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Regulation of PAR-1 in patients undergoing percutaneous coronary intervention: effects of unfractionated heparin and bivalirudin

Roza Badr Eslam, Nina Reiter, Alexandra Kaider, Sabine Eichinger, Irene M. Lang, Simon Panzer
DOI: http://dx.doi.org/10.1093/eurheartj/ehp186 1831-1836 First published online: 24 May 2009

Abstract

Aims We examined the specific effects of unfractionated heparin and bivalirudin on thrombin-inducible platelet PAR-1 in patients undergoing percutaneous coronary intervention (PCI).

Methods and results To simulate in vivo conditions that may precipitate a bleeding event, we added thrombin in vitro to blood samples from 89 patients who had been randomly assigned to receive heparin or bivalirudin for elective PCI and examined thrombin-inducible PAR-1 expression. Thrombin-inducible cleavage of PAR-1 was inhibited by heparin, but not affected by bivalirudin (P = 0.0001). Further, PAR-1 internalization was more effectively inhibited by heparin than bivalirudin (P = 0.002).

Conclusion Heparin has stronger inhibitory effects on thrombin-dependent PAR-1 cleavage and internalization, thus providing a biological explanation for lower clinical bleeding rates with bivalirudin.

  • Bivalirudin
  • Heparin
  • Platelets
  • PAR-1
  • Thrombin
  • Percutaneous coronary intervention

Introduction

Unfractionated heparin, low molecular heparin, and bivalirudin are currently approved anticoagulants for percutaneous coronary intervention (PCI). Bivalirudin, a synthetic congener of hirudin, is a small molecule that inhibits thrombin by specific and direct binding to the catalytic site and to the anion-binding exosite of circulating and clot-bound thrombin. In contrast, heparin binds to antithrombin, which in turn becomes an inhibitor of factor Xa, thereby inhibiting a burst of thrombin generation. Further, heparin residues also bind to thrombin (at a site remote from its reactive centre), and the heparin-antithrombin complex also efficiently inhibits thrombin.

Platelet activation plays a key role in athero-thrombosis. Thrombin, generated during PCI, activates platelets via 3 distinct receptors, protease-activated receptors (PAR)-1 and PAR-4,1,2 and GPIb-alpha.35 There is strong evidence that the PAR-platelet activation pathway is clinically significant. For example, platelet PAR-1 modulation has been shown in patients with stroke.6 Furthermore, recent data have demonstrated that despite effective dual platelet inhibition, thrombin generation during PCI can activate platelets.7 Thrombosis, leukocyte chemotaxis, smooth muscle cell proliferation, and migration may ensue and stimulate vascular remodelling. A phase II trial with an oral PAR-1 antagonist in patients undergoing PCI has been completed (www.clinicaltrials.gov, NCT00132912).

The inhibitory effects of heparin may differ from those of bivalirudin on thrombin-inducible platelet activation. Moreover, heparin may induce platelet activation by its binding to platelet factor 4 and consecutive antibody generation, leading to heparin-induced thrombocytopenia.8 Indeed, in clinical trials, bivalirudin reduced the rate of thrombocytopenia significantly, consecutively also reducing blood tranfusions. Platelet expression of P-selectin, activation of the platelet fibrinogen binding site, and the formation of platelet-monocyte heterotypic aggregates, which significantly amplify the tissue pathway of thrombin generation,9 are increased in heparin, but not in bivalirudin treated patients.1013 These observations are complemented by the differential influence on plasma concentrations of CD40L, an important marker of the platelet inflammatory pathway.14 Systemic inflammation associated with thrombin generation attenuates PAR-1 mediated platelet activation.15 A comprehensive review on key trials evaluating bivalirudin for acute coronary syndrome has been published recently.8 In these clinical studies, bivalirudin was not superior to heparin in the prevention of ischaemic events after PCI, but was associated with less bleeding.1619 This difference was also observed if bivalirudin was compared with heparin in the absence of platelet glycoprotein/IIIa antagonists.20

Prompted by these observations, we tested the hypothesis of differential effects of heparin vs. bivalirudin on thrombin-inducible platelet activation. To simulate the effects of in vivo thrombin generation, we exposed blood samples from patients who had received heparin or bivalirudin to thrombin in vitro. Our data indicate that in contrast to heparin, bivalirudin preserves the platelet PAR-1 response to thrombin.

Patients

The study complied with the Declaration of Helsinki, was approved by the Ethics Committee of the Medical University of Vienna, and all patients gave written informed consent. The study was designed to test the hypothesis of differential effects of heparin vs. bivalirudin on thrombin-inducible platelet activation in a single centre trial. Prior to PCI, patients were randomized to receive either unfractionated heparin (Heparin Immuno, Unterach, Austria, 65 U/kg as a bolus) or bivalirudin (Angiox, Nycomed, Vienna; 0.75 mg/kg followed by an infusion of 1.75 mg/kg per hour during PCI). Patients who had been pre-treated with thrombolytic therapy or platelet glycoprotein IIb/IIIa antagonists were excluded. Prior to and 30 min after the administration of heparin or bivalirudin, trisodium citrate-anticoagulated whole blood (nine parts of whole blood and one part of trisodium citrate 0.108 mol/L) was obtained from the arterial sheath after the initial 3 mL of aspirate had been discarded. All samples were analysed within 30 min to 2 h after blood collection.

Laboratory methods

Regulation of PAR-1 and P-selectin

To differentiate between thrombin-inducible PAR-1 cleavage and its internalization, two specific monoclonal antibodies (mAbs) were employed. MAbs SPAN12 and WEDE15 bind to two distinct epitopes on PAR-1. Clone SPAN12 is directed against a peptide spanning the receptor cleavage site, and binds only to uncleaved PAR-1. Clone WEDE15 recognizes an extracellular epitope on uncleaved as well as on cleaved PAR-1, but not internalized PAR-1.6 In addition, platelet activation was analysed by measuring P-selectin expression. A FACSCalibur flow cytometer (Becton Dickinson, BD, San Jose, CA, USA) with excitation by an argon laser at 488 nm and a red diode laser at 635 nm was used. The instrument was calibrated daily with standard BD Calibrite beads containing specific amounts of ‘mean equivalent soluble fluoresceine molecules’ in combination with the CELLQUestPro software. Trisodium citrate-anticoagulated whole blood was diluted with HEPES buffer to 20 × 109/L platelets. Twenty microlitre aliquots were incubated in the presence of GPRP (1.25 mmol/L, Sigma-Aldrich, Vienna, Austria) with α-thrombin (0.2 or 1 U/mL for the evaluation of binding of the mAb SPAN12 and anti-CD62p or WEDE15, respectively; Sigma-Aldrich) or PBS for 10 min at room temperature. A mixture of mAbs (anti-CD62p, clone CLB-Thromb6, fluorescein isothiocyanate—FITC—labelled; anti-PAR-1, clone SPAN12 or clone WEDE15, both phycoerythrin—PE—labelled; all from Immunotech, Beckman Coulter, Fullerton, CA, USA; the platelet-specific antibody anti-CD41 (anti-glycoprotein IIb), clone HIP8, allophycocyanin—APC—labelled, BD; each 10 µL) or of isotype-matched controls was added. After 15 min, samples were diluted in 500 µL HEPES-paraformaldehyde (0.5%) and acquired immediately. At least 10 000 CD41+ events were acquired for the determination of PE+ signals.

Determination of D-dimer and thrombin generation

D-Dimer was determined from citrated plasma using a commercially available enzyme immunoassay (D-Dimer: Asserachrom® D-Di, Roche, Germany). In vitro thrombin generation was determined in platelet poor plasma by use of a commercially available assay (Technothrombin®TGA, Technoclone, Vienna, Austria) that monitors the fluorescence generated by thrombin cleavage of a fluorogenic substrate over time upon activation of the coagulation cascade by recombinant human tissue factor (final concentration 7.16 pM) and negatively charged phospholipids (3.2 µM). The peak amount of thrombin was used as read out variable.

Statistical analyses

We determined changes induced by heparin or bivalirudin in each individual and used the sign test for statistical evaluations within a group. The Mann–Whitney test was used to compare changes between groups. Data are presented as median and lower and upper quartiles. Two-sided P-values <0.05 were considered statistically significant.

Results

Patients’ characteristics are shown in Table 1. All patients had stable coronary artery disease and all were on aspirin (100 mg per day) and clopidogrel (75 mg per day) for at least 2 weeks. Thirty-seven patients in the heparin group and 41 patients in the bivalirudin group were on lipid-lowering medications. There was no difference between groups with respect to age, history of diabetes, systemic hypertension, or any concomitant disease.

View this table:
Table 1

Clinical characteristics of the study population

Heparin (n = 44)Bivalirudin (n = 45)
Age (median years; Q1–Q3)(65; 58–76)(67; 53–72)
Female/male ratio8/3616/29
CCS I4 (9%)3 (7%)
CCS II11 (25%)11 (24%)
CCS III17 (39%)20 (44%)
Diabetes14 (32%)13 (29%)
Systemic arterial hypertension40 (91%)40 (89%)
Hyperlipidaemia35 (80%)37 (82%)
Active smoker16 (36%)21 (47%)
BMI (median; Q1–Q3)(27; 25–30)(28; 26–31)
Prior PCI24 (55%)22 (49%)
Prior coronary bypass surgery5 (11%)8 (18%)
Procedural data
 Target vessel
  LAD24 (55%)24 (53%)
  RCA19 (43%)23 (51%)
  CX8 (18%)11 (24%)
  LM1 (2%)2 (4%)
  BP0 (0%)2 (4%)
 Number of stents1 ± 12 ± 1
 ACT (median; Q1–Q3)(250; 215–274)(270; 280–292)
  • CCS, Canadian Cardiovascular Angina Classification; BMI, body mass index; PCI, percutaneous coronary intervention; LAD, left artery descending coronary artery; RCA, right coronary artery; CX, circumflex coronary artery; LM, left main coronary artery; BP, bypass graft; ACT, peak activated clotting time.

Effects on PAR-1 cleavage/internalization

At baseline, before anticoagulation, there was no difference of PAR-1 expression between groups (Table 2). Within groups, binding of mAb SPAN12 was not significantly affected by the presence of heparin or bivalirudin (Table 3), but comparison between groups revealed more cleavage of PAR-1 in the bivalirudin group (Figure 1A). By the characteristics of mAb WEDE15 PAR-1 internalization was demonstrable in the bivalirudin group (Table 3). Figure 1A and C demonstrates a higher susceptibility of PAR-1 in the bivalirudin group to endogenous thrombin that was generated during the first 30 min of the PCI. To confirm this observation, thrombin was added in vitro. In the heparin group, both mAbs bound equally well to PAR-1 as in the absence of thrombin indicating that in vitro added thrombin was not able to cleave or induce internalization of the receptor (Table 2 and Figure 1B and D). However, in the bivalirudin samples, in vitro thrombin induced a significant reduction of SPAN12 binding, similar to the extent before anticoagulation (Table 2). This differential responsiveness of samples from the two groups is illustrated in Figure 1B. Further, after thrombin addition, binding of WEDE15 was significantly weaker in the bivalirudin group than in the heparin group (Figure 1D), indicating that PAR-1 was accessible for thrombin only in the bivalirudin group.

Figure 1

The effects of heparin and bivalirudin on PAR-1 and P-selectin expression. Blots show the absolute change between data obtained before and 30 min after the start of anticoagulation, as determined without and after exogenous addition of α-thrombin. Changes within patients’ groups were compared statistically. Clone SPAN12 binds only to uncleaved PAR-1, clone WEDE15 recognizes an extracellular epitope on uncleaved as well as on cleaved PAR-1, but not internalized PAR-1. P-selectin was determined by binding of clone CLB-Thromb6 in samples that were either untreated or treated with α-thrombin (0.2 U/mL). Box plots show the lower and upper quartile of data, whiskers indicate their range, circles outliners, and stars extremes. The dotted line indicates no change.

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Table 2

Expression of the PAR-1 receptor and P-selectin (MFI, mean fluorescence intensity) in patients receiving either unfractionated heparin or bivalirudin

HeparinBivalirudin
MedianQ1–Q3MedianQ1–Q3
Baseline
SPAN120 (MFI)19.916.4–24.419.914.4–26.8
SPAN12thrombin (MFI)13.311.5–17.615.211.4–17.6
WEDE150 (MFI)37.828.6–44.837.831.4–45.5
wEDE15thrombin (MFI)14.611.8–18.814.310.5–18.1
P-selectin0 (MFI)7.26.4–8.57.36.3–8.6
P-selectinthrombin (MFI)112.166.9–147.6109.388.2–130.3
After 30 min
SPAN120 (MFI)19.816.9–25.621.115.5–25.4
SPAN12thrombin (MFI)18.615.6–22.616.212.0–19.7
WEDE150 (MFI)37.728.3–44.937.430.0–44.5
wEDE15thrombin (MFI)33.422.2–39.924.115.9–33.8
P-selectin0 (MFI)7.06.3–8.16.75.9–7.6
P-selectinthrombin (MFI)8.77.3–11.07.06.1–8.1
  • Lower (Q1) and upper quartiles (Q3) are shown. Samples were collected prior to anticoagulation (baseline), and 30 min later.

  • ‘thrombin’ denotes that samples were analysed after in vitro added thrombin.

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Table 3

Flow cytometric determination of PAR-1 expression and P-selectin in patients undergoing percutaneous coronary intervention and randomly assigned to receive either unfractionated heparin or bivalirudin

Heparin (change)Bivalirudin (change)
MedianQ1; Q3P-valueMedianQ1; Q3P-value
SPAN120 (MFI)0.5−0.2; 1.10.233−0.56−0.9; 0.30.11
SPAN12thrombin (MFI)5.42.7; 7.5<0.00010.54−1.3; 3.50.16
WEDE150 (MFI)0.14−0.7; 0.91.0−0.9−1.7; 0.060.0034
WEDE15thrombin (MFI)16.811.4; 22.9<0.00018.91.9; 13.1<0.0001
P-selectin0 (MFI)−0.1−0.4; 0.10.02−0.4−0.97; −0.06<0.0001
P-selectinthrombin (MFI)−100.1−133.1; −46.6<0.0001−102.1−122.2; −80.5<0.0001
  • Data were determined without and after exogenously added thrombin. Differences between data obtained before and after anticoagulation within each group are shown.

Effects of heparin vs. bivalirudin on platelet activation

We tested whether heparin or bivalirudin exerted a differential effect on P-selectin expression. Both anticoagulants reduced the expression of P-selectin and this reduction was more pronounced after bivalirudin (Table 3), but without a statistically significant difference between groups (Figure 1E). Thrombin-inducible expression of P-selectin was inhibited in both groups to a similar extent (Figure 1F).

Thrombin generation

We determined thrombin generation and levels of D-dimers to differentiate if modulation of PAR-1 was due to measurable thrombin generation in one group of patients vs. the other. Levels of thrombin and D-dimer were similar prior to treatment (thrombin: heparin group 193, 110–250 nM; bivalirudin group 169, 65–232 nM; P = 0.2; D-dimer: heparin group 648, 477–1207 ng/mL; bivalirudin group 603, 330–1373 ng/mL; P = 0.7) and at 30 min (thrombin 0, 0–0 nM; vs. 0, 0–0 nM, P = 0.2; D-dimer 556, 379–891 ng/mL vs. 496, 287–1137 ng/mL; P = 0.5).

Discussion

We tested the hypothesis that the clinically observed advantage of bivalirudin over heparin on bleeding is due to differential effects on the platelet thrombin receptor PAR-1. To simulate a real-life scenario of periprocedural vessel injury followed by thrombin generation and platelet activation, we estimated the effects of added thrombin to blood samples from patients who were on dual anti-platelet treatment, were undergoing elective PCI, and who received heparin or bivalirudin in a randomized fashion. At ACT-driven therapeutic doses (Table 1) of either anticoagulant heparin was a slightly stronger inhibitor of thrombin-inducible PAR-1 attenuation. These findings may provide a novel explanation for less bleeding with bivalirudin than heparin.1620

The influence of heparin and bivalirudin on PAR-1 was specifically addressed by studying its thrombin-inducible expression. While PAR-1 remained unchanged in the heparin group, less expression was seen in the bivalirudin group. This PAR-1 attenuation was due to both receptor cleavage and internalization, indicating periprocedural generation of thrombin. It has been shown that thrombin activates PAR-1 in pM concentrations, while higher concentrations are needed to activate PAR-4. The small difference of in vitro inducible thrombin generation between the patients’ groups before anticoagulation does not explain the observed difference of PAR-1 expression, as thrombin generation was slightly higher in the heparin group. As expected, thrombin generation was not detectable after initiation of anticoagulation with heparin or bivalirudin. Higher median levels of D-dimers in the heparin than in the bivalirudin group indicate some activation of coagulation. Therefore, higher levels of P-selectin expression in the heparin-treated patients can be expected. However, all patients were on dual anti-platelet therapy, which possibly obscured a differential effect of bivalirudin vs. heparin on P-selectin expression. It has been shown that bivalirudin inhibits thrombin-inducible expression of P-selectin and activation of platelet glycoprotein IIb/IIIa stronger than heparin. However, this difference between heparin and bivalirudin was only seen with significantly higher concentrations of thrombin.11 PAR-1-induced platelet activation results in platelet aggregation, as PAR-1 and PAR-4 form a heterodimer, susceptible for thrombin.2 A cleaved/internalized PAR-1 is less accessible for further thrombin-induced platelet activation. ADP also leads to platelet aggregation and the combined inhibition of the ADP receptor by clopidogrel and a cleaved/internalized PAR-1 in bivalirudin-treated patients may augment the reduction of in vitro ADP-inducible platelet aggregation response.21

The addition of thrombin accentuated the blocking effects of heparin on thrombin-inducible PAR-1 cleavage/internalization (Table 2 and Figure 1B and D). Our experiments clearly show that not heparin but bivalirudin allowed receptor cleavage illustrated by diminished binding of mAb SPAN12, and receptor internalization illustrated by diminished binding of mAb WEDE15.

Thrombin is a very potent natural platelet activator. Heparin and bivalirudin counteract thrombin, but by different mechanisms. Downregulation of PAR-1 indicates intact active interaction of thrombin with PAR-1, which is preserved in the presence of bivalirudin at therapeutic dosages and results in procoagulatory platelet activity. These findings, together with the shorter half-life of bivalirudin, may explain less bleeding related to PCI,1620 albeit at an increased risk of acute stent thrombosis within 24 h in patients with excessive thrombin, like in acute myocardial infarction.18 Therefore, additional dual antiplatelet therapy is to be considered mandatory. In contrast, complete receptor blockade, e.g. by heparin, is associated with diminished availability of PAR-1, and therefore less platelet response to thrombin.

Funding

This research project received in part financial support from the European Commission under the 6th Framework Programme (Contract No: LSHM-CT-2005-018725, PULMOTENSION).

Conflict of interest: none declared.

Acknowledgements

The authors thank Beate Eichelberger and Daniela Koren for technical assistance, and the staff from the interventional cardiac catheter laboratory of the Medical University of Vienna for their efforts. We would also like to thank Alexander Ostrowerhow, Institute of Microbiology and Genetics, University Vienna, for data managing.

Footnotes

  • I.M.L. and S.P. contributed equally

  • This study was part of the doctorial thesis of NR at the Division of Blood Group Serology.

References

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