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Potentiation of platelet aggregation by heparin in human whole blood is attenuated by P2Y12 and P2Y1 antagonists but not aspirin
Thrombosis Research (2005) 115, 301 – 307
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Potentiation of platelet aggregation by heparin in human whole blood is attenuated by P2Y12 and P2Y1 antagonists but not aspirin Robert F. Storey a,*, Jane A. May b, Stan Heptinstall b a
Cardiovascular Research Unit, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK Cardiovascular Medicine, University Hospital, Queen’s Medical Centre, Nottingham, UK
b
Received 13 July 2004; received in revised form 6 September 2004; accepted 7 September 2004 Available online 30 November 2004
KEYWORDS Platelets; Platelet aggregation inhibitors; P2 receptor; Heparin; Cangrelor
Abstract Introduction: Unfractionated heparin (UFH) potentiates platelet aggregation induced by some agonists. P2Y12 and P2Y1 receptors play a major role in amplifying platelet aggregation. We assessed the ability of cangrelor, a selective P2Y12 antagonist, A2P5P, a selective P2Y1 antagonist, and aspirin to block the potentiating effects of heparin. Materials and methods: Whole blood from healthy human volunteers was anticoagulated with either hirudin or UFH 10 IU/ml. Some tubes anticoagulated with hirudin also contained UFH 1 or 10 IU/ml. The low-molecular-weight heparin dalteparin was also assessed. Platelet aggregation was performed using whole blood single-platelet counting. Dense granule release was assessed using 14C-5HT-labelled platelets. Results: UFH and, to a lesser extent, dalteparin potentiated platelet aggregation induced by ADP, PAF, 5HT, U46619, epinephrine and TRAP in a concentrationdependent manner but inhibited aggregation induced by collagen. Cangrelor effectively opposed the potentiating effects of heparins on sustained aggregation induced by ADP, PAF, 5HT, U46619 and TRAP but had less effect on epinephrineinduced aggregation, whereas A2P5P was more effective at blocking both the initial phase of ADP-induced aggregation and the aggregation response to epinephrine, reflecting the differences in G protein coupling between the agonist receptors.
Abbreviations: UFH, unfractionated heparin; A2P5P, adenosine-2,5-bisphosphate; TRAP, thrombin receptor activating peptide; PAF, platelet-activating factor; 5HT, 5-hydroxytryptamine. * Corresponding author. Tel.: +44 114 2714192; fax: +44 114 2619587. E-mail address: [email protected] (R.F. Storey). 0049-3848/$ - see front matter D 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2004.09.004
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R.F. Storey et al. Aspirin had no effect on potentiation by heparin. Heparins did not increase ADP- or TRAP-induced 14C-5HT release. Conclusions: Heparins potentiate platelet responses to ADP and numerous other agonists. This potentiation is attenuated by cangrelor and A2P5P, and is not mediated by increased dense granule release. ADP receptor antagonists but not aspirin may have potential therapeutic benefits in counteracting the pro-thrombotic effects of heparins. D 2004 Elsevier Ltd. All rights reserved.
Anticoagulation with heparin is used widely in the prevention and management of venous and arterial thrombosis, as well as during procedures such as cardiopulmonary bypass surgery. Whilst heparin inhibits thrombin-induced platelet aggregation [1], it conversely potentiates platelet aggregation induced by a range of platelet agonists including ADP, TRAP, PAF, U46619 and epinephrine [2,3] and these potentiating effects are detectable at therapeutic concentrations of heparin [3]. However, the mechanism whereby heparin potentiates platelet aggregation is unknown, and we sought to elucidate the role of platelet ADP receptors in this phenomenon. ADP may be released from erythrocytes, endothelial cells and platelet dense granules; it plays a major role in haemostasis and the pathogenesis of arterial thrombus by activating platelets, causing granule secretion and aggregation via activation of glycoprotein IIb/IIIa receptors which then bind fibrinogen [4]. The release of ADP by platelets stimulated by other agonists such as thrombin or collagen contributes substantially to platelet aggregation explaining why antagonists of ADP-induced platelet aggregation partially inhibit aggregation by these other platelet aggregating agents [5,6]. There are two receptors for ADP on platelets, P2Y1 and P2Y12 receptors (G-protein coupled receptors), that contribute synergistically to ADP-induced platelet aggregation [7]. A number of specific antagonists to the P2Y1 receptor have been identified, including adenosine-2,5-bisphosphate (A2P5P) [8,9]. Analogues of ATP that bind specifically to the P2Y12 receptor have been developed and one such agent, cangrelor (formerly AR-C69931MX), is currently being developed as an intravenous antithrombotic agent [10,11]. Whole blood single-platelet counting is a measure of microaggregation and, characteristically, there is maximal microaggregation at 10 to 30 s after addition of most agonists (dearlyT aggregation response) followed by either a sustained aggregation response (associated with macroaggregate formation) or reversal of aggregation [5,6]. We previously have described the effects of selective antagonists
of the P2Y1 and P2Y12 receptors on platelet aggregation induced by a range of agonists in human whole blood [6]. Here, we have investigated the role of these receptors in mediating the potentiating effects of heparin on platelet aggregation.
Materials and methods Materials Cangrelor (AR-C69931MX) was provided by AstraZeneca R&D Charnwood, Loughborough, UK. A2P5P, ADP, epinephrine, U46619 (a thromboxane A2 mimetic), thrombin receptor activating peptide (TRAP; SFLLRNPNDKYEPF), aspirin and platelet activating factor (PAF; dissolved in 0.25% w/v human serum albumin) were from Sigma. 5Hydroxytryptamine (5HT; serotonin) was from Amersham International. Hirudin was Revasc (recombinant desulphatohirudin). Saline was 0.9% w/v NaCl supplied by Baxter Healthcare Fixative contained saline with 4.6 mmol/l Na2EDTA, 4.5 mmol/l Na2HPO2, 1.6 mmol/l KH2PO4 and 0.16% w/v formaldehyde, pH 7.4. 14C-5HT was from Amersham (1.85 MBq/ml). Chlorimipramine hydrochloride was from Novartis. Scintillation fluid (fluroscan 2) was from BDH Laboratories Supplies. Heparin sodium (unfractionated heparin) was from Sigma and dalteparin (a low-molecular-weight heparin) was Fragmin from Pfizer.
Preparation of blood Blood was obtained from healthy volunteers using a 19-G needle and plastic syringe. All volunteers denied taking any non-steroidal anti-inflammatory drug in the previous 2 weeks. Aliquots (9-ml) of blood were transferred to polystyrene tubes containing either hirudin (500 Ag in 1 ml 0.9% saline) or UFH (100 IU in 1 ml 0.9% saline). UFH 1 or 10 IU/ml (final concentration) was included with hirudin in some tubes, reflecting a therapeutic concentration and 10-fold greater concentration,
Heparin and platelet ADP receptor antagonism respectively. Aspirin 100 Amol/l (final concentration) was also included with the anticoagulant in some tubes. For the release reaction experiments, 6 Al 14C-5HT (final concentration 0.521 Amol/l; final activity 1.11 kBq/ml) was added to the anticoagulant (for uptake by platelets). The tubes were incubated at 37 8C in a waterbath for a standard 30-min period prior to experimentation.
Whole blood platelet aggregation Platelet aggregation studies were performed using the single-platelet counting technique, as previously described [6]. Saline, cangrelor 1 Amol/l or A2P5P 300 Amol/l was added 2 min prior to agonist. The following agonists were assessed: ADP 0.3–1 Amol/l, PAF 0.1 Amol/l, 5HT 30 Amol/l, U46619 0.3 Amol/l, epinephrine 3 Amol/l, TRAP 3 Amol/l, and collagen 0.5–8 Ag/ml. Concentrations of agonists that yielded submaximal aggregation in the absence of heparin were selected, when appropriate, to demonstrate potentiation. Aliquots of blood were removed after stirring with agonist for 30 s and 4 min and fixed prior to single-platelet counting using an Ultra-Flo 100 Platelet Counter (Becton-Dickinson). 14
C-5HT release measurements
5HT release was determined as previously described [5], using the agonists described in Results. Chlorimipramine (20-Al) was added to 420-Al aliquots of whole blood containing 14 C-5HTlabelled platelets, as well as 40-Al saline and/or P2Y antagonist as indicated in Results. Agonist (20 Al) was then added and samples stirred for 4 min before the reaction was terminated by placing on ice as well as adding 50-Al aspirin solution (1.26 mmol/l). Samples were centrifuged at 1500g for 10 min and duplicate 50-Al samples of supernatant plasma mixed with scintillation fluid prior to analysis in a scintillation counter. One hundred percent release was estimated using duplicate 50Al samples of saline solution of 14C-5HT at the same concentration as that added to the whole blood sample, with adjustment of the results according to the packed cell volume of the blood sample to derive the initial plasma 14C-5HT concentration.
Data analysis Data are mean of six experiments and are expressed as mean and standard deviation. Signifi-
303 cance was attributed to p values b0.05 and determined using ANOVA (SPSS software).
Results Effect of P2Y1 and P2Y12 antagonists on potentiation of ADP-induced platelet aggregation by heparin Heparin potentiated both the early aggregation response to ADP 0.3 Amol/l and the sustained response, 4 min after addition of ADP (Fig. 1). The P2Y1 antagonist A2P5P and the P2Y12 antagonist cangrelor both inhibited the aggregation response to ADP and the combination of both antagonists was required to prevent potentiation of the early aggregation response by heparin, whereas potentiation of the sustained aggregation response was abolished by cangrelor alone. Aspirin had no inhibitory effect. Heparin increased the concentration of cangrelor required to achieve its inhibitory effect (Fig. 2).
Effect of P2Y1 and P2Y12 antagonists on potentiation by heparin of platelet aggregation induced by other agonists Heparin potentiated platelet aggregation induced by PAF and epinephrine (Fig. 3). The potentiating effect of heparin on PAF-induced aggregation was attenuated by the combination of A2P5P and cangrelor whilst the potentiation of epinephrineinduced aggregation was relatively resistant to the effects of these inhibitors. Heparin also potentiated platelet aggregation induced by 5HT and the thromboxane A2 analogue U46619, this potentiation being markedly attenuated by the combination of A2P5P and cangrelor (data not illustrated). In all cases, aspirin had no inhibitory effect. Heparin potentiated both early and sustained TRAP-induced aggregation in a concentration-dependent manner (Fig. 4). Potentiation of the early aggregation response to TRAP was attenuated by cangrelor whilst potentiation of the sustained aggregation response was abolished by cangrelor. Consistent with previous studies [12], collagen-induced aggregation was inhibited by heparin (and, to a lesser extent, dalteparin): for example, aggregation 4 min after the addition of collagen 0.5 Ag/ml was 86F3%, 66F7% and 56F7% for hirudin, dalteparin 5 IU/ml and heparin 10 IU/ml anticoagulation, respectively ( pb0.05 for both dalteparin and heparin vs. hirudin).
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Potentiation of platelet aggregation by the low-molecular-weight heparin dalteparin Like unfractionated heparin, dalteparin also potentiated platelet aggregation induced by ADP, TRAP, PAF and epinephrine (Fig. 5) but, unlike unfractionated heparin, a significant effect was
Figure 2 Mean percentage inhibition by cangrelor of platelet aggregation measured by whole blood singleplatelet counting 4 min after addition of ADP 10 Amol/ l, comparing whole blood anticoagulated with either hirudin or hirudin with UFH 10 IU/ml. Data are meanFS.D. (n=6). *pb0.05 for effect of UFH.
only seen at a supratherapeutic concentration of the drug.
Effect of heparins on dense granule release There was no potentiation by either unfractionated heparin or dalteparin of 14C-5HT release from 14C5HT-labelled platelets induced by ADP 30 and 100 Amol/l or TRAP 3 Amol/l, despite potentiation of platelet aggregation by heparins in the same experiment, indicating that the potentiation of platelet aggregation is not mediated by increased dense granule release (data not illustrated).
Discussion
Figure 1 Platelet aggregation induced by ADP 0.3 Amol/l after (A) 30 s and (B) 4 min in whole blood anticoagulated with either hirudin alone or hirudin plus UFH 10 IU/ml, assessing the effects of pre-incubation for 2 min with either saline, cangrelor 1 Amol/l, A2P5P 300 Amol/l or the combination of cangrelor 1 Amol/l with A2P5P 300 Amol/l and pre-incubation for 30 min with aspirin 100 Amol/l (n=6). *pb0.05 for effect of cangrelor or A2P5P or their interaction. ypb0.05 for effect of UFH vs. hirudin. zpb0.05 for effect of UFH in presence of cangrelor or A2P5P. No significant effect of aspirin was seen alone or in combination.
We have shown that both unfractionated and lowmolecular-weight heparins have substantial potentiating effects on platelet aggregation induced by a wide range of platelet agonists, with the exception of thrombin (not studied here) and collagen, confirming the findings of previous investigations that have used different methodology to assess the effects of heparin [2,3,13]. The lack of significant effect of the low-molecular-weight heparin dalteparin at a therapeutic concentration is also consistent with lack of detectable effect of enoxaparin administration [3], though we have demonstrated that a supratherapeutic concentration of dalteparin has similar, though weaker, effects compared to unfractionated heparin. Previous studies have used apyrase to assess the role of ADP in the potentiation of platelet
Heparin and platelet ADP receptor antagonism
305 shown that potentiation by heparin of activation mediated via this receptor plays a major role in the effects of heparin on sustained platelet aggregation. Both the P2Y12 receptor and the a2 adrenergic receptor, by which epinephrine activates platelets, are linked to Gi and the inhibition of adenylate cyclase [15]. The finding that potentiation of epinephrine-induced aggregation by heparin is resistant to the effects of the P2Y12 receptor antagonist cangrelor indicates that heparin is acting downstream of the P2Y12 receptor. The receptors for PAF, 5HT, TRAP and U46619 are linked to Gaq and the activation of phospholipase C [16]. We have shown that potentiation of the early aggregation response to these agonists by heparin occurs despite P2Y12 and P2Y1 receptor
Figure 3 Platelet aggregation measured by singleplatelet counting 4 min after addition of (A) PAF 0.1 Amol/l or (B) epinephrine 10 Amol/l in hirudin-anticoagulated whole blood incubated for 30 min with either saline control, aspirin 100 Amol/l, UFH 10 IU/ml or a combination of aspirin 100 Amol/l and UFH 10 IU/ml (n=6). Samples were pre-incubated for 2 min with either saline control, cangrelor 1 Amol/l, A2P5P 300 Amol/l, or a combination of cangrelor 1 Amol/l and A2P5P 300 Amol/ l. *pb0.05 for effect of cangrelor or A2P5P or their interaction. ypb0.05 for effect of UFH vs. hirudin. zpb0.05 for effect of UFH in presence of cangrelor or A2P5P. No significant effect of aspirin was seen alone or in combination.
aggregation by heparin and the findings have been inconsistent [2,13]. Developments in the understanding of ADP-induced platelet activation and availability of selective antagonists of the receptors involved in the process have allowed us to define the role of ADP more precisely. The P2Y12 receptor plays a central role in amplifying and sustaining platelet aggregation induced by numerous platelet agonists [5,14] and we have
Figure 4 Platelet aggregation induced by TRAP 3 Amol/ l, comparing anticoagulation of whole blood with hirudin, UFH 10 IU/ml, or hirudin with addition of UFH 1 or 10 IU/ ml (incubated for 30 min at 37 8C). Platelet aggregation is assessed by single-platelet counting at (A) 30 s and (B) 4 min after addition of TRAP after pre-incubation for 2 min with either saline control or cangrelor 1 Amol/l. Data are meanFS.D. (n=6). *pb0.05 for effect of UFH vs. hirudin. ypb0.05 for effect of cangrelor vs. saline.
306
Figure 5 Platelet aggregation measured by singleplatelet counting 4 min after addition of either ADP 0.3 Amol/l, TRAP 3 Amol/l, PAF 0.3 Amol/l or epinephrine 3 IU/ml in whole blood anticoagulated with either hirudin, UFH 10 IU/ml, hirudin plus the low-molecular weight heparin dalteparin 5 IU/ml, hirudin plus dalteparin 0.5 IU/ml or dalteparin 5 IU/ml alone. Data are meanFS.D. (n=6). *pb0.05 for effect of UFH or dalteparin vs. hirudin.
blockade. We have previously found that the high concentrations of cangrelor and A2P5P completely block the respective receptor activation by ADP at a concentration of 0.3 Amol/l [5], and, assuming that receptor blockade was complete in these experiments, it is likely that heparin is also acting downstream of (or at the level of) Gaq. Currently, there are several P2Y12 receptor antagonists either in clinical use or in clinical development [17]. The thienopyridines ticlopidine and clopidogrel act, via hepatic metabolites, on the P2Y12 receptor and have a proven role in the management and prevention of arterial thrombosis [17,18]. Phase II studies have shown cangrelor to be a well-tolerated intravenous antithrombotic agent in patients with acute coronary syndromes [10,11] and it yields more effective receptor blockade than clopidogrel [19]. Further studies of cangrelor and AZD6140, an oral, reversibly acting P2Y12 antagonist, are planned. Our findings here indicate that such P2Y12 antagonists may play a useful role in counteracting the prothrombotic properties of heparin. Indeed, clopidogrel has been studied in combination with heparin, as well as aspirin, in patients with acute coronary syndromes and has been found to reduce the combined incidence of myocardial infarction, stroke and cardiovascular death [18,20]. Opposition by heparin of the inhibitory effects of cangrelor on platelet aggregation may require consideration of dose adjustment of cangrelor in settings where significant plasma concentrations of unfractionated heparin are achieved. The possibility of developing P2Y1 recep-
R.F. Storey et al. tor antagonists for clinical use remains to be explored. Aspirin fails to inhibit platelet aggregation induced by numerous agonists, with the notable exception of collagen, when physiological divalent cation levels are maintained by the use of a direct thrombin inhibitor such as hirudin [5]. This study demonstrates that the potentiation of platelet aggregation by heparin is independent of the cyclo-oxygenase pathway. Thus, co-administration of aspirin with heparin, as frequently occurs in the management of arterial interventions and acute coronary syndromes, fails to provide any protection against the deleterious effects of heparin, which may be of particular importance in these settings. In conclusion, heparins potentiate platelet aggregation induced by ADP and numerous other agonists, apart from thrombin and collagen, and P2Y receptors play an important role in this potentiation. The potentiation is not mediated by increased thromboxane A2 secretion or dense granule release. ADP receptor antagonists but not aspirin may have potential therapeutic benefits in counteracting the pro-thrombotic effects of heparins.
Acknowledgements We are grateful to AstraZeneca R&D Charnwood for the gift of cangrelor (AR-C69931MX).
References [1] Eika C. Inhibition of thrombin induced aggregation of human platelets by heparin. Scand J Haematol 1971;8: 216 – 22. [2] Salzman EW, Rosenberg RD, Smith MH, Lindon JN, Favreau L. Effect of heparin and heparin fractions on platelet aggregation. J Clin Invest 1980;65:64 – 73. [3] Xiao Z, Theroux P. Platelet activation with unfractionated heparin at therapeutic concentrations and comparisons with a low-molecular-weight heparin and with a direct thrombin inhibitor. Circulation 1998;97:251 – 6. [4] Gachet C, Hechler B, Leon C, Vial C, Ohlmann P, Cazenave J-P. Purinergic receptors on blood platelets. Platelets 1996;7:261 – 7. [5] Storey RF, Sanderson HM, White AE, May JA, Cameron KE, Heptinstall S. The central role of the P2T receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity. Br J Haematol 2000; 110:925 – 34. [6] Storey RF, Newby LJ, Heptinstall S. Effects of P2Y1 and P2Y12 receptor antagonists on platelet aggregation induced by different agonists in human whole blood. Platelets 2001; 12:443 – 7. [7] Jin J, Kunapuli SP. Coactivation of two different G proteincoupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A 1998;95:8070 – 4.
Heparin and platelet ADP receptor antagonism [8] Sun B, Li J, Okahara K, Kambayashi J. P2X1 purinoceptor in human platelets. Molecular cloning and functional characterization after heterologous expression. J Biol Chem 1998; 273:11544 – 7. [9] Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADPinduced platelet activation: II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 1998;273:2030 – 4. [10] Storey RF, Oldroyd KG, Wilcox RG. Open multicentre study of the P2T receptor antagonist AR-C69931MX assessing safety, tolerability and activity in patients with acute coronary syndromes. Thromb Haemost 2001;85:401 – 7. [11] Jacobsson F, Swahn E, Wallentin L, Dellborg M. Safety profile and tolerability of intravenous AR-C69931MX, a new antiplatelet drug, in unstable angina pectoris and non-Qwave myocardial infarction. Clin Ther 2002;24:752 – 65. [12] Paolini R, Casonato A, Boeri G, Luzzatto G, Girolami A, Sasahara AA, et al. Effect of recombinant-tissue plasminogen activator, low molecular weight urokinase and unfractionated heparin on platelet aggregation. J Med 1993;24:113 – 30. [13] Eika C. On the mechanism of platelet aggregation induced by heparin, protamine and polybrene. Scand J Haematol 1972;9:248 – 57. [14] Trumel C, Payrastre B, Plantavid M, Hechler B, Vial C, Presek P, et al. A key role of adenosine diphosphate in the
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irreversible platelet aggregation induced by the PAR-1 activating peptide through the late activation of phosphoinositide 3-kinase. Blood 1999;94:4156 – 65. Daniel JL, Dangelmaier C, Jin J, Kim YB, Kunapuli SP. Role of intracellular signaling events in ADP-induced platelet aggregation. Thromb Haemost 1999;82:1322 – 6. Offermanns S, Toombs CF, Hu Y-H, Simon MI. Defective platelet activation in Gaq-deficient mice. Nature 1997; 389:183 – 6. Storey RF. The platelet P2Y12 receptor as a therapeutic target in cardiovascular disease. Platelets 2001;12:197 – 209. The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators RF. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 2001; 345:494 – 502. Storey RF, Wilcox RG, Heptinstall S. Comparison of the pharmacodynamic effects of the platelet ADP receptor antagonists clopidogrel and AR-C69931MX in patients with ischaemic heart disease. Platelets 2002;13:407 – 13. Mehta SR, Yusuf S, Peters RJG, Bertrand ME, Lewis BS, Natarajan MK, et al. Effects of pretreatment with clopidogrel and aspirin followed by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet 2001;358:527 – 33.
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Potentiation of platelet aggregation by heparin in human whole blood is attenuated by P2Y12 and P2Y1 antagonists but not aspirin Robert F. Storey a,*, Jane A. May b, Stan Heptinstall b a
Cardiovascular Research Unit, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK Cardiovascular Medicine, University Hospital, Queen’s Medical Centre, Nottingham, UK
b
Received 13 July 2004; received in revised form 6 September 2004; accepted 7 September 2004 Available online 30 November 2004
KEYWORDS Platelets; Platelet aggregation inhibitors; P2 receptor; Heparin; Cangrelor
Abstract Introduction: Unfractionated heparin (UFH) potentiates platelet aggregation induced by some agonists. P2Y12 and P2Y1 receptors play a major role in amplifying platelet aggregation. We assessed the ability of cangrelor, a selective P2Y12 antagonist, A2P5P, a selective P2Y1 antagonist, and aspirin to block the potentiating effects of heparin. Materials and methods: Whole blood from healthy human volunteers was anticoagulated with either hirudin or UFH 10 IU/ml. Some tubes anticoagulated with hirudin also contained UFH 1 or 10 IU/ml. The low-molecular-weight heparin dalteparin was also assessed. Platelet aggregation was performed using whole blood single-platelet counting. Dense granule release was assessed using 14C-5HT-labelled platelets. Results: UFH and, to a lesser extent, dalteparin potentiated platelet aggregation induced by ADP, PAF, 5HT, U46619, epinephrine and TRAP in a concentrationdependent manner but inhibited aggregation induced by collagen. Cangrelor effectively opposed the potentiating effects of heparins on sustained aggregation induced by ADP, PAF, 5HT, U46619 and TRAP but had less effect on epinephrineinduced aggregation, whereas A2P5P was more effective at blocking both the initial phase of ADP-induced aggregation and the aggregation response to epinephrine, reflecting the differences in G protein coupling between the agonist receptors.
Abbreviations: UFH, unfractionated heparin; A2P5P, adenosine-2,5-bisphosphate; TRAP, thrombin receptor activating peptide; PAF, platelet-activating factor; 5HT, 5-hydroxytryptamine. * Corresponding author. Tel.: +44 114 2714192; fax: +44 114 2619587. E-mail address: [email protected] (R.F. Storey). 0049-3848/$ - see front matter D 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2004.09.004
302
R.F. Storey et al. Aspirin had no effect on potentiation by heparin. Heparins did not increase ADP- or TRAP-induced 14C-5HT release. Conclusions: Heparins potentiate platelet responses to ADP and numerous other agonists. This potentiation is attenuated by cangrelor and A2P5P, and is not mediated by increased dense granule release. ADP receptor antagonists but not aspirin may have potential therapeutic benefits in counteracting the pro-thrombotic effects of heparins. D 2004 Elsevier Ltd. All rights reserved.
Anticoagulation with heparin is used widely in the prevention and management of venous and arterial thrombosis, as well as during procedures such as cardiopulmonary bypass surgery. Whilst heparin inhibits thrombin-induced platelet aggregation [1], it conversely potentiates platelet aggregation induced by a range of platelet agonists including ADP, TRAP, PAF, U46619 and epinephrine [2,3] and these potentiating effects are detectable at therapeutic concentrations of heparin [3]. However, the mechanism whereby heparin potentiates platelet aggregation is unknown, and we sought to elucidate the role of platelet ADP receptors in this phenomenon. ADP may be released from erythrocytes, endothelial cells and platelet dense granules; it plays a major role in haemostasis and the pathogenesis of arterial thrombus by activating platelets, causing granule secretion and aggregation via activation of glycoprotein IIb/IIIa receptors which then bind fibrinogen [4]. The release of ADP by platelets stimulated by other agonists such as thrombin or collagen contributes substantially to platelet aggregation explaining why antagonists of ADP-induced platelet aggregation partially inhibit aggregation by these other platelet aggregating agents [5,6]. There are two receptors for ADP on platelets, P2Y1 and P2Y12 receptors (G-protein coupled receptors), that contribute synergistically to ADP-induced platelet aggregation [7]. A number of specific antagonists to the P2Y1 receptor have been identified, including adenosine-2,5-bisphosphate (A2P5P) [8,9]. Analogues of ATP that bind specifically to the P2Y12 receptor have been developed and one such agent, cangrelor (formerly AR-C69931MX), is currently being developed as an intravenous antithrombotic agent [10,11]. Whole blood single-platelet counting is a measure of microaggregation and, characteristically, there is maximal microaggregation at 10 to 30 s after addition of most agonists (dearlyT aggregation response) followed by either a sustained aggregation response (associated with macroaggregate formation) or reversal of aggregation [5,6]. We previously have described the effects of selective antagonists
of the P2Y1 and P2Y12 receptors on platelet aggregation induced by a range of agonists in human whole blood [6]. Here, we have investigated the role of these receptors in mediating the potentiating effects of heparin on platelet aggregation.
Materials and methods Materials Cangrelor (AR-C69931MX) was provided by AstraZeneca R&D Charnwood, Loughborough, UK. A2P5P, ADP, epinephrine, U46619 (a thromboxane A2 mimetic), thrombin receptor activating peptide (TRAP; SFLLRNPNDKYEPF), aspirin and platelet activating factor (PAF; dissolved in 0.25% w/v human serum albumin) were from Sigma. 5Hydroxytryptamine (5HT; serotonin) was from Amersham International. Hirudin was Revasc (recombinant desulphatohirudin). Saline was 0.9% w/v NaCl supplied by Baxter Healthcare Fixative contained saline with 4.6 mmol/l Na2EDTA, 4.5 mmol/l Na2HPO2, 1.6 mmol/l KH2PO4 and 0.16% w/v formaldehyde, pH 7.4. 14C-5HT was from Amersham (1.85 MBq/ml). Chlorimipramine hydrochloride was from Novartis. Scintillation fluid (fluroscan 2) was from BDH Laboratories Supplies. Heparin sodium (unfractionated heparin) was from Sigma and dalteparin (a low-molecular-weight heparin) was Fragmin from Pfizer.
Preparation of blood Blood was obtained from healthy volunteers using a 19-G needle and plastic syringe. All volunteers denied taking any non-steroidal anti-inflammatory drug in the previous 2 weeks. Aliquots (9-ml) of blood were transferred to polystyrene tubes containing either hirudin (500 Ag in 1 ml 0.9% saline) or UFH (100 IU in 1 ml 0.9% saline). UFH 1 or 10 IU/ml (final concentration) was included with hirudin in some tubes, reflecting a therapeutic concentration and 10-fold greater concentration,
Heparin and platelet ADP receptor antagonism respectively. Aspirin 100 Amol/l (final concentration) was also included with the anticoagulant in some tubes. For the release reaction experiments, 6 Al 14C-5HT (final concentration 0.521 Amol/l; final activity 1.11 kBq/ml) was added to the anticoagulant (for uptake by platelets). The tubes were incubated at 37 8C in a waterbath for a standard 30-min period prior to experimentation.
Whole blood platelet aggregation Platelet aggregation studies were performed using the single-platelet counting technique, as previously described [6]. Saline, cangrelor 1 Amol/l or A2P5P 300 Amol/l was added 2 min prior to agonist. The following agonists were assessed: ADP 0.3–1 Amol/l, PAF 0.1 Amol/l, 5HT 30 Amol/l, U46619 0.3 Amol/l, epinephrine 3 Amol/l, TRAP 3 Amol/l, and collagen 0.5–8 Ag/ml. Concentrations of agonists that yielded submaximal aggregation in the absence of heparin were selected, when appropriate, to demonstrate potentiation. Aliquots of blood were removed after stirring with agonist for 30 s and 4 min and fixed prior to single-platelet counting using an Ultra-Flo 100 Platelet Counter (Becton-Dickinson). 14
C-5HT release measurements
5HT release was determined as previously described [5], using the agonists described in Results. Chlorimipramine (20-Al) was added to 420-Al aliquots of whole blood containing 14 C-5HTlabelled platelets, as well as 40-Al saline and/or P2Y antagonist as indicated in Results. Agonist (20 Al) was then added and samples stirred for 4 min before the reaction was terminated by placing on ice as well as adding 50-Al aspirin solution (1.26 mmol/l). Samples were centrifuged at 1500g for 10 min and duplicate 50-Al samples of supernatant plasma mixed with scintillation fluid prior to analysis in a scintillation counter. One hundred percent release was estimated using duplicate 50Al samples of saline solution of 14C-5HT at the same concentration as that added to the whole blood sample, with adjustment of the results according to the packed cell volume of the blood sample to derive the initial plasma 14C-5HT concentration.
Data analysis Data are mean of six experiments and are expressed as mean and standard deviation. Signifi-
303 cance was attributed to p values b0.05 and determined using ANOVA (SPSS software).
Results Effect of P2Y1 and P2Y12 antagonists on potentiation of ADP-induced platelet aggregation by heparin Heparin potentiated both the early aggregation response to ADP 0.3 Amol/l and the sustained response, 4 min after addition of ADP (Fig. 1). The P2Y1 antagonist A2P5P and the P2Y12 antagonist cangrelor both inhibited the aggregation response to ADP and the combination of both antagonists was required to prevent potentiation of the early aggregation response by heparin, whereas potentiation of the sustained aggregation response was abolished by cangrelor alone. Aspirin had no inhibitory effect. Heparin increased the concentration of cangrelor required to achieve its inhibitory effect (Fig. 2).
Effect of P2Y1 and P2Y12 antagonists on potentiation by heparin of platelet aggregation induced by other agonists Heparin potentiated platelet aggregation induced by PAF and epinephrine (Fig. 3). The potentiating effect of heparin on PAF-induced aggregation was attenuated by the combination of A2P5P and cangrelor whilst the potentiation of epinephrineinduced aggregation was relatively resistant to the effects of these inhibitors. Heparin also potentiated platelet aggregation induced by 5HT and the thromboxane A2 analogue U46619, this potentiation being markedly attenuated by the combination of A2P5P and cangrelor (data not illustrated). In all cases, aspirin had no inhibitory effect. Heparin potentiated both early and sustained TRAP-induced aggregation in a concentration-dependent manner (Fig. 4). Potentiation of the early aggregation response to TRAP was attenuated by cangrelor whilst potentiation of the sustained aggregation response was abolished by cangrelor. Consistent with previous studies [12], collagen-induced aggregation was inhibited by heparin (and, to a lesser extent, dalteparin): for example, aggregation 4 min after the addition of collagen 0.5 Ag/ml was 86F3%, 66F7% and 56F7% for hirudin, dalteparin 5 IU/ml and heparin 10 IU/ml anticoagulation, respectively ( pb0.05 for both dalteparin and heparin vs. hirudin).
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Potentiation of platelet aggregation by the low-molecular-weight heparin dalteparin Like unfractionated heparin, dalteparin also potentiated platelet aggregation induced by ADP, TRAP, PAF and epinephrine (Fig. 5) but, unlike unfractionated heparin, a significant effect was
Figure 2 Mean percentage inhibition by cangrelor of platelet aggregation measured by whole blood singleplatelet counting 4 min after addition of ADP 10 Amol/ l, comparing whole blood anticoagulated with either hirudin or hirudin with UFH 10 IU/ml. Data are meanFS.D. (n=6). *pb0.05 for effect of UFH.
only seen at a supratherapeutic concentration of the drug.
Effect of heparins on dense granule release There was no potentiation by either unfractionated heparin or dalteparin of 14C-5HT release from 14C5HT-labelled platelets induced by ADP 30 and 100 Amol/l or TRAP 3 Amol/l, despite potentiation of platelet aggregation by heparins in the same experiment, indicating that the potentiation of platelet aggregation is not mediated by increased dense granule release (data not illustrated).
Discussion
Figure 1 Platelet aggregation induced by ADP 0.3 Amol/l after (A) 30 s and (B) 4 min in whole blood anticoagulated with either hirudin alone or hirudin plus UFH 10 IU/ml, assessing the effects of pre-incubation for 2 min with either saline, cangrelor 1 Amol/l, A2P5P 300 Amol/l or the combination of cangrelor 1 Amol/l with A2P5P 300 Amol/l and pre-incubation for 30 min with aspirin 100 Amol/l (n=6). *pb0.05 for effect of cangrelor or A2P5P or their interaction. ypb0.05 for effect of UFH vs. hirudin. zpb0.05 for effect of UFH in presence of cangrelor or A2P5P. No significant effect of aspirin was seen alone or in combination.
We have shown that both unfractionated and lowmolecular-weight heparins have substantial potentiating effects on platelet aggregation induced by a wide range of platelet agonists, with the exception of thrombin (not studied here) and collagen, confirming the findings of previous investigations that have used different methodology to assess the effects of heparin [2,3,13]. The lack of significant effect of the low-molecular-weight heparin dalteparin at a therapeutic concentration is also consistent with lack of detectable effect of enoxaparin administration [3], though we have demonstrated that a supratherapeutic concentration of dalteparin has similar, though weaker, effects compared to unfractionated heparin. Previous studies have used apyrase to assess the role of ADP in the potentiation of platelet
Heparin and platelet ADP receptor antagonism
305 shown that potentiation by heparin of activation mediated via this receptor plays a major role in the effects of heparin on sustained platelet aggregation. Both the P2Y12 receptor and the a2 adrenergic receptor, by which epinephrine activates platelets, are linked to Gi and the inhibition of adenylate cyclase [15]. The finding that potentiation of epinephrine-induced aggregation by heparin is resistant to the effects of the P2Y12 receptor antagonist cangrelor indicates that heparin is acting downstream of the P2Y12 receptor. The receptors for PAF, 5HT, TRAP and U46619 are linked to Gaq and the activation of phospholipase C [16]. We have shown that potentiation of the early aggregation response to these agonists by heparin occurs despite P2Y12 and P2Y1 receptor
Figure 3 Platelet aggregation measured by singleplatelet counting 4 min after addition of (A) PAF 0.1 Amol/l or (B) epinephrine 10 Amol/l in hirudin-anticoagulated whole blood incubated for 30 min with either saline control, aspirin 100 Amol/l, UFH 10 IU/ml or a combination of aspirin 100 Amol/l and UFH 10 IU/ml (n=6). Samples were pre-incubated for 2 min with either saline control, cangrelor 1 Amol/l, A2P5P 300 Amol/l, or a combination of cangrelor 1 Amol/l and A2P5P 300 Amol/ l. *pb0.05 for effect of cangrelor or A2P5P or their interaction. ypb0.05 for effect of UFH vs. hirudin. zpb0.05 for effect of UFH in presence of cangrelor or A2P5P. No significant effect of aspirin was seen alone or in combination.
aggregation by heparin and the findings have been inconsistent [2,13]. Developments in the understanding of ADP-induced platelet activation and availability of selective antagonists of the receptors involved in the process have allowed us to define the role of ADP more precisely. The P2Y12 receptor plays a central role in amplifying and sustaining platelet aggregation induced by numerous platelet agonists [5,14] and we have
Figure 4 Platelet aggregation induced by TRAP 3 Amol/ l, comparing anticoagulation of whole blood with hirudin, UFH 10 IU/ml, or hirudin with addition of UFH 1 or 10 IU/ ml (incubated for 30 min at 37 8C). Platelet aggregation is assessed by single-platelet counting at (A) 30 s and (B) 4 min after addition of TRAP after pre-incubation for 2 min with either saline control or cangrelor 1 Amol/l. Data are meanFS.D. (n=6). *pb0.05 for effect of UFH vs. hirudin. ypb0.05 for effect of cangrelor vs. saline.
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Figure 5 Platelet aggregation measured by singleplatelet counting 4 min after addition of either ADP 0.3 Amol/l, TRAP 3 Amol/l, PAF 0.3 Amol/l or epinephrine 3 IU/ml in whole blood anticoagulated with either hirudin, UFH 10 IU/ml, hirudin plus the low-molecular weight heparin dalteparin 5 IU/ml, hirudin plus dalteparin 0.5 IU/ml or dalteparin 5 IU/ml alone. Data are meanFS.D. (n=6). *pb0.05 for effect of UFH or dalteparin vs. hirudin.
blockade. We have previously found that the high concentrations of cangrelor and A2P5P completely block the respective receptor activation by ADP at a concentration of 0.3 Amol/l [5], and, assuming that receptor blockade was complete in these experiments, it is likely that heparin is also acting downstream of (or at the level of) Gaq. Currently, there are several P2Y12 receptor antagonists either in clinical use or in clinical development [17]. The thienopyridines ticlopidine and clopidogrel act, via hepatic metabolites, on the P2Y12 receptor and have a proven role in the management and prevention of arterial thrombosis [17,18]. Phase II studies have shown cangrelor to be a well-tolerated intravenous antithrombotic agent in patients with acute coronary syndromes [10,11] and it yields more effective receptor blockade than clopidogrel [19]. Further studies of cangrelor and AZD6140, an oral, reversibly acting P2Y12 antagonist, are planned. Our findings here indicate that such P2Y12 antagonists may play a useful role in counteracting the prothrombotic properties of heparin. Indeed, clopidogrel has been studied in combination with heparin, as well as aspirin, in patients with acute coronary syndromes and has been found to reduce the combined incidence of myocardial infarction, stroke and cardiovascular death [18,20]. Opposition by heparin of the inhibitory effects of cangrelor on platelet aggregation may require consideration of dose adjustment of cangrelor in settings where significant plasma concentrations of unfractionated heparin are achieved. The possibility of developing P2Y1 recep-
R.F. Storey et al. tor antagonists for clinical use remains to be explored. Aspirin fails to inhibit platelet aggregation induced by numerous agonists, with the notable exception of collagen, when physiological divalent cation levels are maintained by the use of a direct thrombin inhibitor such as hirudin [5]. This study demonstrates that the potentiation of platelet aggregation by heparin is independent of the cyclo-oxygenase pathway. Thus, co-administration of aspirin with heparin, as frequently occurs in the management of arterial interventions and acute coronary syndromes, fails to provide any protection against the deleterious effects of heparin, which may be of particular importance in these settings. In conclusion, heparins potentiate platelet aggregation induced by ADP and numerous other agonists, apart from thrombin and collagen, and P2Y receptors play an important role in this potentiation. The potentiation is not mediated by increased thromboxane A2 secretion or dense granule release. ADP receptor antagonists but not aspirin may have potential therapeutic benefits in counteracting the pro-thrombotic effects of heparins.
Acknowledgements We are grateful to AstraZeneca R&D Charnwood for the gift of cangrelor (AR-C69931MX).
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