levels of sCD40L neither changed in the aspirin nor in the aspirin + ximelagatran group after 1 week. TF activity, analysed as part of the lag time of thrombin generation in PPP 1 week after the start of study treatment, was significantly prolonged in the aspirin + ximelagatran group to 31.7 ± 4.0 minutes compared to 14.5 ± 1.0 minutes in the aspirin group (p b 0.001) (Fig. 5).
Discussion PMAs, as a sensitive marker for platelet activation, are formed through interactions of receptors, among others GpIIb/IIIa, P-selectin and CD40L, with their respective counter-receptors on monocytes. Formed PMAs are increased at acute MI and correlate to the severity of atherosclerosis [1,30,31]. P-selectin is important for the formation and stability of PMAs, and in the present study the DTIs reduced the platelet P-selectin expression in accordance with previous results [11,22,32]. We found that in thrombin-stimulation of whole blood, DTIs reduced the amount of bound platelets per monocyte and the expression of Pselectin within these aggregates. Moreover, the DTIs also effectively reduced TF production induced by thrombin. The expression of P-
Fig. 3. Microparticles in stimulated whole blood and the effect of DTIs. Results are presented as number of microparticles per 10000 platelets in relation to the amount in the stimulated sample in the same experiment (n = 6). ***p-value≤ 0.001, **p-value ≤ 0.005 and *p-valueb 0.05.
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Fig. 4. The effect of DTIs on tissue factor activity, determined as the time to start of thrombin generation (lag time) in platelet poor plasma from stimulated whole blood. Results are presented as relative change of lag time in relation to the stimulated sample in the same experiment. (n = 3). *p-value b 0.05.
selectin binding to PSGL-1 and the formation of PMAs are important for the induction of TF production [5,11]. DTIs have been found to reduce the platelet activity as evaluated by flow cytometry, compared to unfractionated heparin at percutanous coronary interventions [33,34]. In the present study, treatment with an oral DTI tended to diminish the increase of sP-selectin after MI. Upon treatment with DTIs, the reduced number of platelets adhered to monocytes and subsequent signalling events induced by cellular cross-talk therein may be beneficial during long-term treatment in patients with increased risk of new atherothrombotic events. Activated platelets induce inflammatory processes including upregulation of adhesion molecules and production of inflammatory cytokines in neutrophils [35]. Formed PGAs are related to the levels of C-reactive protein and IL-6 in patients with coronary artery disease and, though debated, neutrophils have been described as producing TF [36–38]. The formation of PGAs, the amount of platelets adhered per granulocyte and the P-selectin expression within these aggregates were inhibited by melagatran and dabigatran to an even higher extent compared to their effect on PMAs. These results may be explained by an increased reversibility in PGA formation [39]. Moreover, monocytes could be preferred over granulocytes by the limited amount of activated platelets [2]. The activation of platelets through ADP targeting the P2Y12 receptor is another mechanism for the formation of PMAs [40]. The DTIs had no effect on the formed PMAs and PGAs or the P-selectin expression within these aggregates upon ADP-stimulation, and there was even a tendency to increased formation of PMAs by the thrombin inhibitors. Moreover, the DTIs were not able to decrease the ADP-
Fig. 5. The effect on TF activity of an oral DTI in patients with recent myocardial infarction. Results represent the lag time in patients treated with aspirin (n = 12) or ximelagatran together with aspirin (n = 12) for 1 week. ***p-value ≤ 0.001.
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induced TF production. In clinical trials where DTIs were used without any complementary antiplatelet therapy, there have been concerns about increased risk of MI and elevation of markers for platelet activity [23,41]. The lack of effect of the DTIs on ADP-induced formation of platelet-leukocyte aggregates indicates a need for a combination of therapies interacting with different mechanisms of platelet activation, even though this hypothesis must be further investigated. Upon activation, bloodborne TF originates from, among other sources, platelet and monocyte- derived MPs [13,42]. The DTIs effectively lead to diminished formation of free MPs and also TF expression within the PGAs upon both thrombin and ADP stimulation in the present study. Both adherence of monocyte-derived MPs to platelets and TF expression in neutrophils are dependent of P-selectin, which was effectively diminished by the DTIs [38,43]. Another explanation may be that the available amount of MPs for incorporation in granulocytes through phagocytosis is reduced by the thrombin inhibitors. In contrast, the DTIs did not affect the TF expression within the formed PMAs. These results may indicate that the early TF expression within the formed PMAs depends on decrypted TF, or that the TF presenting MPs prefer adherence to monocytes, and thus TF will only adhere to granulocytes when expressed in high amounts [2]. The use of lag time in the CAT assay as a method for determination of change of endogenous TF activity has recently been described [44]. In our experimental model DTIs prolonged the lag time in accordance with the diminished amount of MPs. Similar results were found in the post-MI patients, where oral treatment with ximelagatran for 1 week significantly prolonged the lag time. An explanation for these results may be that oral treatment with DTI as secondary prevention has the capacity to reduce the amount of circulating procoagulant MPs and the production of TF within the formed PMAs in patients with recent MI. Thrombin also induced, to a lower degree, the formation of aggregates between platelets and lymphocytes. The interaction between platelets with natural killer cells and T-cytolytic cells are more pronounced than interaction with B-cells, and a limitation of the results in the present study is the lack of classification of lymphocytes [45]. To effectively reduce PLA formation, there is a need for combined blocking of several adhesion molecules [46]. The DTIs did not affect PLA formation even though platelet P-selectin expression was reduced in single platelets. CD40L has been described as an important receptor in the platelet-lymphocyte interaction, and in the present study DTIs neither diminish CD40L in single platelets nor within the aggregates [47]. We conclude that DTIs reduce TF production and activity through diminished adherence of platelets to monocytes and granulocytes upon thrombin stimulation. Oral DTIs also affect the TF activity in a post-MI population, which partly may depend on a diminished amount of circulating procoagulant microparticles. These effects could contribute to the clinical benefit with reduced risk of new thromboembolic events found in clinical trials. In contrast, the DTIs did not have an effect on ADP-induced PMA formation or TF production. Further studies of the importance of a combined treatment targeting different platelet and coagulant mechanisms in clinical settings are warranted. Conflict of interest statement None Acknowledgement We acknowledge Birgitta Fahlström, biomedical analyst, and Helena Vretman, research engineer, for excellent laboratory assistance. This work was supported by Swedish Heart-Lung Foundation,
Swedish Research Council and the Uppsala County Association Against Heart and Lung Diseases.
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Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression Christina Christersson a,b,⁎, Matilda Johnell b, Agneta Siegbahn b,⁎ a b
Department of Medical Sciences, Cardiology, Uppsala University, Sweden Department of Medical Sciences, Clinical Chemistry, Uppsala University, Sweden
a r t i c l e
i n f o
Article history: Received 16 December 2009 Received in revised form 9 March 2010 Accepted 30 March 2010 Available online 7 May 2010 Keywords: Direct thrombin inhibitors Myocardial infarction Platelet-monocyte aggregates Tissue factor
a b s t r a c t Introduction: High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs). Material and Methods: DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI. Results: In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p = 0.02) and to granulocytes (p = 0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p = 0.02-0.002). In the MI population, the DTI reduced the TF activity (p b 0.001). Conclusion: DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production. © 2010 Elsevier Ltd. All rights reserved.
Introduction Activated platelets form aggregates with leukocytes and increased levels of these aggregates, in particular platelet-monocyte aggregates (PMAs), have been found in patients with stable angina and early in acute myocardial infarction (MI) [1–3]. P-selectin binding to the counter receptor P-selectin glycoprotein ligand (PSGL)-1 on leukocytes is important for the stability of the formed aggregates [4]. Cellular signalling is also induced in monocytes leading to production of tissue factor (TF) and several proinflammatory cytokines with an enhancement of the inflammatory response [5–8]. Agonists such as thrombin and ADP both induce platelet activity generating thrombus formation. Several interactions between these agonists have been described, among others the ADP receptor (P2Y12), inducing activa-
⁎ Corresponding authors. Christersson is to be contacted at Department of Cardiology, Uppsala University Hospital, S-751 85 Uppsala, Sweden. Tel.: +46 18 611 90 68; fax: +46 18 50 66 38. Siegbahn, Department of Clinical Chemistry, Uppsala University Hospital, S-751 85 Uppsala, Sweden. E-mail addresses: [email protected] (C. Christersson), [email protected] (A. Siegbahn). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.03.019
tion of the thrombin receptor (PAR-1) [9,10]. TF, the main initiator of coagulation, is immediately presented to the blood stream upon activation through cell interactions involving monocytes and platelets without any increase of TF mRNA [11]. However, the source of the bloodborne TF is debated. During activation, platelets and monocytes expose encrypted TF, and activated platelets also induce formation of procoagulant microparticles (MPs) with TF on their surfaces [11–14]. Besides monocytes platelets activated by thrombin or ADP have been found to induce TF production [15,16]. Patients with MI have increased TF mRNA in platelets compared to controls [17]. However, the platelet contribution to TF-expression is contradictory with other groups that could not confirm the TF production or activity in platelets [18,19]. Oral direct thrombin inhibitors (DTIs) have been investigated in clinical trials as additional treatment after MI, and lead to long-term reduction of thrombin generation and fibrin turnover [20]. Oral DTIs also reduce the platelet P-selectin expression in atrial fibrillation and in experimental studies [21,22]. The aim of this study was to investigate whether DTIs could modulate the formation of plateletleukocyte aggregates, MPs and TF expression.
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Materials and Methods Reagents and antibodies The fibrin polymerization inhibitor Pephabloc FG® was purchased from Pentapharm (Basel, Switzerland). The commercial direct thrombin inhibitor melagatran® (Astrazeneca, Mölndal, Sweden) and dabigatran (a kind gift from Boehringer Ingelheim, Biberach, Germany) were used in the experiments with whole blood from healthy volunteers. Human α-thrombin and ADP were purchased from Enzyme Research Laboratories (South Bend, IN) and Chromolog Corporation (Havertown, PA), respectively. Anti-CD45-antibodies (Ab) conjugated with phycoerythrin (PE) or fluorescein isothiocyanate (FITC), anti-CD14PEAb, anti-CD16PEAb and negative control mouse IgG were from DakoCytomation (Glostrup, Denmark). AntiCD42aPEAb, anti-CD154PE Ab and Annexin VPE/FITC were purchased from BD Pharmingen (San José, CA). Anti-CD62PFITCAb and antiCD142FITCAb (4508CJ) were from Serotec ltd (Oxford, UK) and American Diagnostics (Greenwich, CA), respectively. Size Standard beads 1.97 μm, 1.48 μm, 1.30 μm and 1.01 μm were from Bangs Laboratories (Fishers, IN). MP-reagent, Thrombin Calibrator and FluCa-kit were from Thrombinoscope (Maastricht, the Netherlands). Anti-TF Ab, TF8-5G9, was a kind gift from Dr. J.H. Morrissey (UrbanaChampaign, IL). Whole blood collection and activation Blood was drawn from healthy volunteers with a 21G needle at no stasis into BD Vacutainer® tubes containing 3.8% citrate. To minimize platelet aggregation we used polypropylene tubes. 1-1.5 mg/mL Pephabloc FG®, a fibrin polymerization inhibitor, was added to all samples before stimulation in order to avoid clotting and platelet aggregation upon activation. Whole blood was stimulated with 5 nM thrombin or 20 μM ADP. The concentrations of agonists were chosen to reach maximal platelet expression of P-selectin. Whole blood was stimulated up to 1 hour at 37 °C, gently rotated after 30 minutes of incubation both with and without 30 minutes preincubation with 1 µM melagatran or 300 ng/mL dabigatran before stimulation. The concentrations of the direct thrombin inhibitors were chosen from the plasma concentrations found in clinical trials [20,23]. The study was approved by the local ethics committee at Uppsala University and followed the regulations of the Helsinki declaration. Platelet activity, formation of platelet-leukocyte aggregates and analysis of microparticles
granulocytes was verified by their CD14 and CD16 expression, respectively. The amount of platelets per leukocyte was detected by the mean fluorescence intensity (MFI) of CD42a-positive cells in the respective population. The amount of platelet-leukocyte aggregates positively stained for P-selectin, CD40L or TF and the MFI for these antigens was also analysed. MPs were defined as particles b1 μm, using size standard beads as reference limit, and positively stained for annexin V. The number of MPs was calculated as the number of MPs/10000 platelets in the same sample. RNA preparation, cDNA synthesis and mRNA quantification The total RNA from 2 mL of whole blood stimulated as described above was prepared by using QIamp®RNA Blood Mini Kits (QIAGEN, KJ Venlo, the Netherlands). Reverse transcription using oligoDT (Invitrogen Corporation, Carlsbad, CA) was used for cDNA synthesis and real time quantitative polymerase chain reaction (PCR) was carried out with the TaqMan real-time PCR assay (ABI PRISM™ 7000, Applied Biosystems, Foster City, CA). TF mRNA was analysed as previously described and ß 2 microglobulin was used as a housekeeping gene [25]. Fluorogenic analysis of tissue factor activity Platelet poor plasma (PPP) was prepared from citrated whole blood pre-incubated both with and without the DTIs before stimulation with thrombin or ADP for 15 minutes. Thrombin generation assay was performed using the Calibrated Automated Thrombogram (Thrombinoscope) measured in a 96-well plate fluorometer (Fluoroskan Ascent®, ThermoScientific, Waltham, MA) as previously described [26]. Briefly, 80 µL plasma was mixed with 20 µL of standardized reagent with 4 µM phospholipids final concentration to determine the activity of endogenous TF. PPP prepared from whole blood without direct thrombin inhibitors from the same donor was used for the Thrombin Calibrator. The fluorometric measurements were performed after automated addition of 20 µL of a fluorescent substrate (Z-Gly-Gly-Arg-AMC) and calcium chloride. The thrombin generation process was followed for 60 minutes. The initial phase of thrombin generation, the lag time, as a measurement for TF activity, was determined for each condition. In control experiments the contribution of TF to thrombin generation was assessed by preincubation of PPP at room temperature for 20 min with 10 µg/mL of the neutralising anti-TF Ab, TF8-5G9. Laboratory methods in the patient population
Flow cytometry was performed using an Epics XL-MCL (Beckman Coulter, Fullerton, CA). P-selectin and CD40 ligand (CD40L) were detected on platelets in whole blood labelled with anti-CD62PFITC and anti-CD154FITC Ab. Irrelevant FITC-conjugated isotype Ab was used as control. Stimulated or control whole blood was incubated with Ab 20 minutes in the dark at room temperature and fixed with cold 0.2% para-formaldehyde /hepes saline. No washing steps were used. The samples were diluted 1:20 in PBS before analysis. Single platelets were identified by their forward and side scatter characteristics, and the percentage of positive cells was determined. Platelet-leukocyte aggregate formation was determined in stimulated or control whole blood incubated with anti-CD45FITC or PE combined with anti-CD42aPE, anti-CD62PFITC, anti-CD154PE, antiCD142FITC or an irrelevant isotype AbFITC/PE. The samples were incubated for 20 minutes in the dark at room temperature and fixed in cold 0.5% formaldehyde in 0.9% NaCl [24]. For analysis, at least 5000 leukocytes were evaluated and CD45-positive cells were gated by the fluorescentdetection channel versus side scatter. The amount of monocytes, granulocytes and lymphocytes with conjugated platelets was determined. In separate experiments, the identity of monocytes and
In order to evaluate the in vitro results, we analysed plasma from 24 patients in the ESTEEM-trial. The ESTEEM-trial was a phase II study for safety and efficacy of the oral direct thrombin inhibitor ximelagatran [27]. Patients were randomized to one of four doses of ximelagatran together with aspirin or aspirin alone as antithrombotic treatment. Soluble (s)P-selectin and sCD40L were analysed by immunoassay (R&D Systems, Minneapolis, MN and Bender MedSystems GmbH, Vienna, Austria for sP-selectin and CD40L respectively) in plasma collected at randomization before the start of study treatment and after 1 week of treatment. The procoagulant TF activity after 1 week of treatment was calculated as described above, and PPP from the randomization visit, before the start of the study drug was used for calibration of each patient. Statistical analysis In the in vitro whole blood experiments results were described by mean or mean ± SEM if not stated otherwise. The MFI in each population was calculated as the MFI in the positive population/MFI of
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the isotype-Ab in the same sample. The student's t-test for dependent samples was performed to determine statistical significance between different data sets. The number of experiments in each analysis was 38. The results from the patient population were described by mean ± SEM. The Wilcoxon signed ranks test and Mann-Whitney U test were used for within and between group comparisons. Results Platelet activation and formation of platelet-leukocyte aggregates in vitro In order to investigate the formation and function of plateletleukocyte aggregates, we used an experimental model where whole blood was stimulated with thrombin or ADP for 15 and 60 minutes. Incubated whole blood without stimulation was used as control at the different time periods. In whole blood stimulated with thrombin for 15 minutes, 57.0 ± 6.0% of the single platelets expressed P-selectin, which was 7.9 ± 1.3 times more than in control whole blood (p = 0.003) where 8.1 ± 0.8% expressed P-selectin.. The amount of single platelets expressing CD40L also increased by 2.5 ± 0.4 times (p = 0.01) from 1.8 ± 0.1% to 4.8 ± 0.6%. Upon thrombin stimulation, 18.3 ± 3.1% of the monocytes formed aggregates with platelets compared to 10.5 ± 2.1% in control blood (p = 0.002,) and the amount of adhered platelets to each monocyte reflected by MFI for CD42a increased 2.4 ± 0.2 times (p = 0.002). Thrombin also induced an elevation of the amount of platelet-granulocyte aggregates (PGA) from 38.5 ± 4.1% to 85.0 ± 2.6% (p b 0.001) together with a 3.7 ± 0.5 times increase of MFI for CD42a on each granulocyte (p = 0.002). Moreover, thrombin increased the amount of formed plateletlymphocyte aggregates (PLAs) from 3.2 ± 0.4% in control blood to 5.1 ± 0.8% (p = 0.01), and the MFI for CD42a on each lymphocyte increased by 1.6 ± 0.1 times compared to control (p = 0.07). In ADP-stimulated whole blood, 21.7 ± 3.0% of the single platelets expressed P-selectin, an increase by 2.6 ± 0.3 times compared to control blood (p = 0.004) where 8.1 ± 0.8% expressed P-selectin. ADPstimulation also tended to increase the expression of CD40L on free platelets by 1.3 ± 0.1 times compared to control (p = 0.06). The amount of formed PMAs, PGAs or PLAs did not increase significantly upon ADP-stimulation. However, the amount of platelets bound to leukocytes analysed as MFI for CD42a increased for monocytes by 1.3 ± 0.1 (p = 0.06) times to 4.16 ± 0.87, for granulocytes 2.7 ± 0.6 (p = 0.03) times to 11.30 ± 2.70 and for lymphocytes 1.2 ± 0.1 times to 2.90 ± 0.37, compared to control whole blood. Melagatran and dabigatran diminish the formation of platelet-leukocyte aggregates in vitro Whole blood was pre-incubated with melagatran or dabigatran before stimulation with thrombin for 15 minutes. Melagatran and dabigatran inhibited induction of P-selectin expression in single platelets induced by thrombin stimulation by 86 ± 3% and 81 ± 3%, respectively (p b 0.001). The DTIs also decreased the CD40L expression in single platelets upon thrombin stimulation by 44 ± 9% and 46 ± 7% for melagatran and dabigatran, respectively (p = 0.001). DTIs added to unstimulated whole blood had no effect on the amount of formed PMAs. However, preincubation with both melagatran and dabigatran significantly reduced the number of adhered platelets to each monocyte upon thrombin stimulation (Fig. 1A). Melagatran also inhibited the amount of PMA expressing P-selectin to 18 ± 4% compared to 28 ± 4% in stimulated whole blood without the DTI, a reduction by 44±11% (p = 0.008). However, the DTIs had no significant effect on the CD40L expression within the PMAs induced by thrombin-stimulation (p = 0.6 and p = 0.1 for melagatran and dabigatran respectively). The amount of PGAs was significantly reduced by 50 ± 5% and 36 ± 6% by preincubation with melagatran
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and dabigatran, respectively, before thrombin stimulation (p = 0.001), and the amount of platelets bound per granulocyte also decreased significantly (Fig. 1B). Furthermore, the amount of PGAs expressing P-selectin was reduced to 24 ± 4% by melagatran compared to 91 ± 3% in thrombin stimulated whole blood (p b 0.001). The PGAs expressing CD40L also decreased from 44 ± 3% to 11 ± 3% by melagatran treatment (p b 0.001). Dabigatran also reduced the amount of PGAs expressing P-selectin and CD40L similar to melagatran (p = 0.001 and p = 0.004, respectively). In whole blood pre-incubated with melagatran or dabigatran before ADP-stimulation, the number of platelets adhered to monocytes tended to increase (p = 0.06) and was significantly elevated on granulocytes in comparison to ADP-stimulated blood without the DTIs (Fig. 1D-E). No effect was recorded of either the Pselectin or the CD40L expression within PGAs by preincubation with the DTIs upon ADP-stimulation (p = 0.4). Neither the number of formed PLAs nor the amount of platelets bound per lymphocyte at thrombin- or ADP- stimulation was affected by preincubation with DTIs (Fig. 1C-F). Tissue factor mRNA in whole blood and the effect of direct thrombin inhibitors In this experimental model, TF mRNA was determined in unstimulated lysed control blood after 1 hour or in blood stimulated by thrombin or ADP for 1 hour. In the control blood a low concentration of TF mRNA was found, 0.2 ± 0.08 arb units. Both agonists increased the TF mRNA concentration 2.4 ± 0.8 times upon thrombin stimulation and 2.6 ± 0.7 times upon ADP stimulation (p = 0.02 and p = 0.03, respectively). TF mRNA in thrombinstimulated whole blood preincubated with melagatran was reduced by 44 ± 8% (p = 0.002) to 0.25 ± 0.09 arb units, and by dabigatran 24 ± 8% (p = 0.04) to 0.34 ± 0.12 arb units compared to thrombin stimulation alone (Fig. 2). In contrast, neither melagatran nor dabigatran had any inhibitory effect regarding ADP-induced TF production. Direct thrombin inhibitors reduce TF expression in whole blood MPs, platelets and monocytes have been described as the sources of bloodborne TF [12,28,29]. We investigated the possible effects of DTIs regarding this type of TF, rapidly expressed upon activation without increase of mRNA levels. In control whole blood, there were 191 ± 33 circulating MPs/10000 single platelets, all expressing TF. In thrombin-stimulated whole blood, the amount of free MPs increased to 444 ± 96 MPs/10000 platelets (Fig. 3). Stimulation with 20 μM ADP increased the amount of MPs to 449 ± 88/10000 platelets. Preincubation with both melagatran and dabigatran, before thrombin-stimulation, decreased the amount of TF presenting MPs by 42 ± 12% (p = 0.02) to 204 ± 17/10000 platelets and 36 ± 12% (p = 0.03) to 231 ± 13/10000 platelets respectively. The MPs formed at ADP stimulation were also inhibited by melagatran with 21 ± 7% (p = 0.03) to 359 ± 72/10000 platelets, and by dabigatran with 39 ± 6% (p = 0.002) to 275 ± 46/10000 platelets (Fig. 3). The TF expression within the formed PMAs after15 minutes of thrombin or ADP stimulation was not affected by preincubation with the DTIs. However, TF expression measured within the PGAs after thrombin and ADP stimulation was lower in whole blood preincubated with melagatran (p = 0.005 and p = 0.007, respectively) than without the thrombin inhibitor (data not shown). Effect of direct thrombin inhibitors on TF activity The TF activity, determined as part of the lag time to start of thrombin generation, after addition of a standardised concentration of
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Fig. 1. The effect of DTIs on the formation of platelet-monocyte aggregates (A, D), platelet-granulocyte aggregates (B, E) and platelet-lymphocyte aggregates (C, F) in stimulated whole blood (n = 6). ***p-value ≤ 0.001 ,**p-value ≤ 0.005 and *p-value b 0.05.
phospholipids and calcium chloride in the CAT assay was 13.6 ± 1.6 minutes in plasma from control whole blood. In control experiments addition of the neutralizing anti-TF Ab prolonged the lag time by 4.53 ± 1.56 minutes (p = 0.001). The lag time in plasma from thrombin and ADP-stimulated whole blood was 5.4 ± 0.2 and 12.7 ± 0.3 minutes, respectively. Pre-treatment with melagatran before stimulation prolonged the lag time for thrombin stimulation to 24.7 ± 3.2 minutes (p = 0.02) and ADP stimulation to 53.4 ± 6.6 minutes (p = 0.03). Similar results were found after pre-treatment with dabigatran (Fig. 4).
The effect of an oral direct thrombin inhibitor after a myocardial infarction To evaluate the effect of oral direct thrombin inhibition in vivo, we analysed samples from 24 patients with recent MI randomised to 160 mg aspirin (n= 12) or aspirin in combination with 60 mg of the DTI, ximelagatran (n= 12) as antithrombotic treatment in the ESTEEM-trial [27]. Samples were collected at randomisation within 14 days after the acute MI, before the start of study treatment, and after 1 week of treatment, in the morning before intake of the study drug. SP-selectin
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Fig. 2. The effect of DTIs on TF production in stimulated whole blood. Results are presented relative to the TF mRNA concentrations of the thrombin- or ADP-stimulated samples in the same experiment (n = 6). **p-value ≤ 0.005 and *p-value b 0.05.
was significantly increased in the total population cohort after 1 week (p= 0.005). There was an elevation of sP-selectin by 29% (p= 0.02), from 29 ± 4 ng/mL to 36 ± 3 ng/mL within the aspirin group. A diminished, non significant, increase of sP-selectin by 14% (p= 0.05) was recorded within the aspirin + ximelagatran group, from 30± 3 ng/ mL to 33 ± 3 ng/mL. The levels of sCD40L neither changed in the aspirin nor in the aspirin + ximelagatran group after 1 week. TF activity, analysed as part of the lag time of thrombin generation in PPP 1 week after the start of study treatment, was significantly prolonged in the aspirin + ximelagatran group to 31.7 ± 4.0 minutes compared to 14.5 ± 1.0 minutes in the aspirin group (p b 0.001) (Fig. 5).
Discussion PMAs, as a sensitive marker for platelet activation, are formed through interactions of receptors, among others GpIIb/IIIa, P-selectin and CD40L, with their respective counter-receptors on monocytes. Formed PMAs are increased at acute MI and correlate to the severity of atherosclerosis [1,30,31]. P-selectin is important for the formation and stability of PMAs, and in the present study the DTIs reduced the platelet P-selectin expression in accordance with previous results [11,22,32]. We found that in thrombin-stimulation of whole blood, DTIs reduced the amount of bound platelets per monocyte and the expression of Pselectin within these aggregates. Moreover, the DTIs also effectively reduced TF production induced by thrombin. The expression of P-
Fig. 3. Microparticles in stimulated whole blood and the effect of DTIs. Results are presented as number of microparticles per 10000 platelets in relation to the amount in the stimulated sample in the same experiment (n = 6). ***p-value≤ 0.001, **p-value ≤ 0.005 and *p-valueb 0.05.
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Fig. 4. The effect of DTIs on tissue factor activity, determined as the time to start of thrombin generation (lag time) in platelet poor plasma from stimulated whole blood. Results are presented as relative change of lag time in relation to the stimulated sample in the same experiment. (n = 3). *p-value b 0.05.
selectin binding to PSGL-1 and the formation of PMAs are important for the induction of TF production [5,11]. DTIs have been found to reduce the platelet activity as evaluated by flow cytometry, compared to unfractionated heparin at percutanous coronary interventions [33,34]. In the present study, treatment with an oral DTI tended to diminish the increase of sP-selectin after MI. Upon treatment with DTIs, the reduced number of platelets adhered to monocytes and subsequent signalling events induced by cellular cross-talk therein may be beneficial during long-term treatment in patients with increased risk of new atherothrombotic events. Activated platelets induce inflammatory processes including upregulation of adhesion molecules and production of inflammatory cytokines in neutrophils [35]. Formed PGAs are related to the levels of C-reactive protein and IL-6 in patients with coronary artery disease and, though debated, neutrophils have been described as producing TF [36–38]. The formation of PGAs, the amount of platelets adhered per granulocyte and the P-selectin expression within these aggregates were inhibited by melagatran and dabigatran to an even higher extent compared to their effect on PMAs. These results may be explained by an increased reversibility in PGA formation [39]. Moreover, monocytes could be preferred over granulocytes by the limited amount of activated platelets [2]. The activation of platelets through ADP targeting the P2Y12 receptor is another mechanism for the formation of PMAs [40]. The DTIs had no effect on the formed PMAs and PGAs or the P-selectin expression within these aggregates upon ADP-stimulation, and there was even a tendency to increased formation of PMAs by the thrombin inhibitors. Moreover, the DTIs were not able to decrease the ADP-
Fig. 5. The effect on TF activity of an oral DTI in patients with recent myocardial infarction. Results represent the lag time in patients treated with aspirin (n = 12) or ximelagatran together with aspirin (n = 12) for 1 week. ***p-value ≤ 0.001.
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induced TF production. In clinical trials where DTIs were used without any complementary antiplatelet therapy, there have been concerns about increased risk of MI and elevation of markers for platelet activity [23,41]. The lack of effect of the DTIs on ADP-induced formation of platelet-leukocyte aggregates indicates a need for a combination of therapies interacting with different mechanisms of platelet activation, even though this hypothesis must be further investigated. Upon activation, bloodborne TF originates from, among other sources, platelet and monocyte- derived MPs [13,42]. The DTIs effectively lead to diminished formation of free MPs and also TF expression within the PGAs upon both thrombin and ADP stimulation in the present study. Both adherence of monocyte-derived MPs to platelets and TF expression in neutrophils are dependent of P-selectin, which was effectively diminished by the DTIs [38,43]. Another explanation may be that the available amount of MPs for incorporation in granulocytes through phagocytosis is reduced by the thrombin inhibitors. In contrast, the DTIs did not affect the TF expression within the formed PMAs. These results may indicate that the early TF expression within the formed PMAs depends on decrypted TF, or that the TF presenting MPs prefer adherence to monocytes, and thus TF will only adhere to granulocytes when expressed in high amounts [2]. The use of lag time in the CAT assay as a method for determination of change of endogenous TF activity has recently been described [44]. In our experimental model DTIs prolonged the lag time in accordance with the diminished amount of MPs. Similar results were found in the post-MI patients, where oral treatment with ximelagatran for 1 week significantly prolonged the lag time. An explanation for these results may be that oral treatment with DTI as secondary prevention has the capacity to reduce the amount of circulating procoagulant MPs and the production of TF within the formed PMAs in patients with recent MI. Thrombin also induced, to a lower degree, the formation of aggregates between platelets and lymphocytes. The interaction between platelets with natural killer cells and T-cytolytic cells are more pronounced than interaction with B-cells, and a limitation of the results in the present study is the lack of classification of lymphocytes [45]. To effectively reduce PLA formation, there is a need for combined blocking of several adhesion molecules [46]. The DTIs did not affect PLA formation even though platelet P-selectin expression was reduced in single platelets. CD40L has been described as an important receptor in the platelet-lymphocyte interaction, and in the present study DTIs neither diminish CD40L in single platelets nor within the aggregates [47]. We conclude that DTIs reduce TF production and activity through diminished adherence of platelets to monocytes and granulocytes upon thrombin stimulation. Oral DTIs also affect the TF activity in a post-MI population, which partly may depend on a diminished amount of circulating procoagulant microparticles. These effects could contribute to the clinical benefit with reduced risk of new thromboembolic events found in clinical trials. In contrast, the DTIs did not have an effect on ADP-induced PMA formation or TF production. Further studies of the importance of a combined treatment targeting different platelet and coagulant mechanisms in clinical settings are warranted. Conflict of interest statement None Acknowledgement We acknowledge Birgitta Fahlström, biomedical analyst, and Helena Vretman, research engineer, for excellent laboratory assistance. This work was supported by Swedish Heart-Lung Foundation,
Swedish Research Council and the Uppsala County Association Against Heart and Lung Diseases.
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