Published online before print December 22, 2008, doi: 10.1161/CIRCULATIONAHA.107.762690
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2009;119:116-122.)
© 2009 American Heart Association, Inc.
Vascular Medicine |
Platelet P2Y12 Receptor Influences the Vessel Wall Response to Arterial Injury and Thrombosis
From the Cardiovascular Research Unit, University of Sheffield, Sheffield, UK.
Correspondence to Dr Robert F. Storey, Cardiovascular Research Unit, School of Medicine and Biomedical Sciences, Beech Hill Rd, Sheffield, S10 2RX, UK. E-mail r.f.storey{at}sheffield.ac.uk
Received January 9, 2008; accepted October 14, 2008.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— Platelets are believed to play an important role in atherogenesis and the vessel response to vascular injury. The P2Y12 receptor (P2Y12) plays a central role in amplifying platelet aggregation, dense granule and
-granule secretion, P-selectin expression, microparticle formation, and procoagulant membrane changes, regardless of the activating stimulus. We hypothesized that P2Y12 deficiency might reduce the vessel wall response to vascular injury as well as thrombosis in murine vascular injury models. Methods and Results— P2Y12-deficient (–/–) mice and littermate controls (+/+) were bred on a C57 BL/6 background. In vivo murine models of arterial injury were employed alone and in combination with bone marrow transplantation to investigate the role of P2Y12 in the vessel wall response to arterial injury and thrombosis. At 21 days after ferric chloride injury, neointima formation in P2Y12–/– arteries was significantly less than that observed in control strain arteries (P<0.025). In agreement with this, the intima-media ratio was significantly greater in femoral wire-injured arteries from P2Y12+/+ compared with P2Y12–/– animals (P<0.05). Bone marrow transplantation was used to examine the importance of vessel wall P2Y12 versus platelet P2Y12. Analysis of arterial sections from chimeric animals at 21 days after injury revealed a smaller intima-media ratio in –/– to +/+ animals than in the positive (+/+ to +/+) control group (P<0.01).
Conclusions— These data demonstrate a role for platelet P2Y12 in the vessel wall response to arterial injury and thrombosis. This illustrates the manner in which platelets may contribute to atherogenesis and restenosis.
Key Words: arterial thrombosis • platelets • restenosis • muscle, smooth
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Accumulating evidence supports important roles for the platelet in atherogenesis and restenosis after percutaneous coronary intervention (PCI). Vessel injury and atherogenesis can result in luminal exposure of thrombogenic subendothelial components such as tissue factor and von Willebrand factor, resulting in platelet degranulation and release of several platelet activators, including ADP. The ensuing platelet activation, adhesion, and aggregation are considered the initial steps in thrombus formation.1–3 Once recruited to or in the proximity of the vessel wall, platelets may release or express a plethora of proinflammatory molecules, including platelet factor 4, macrophage inflammatory protein 1-
, Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), CD40 ligand, interleukin-1, transforming growth factor-β, and P-selectin.4–8 Two G-protein–coupled receptors for ADP exist on platelets, P2Y1 and P2Y12.9–11 ADP-induced platelet aggregation is initiated by the P2Y1 receptor and amplified and sustained in a synergistic manner by the P2Y12 receptor. The P2Y12 receptor also amplifies platelet aggregation induced by other agonists such as thrombin and consequently plays an important role in thrombogenesis and thrombus stability.12–14 P2Y12 receptor–deficient mice (P2Y12–/–) have enhanced bleeding times and a poor platelet aggregation response to ADP.11 P2Y12 receptors have also been reported on vascular smooth muscle cells, where activation is associated with vasospasm, but their pathophysiological significance with regard to the vessel response to injury is currently uncertain.15
Clinical Perspective p 122
Given the evidence for the platelet as a key mediator of atherogenesis and restenosis and the importance of the P2Y12 receptor in the regulation of platelet activity and proinflammatory platelet responses, we hypothesized that P2Y12 receptor deficiency may attenuate the vessel wall response after injury. In this study, we profiled the responses of platelets in P2Y12 receptor–deficient and control strain mice and then addressed the role of P2Y12 in early thrombus generation and late neointima formation by examining the response of P2Y12–/– and wild-type arteries to ferric chloride (FeCl3) and wire injury.16,17 The contribution of platelet versus vascular smooth muscle cell P2Y12 was examined with the use of bone marrow transplantation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Genotyping, tissue preparation, histology, morphometric analysis, and immunohistochemistry were performed as described in the online-only Data Supplement.
Mouse Strains Used
P2Y12–/– mice11 and their genetically matched wild-type strain, P2Y12+/+, were obtained from an in-house colony, derived from breeding pairs provided by Dr M. Chintala (Schering-Plough Research Institute). All animals were housed in a controlled environment with a 12-hour light/dark cycle at 22°C and fed standard laboratory chow. In some groups, mice were gavaged with 20 mg/kg clopidogrel in 200 µL water or 200 µL sterile water (control). For long-term treatments, mice were gavaged with clopidogrel or water 24 hours and 1 to 2 hours before surgery and each day thereafter for 21 days. For short-term treatment experiments, mice were gavaged with clopidogrel 4 hours before and 24 hours after surgery. All experiments were performed in accordance with UK legislation under the Animals Scientific Procedures Act (1986). Male mice aged 8 to 12 weeks (
25 g in weight) underwent the procedure in each respective group.
Preparation of Blood
Blood (900 µL) was withdrawn by cardiac puncture into 100 µL hirudin (5 µg mL–1; 900 anti-IIa units mL–1), then immediately transferred to a preheated water bath at 37°C.
Platelet Aggregation in Whole Blood
Blood (0.5 to 1 mL) was withdrawn from 1 mouse into hirudin as described above. In initial experiments, we found that using the coagonist 3 µmol/L 5-hydroxytryptamine with each concentration of ADP gave an enhanced aggregation response that was sensitive to P2Y12 inhibition. Before blood withdrawal, the following tubes containing agonists and stir bars were prepared (and placed on ice) to test for platelet aggregation: EDTA (4 µL 20 mmol/L EDTA+16 µL blood) and 30 µmol/L ADP+3 µmol/L 5-hydroxytryptamine (10 µL each agonist+220 µL blood). Tubes were placed into a heated (37°C) stir block (stirring speed 800 rpm), as designed at the University of Sheffield, and aliquots of blood (220 µL) were placed into each of the tubes for 4 minutes, after which 500 µL of fixative solution was added. Fixed samples were counted with a KX21 Hematology Analyzer (Sysmex Corporation, Japan), and the percent aggregation was calculated as percent loss of single platelets compared with total single-platelet count (EDTA sample). The use of the KX21 analyzer was validated for murine single-platelet counting by comparison with flow cytometric assessment of single-platelet counts.
P-Selectin Expression of Platelets in Whole Blood
Before blood withdrawal, flow cytometry tubes were also prepared. Tubes contained 2 µL PAR-4 thrombin receptor activating peptide (TRAP) (0.3, 1, 3, or 10 mmol/L), 2 µL saline, 10 µL FITC anti-CD62P, and 31 µL PBS. PAR-4 TRAP was used to provide strong agonist stimulation via this platelet thrombin receptor, as may occur with the generation of thrombin consequent to arterial injury, with amplification of the response occurring via activation of P2Y12 by ADP released from platelet dense granules. Aliquots of blood (5 µL) were added to each tube and incubated for 20 minutes at room temperature without light. After incubation, 2 mL FACSFlow (BD Biosciences, San Jose, Calif) was added, and the sample was then analyzed by flow cytometry. Nonspecific binding was determined with the use of rat anti-mouse immunoglobulin G–FITC in place of the CD62P antibody. A gate was applied to the platelet region, and 2000 platelet events were collected. P-selectin expression was determined as the median fluorescence of the entire platelet population, and the results were expressed as an increase over baseline values.
FeCl3 Injury
Mice were anesthetized by intraperitoneal injection (0.01 mL/g) of Hypnorm solution (fentanyl citrate 0.02 mg/mL; fluanisone 1.25 mg/mL) and midazolam (2.5 mg/mL), and the right common carotid artery was exposed with minimal dissection. A 1x2-mm piece of filter paper soaked in 10% FeCl3 solution was placed on the common carotid artery for 3 minutes. The external surface of the artery was then washed with saline, the dermis was closed and sutured, and the animals were allowed to recover. All animals recovered and showed no signs of a stroke.
Wire Injury
Male mice were anesthetized by inhalational isoflurane (maintenance rate of 1.5%), and endoluminal injury to the common femoral artery was performed with a method similar to that of Sata et al.18 A 0.12-mm wire was inserted distal to the muscular branch artery and passed 6 times back and forth along the common femoral artery. The arteriotomy site was subsequently ligated. Control sham-operated arteries underwent dissection, temporary clamping, arteriotomy, and ligature, without passing of the wire. Arteries were perfusion fixed and processed into paraffin wax at 21 days after injury. Sequential sections, 100 µm apart, were cut from the entire length of femoral artery from the point of ligation. Only sections proximal to the muscular branch (ie, that were exposed to flow conditions, to remove the effect of ligation injury) were used in the analysis.
Bone Marrow Transplantation
Female donor mice were killed, and their femurs and tibias were removed under aseptic conditions. Marrow cavities were flushed, and single-cell suspensions were prepared, washed, and resuspended in Hanks’ balanced salt solution before transplantation. Male mice, aged 4 to 6 weeks, received a lethal dose of whole body irradiation (1100 rad, split into 2 doses, 4 hours apart). Irradiated mice then received 1 to 2x106 cells in Hank’s buffered salt solution via tail-vein injection. FeCl3 injury was performed 5 weeks after bone marrow transfer. This method has previously been used successfully by our group.19 To further confirm the efficiency of the bone marrow transplantation, blood from selected mice was profiled for P-selectin expression in response to PAR-4 TRAP (as above; see online-only Data Supplement). Two types of chimeric mice were created: P2Y12+/+ mice with P2Y12–/– bone marrow and P2Y12–/– mice with P2Y12+/+ bone marrow. Control groups of P2Y12+/+ to P2Y12+/+ and P2Y12–/– to P2Y12 –/– transfers were also performed.
Statistical Analysis
Data are expressed as means and presented either with SEM or as individual data. Data were analyzed with the use of SPSS (version 15.0; SPSS Inc, Chicago, Ill). Levene’s test was used to assess equality of variance, and where the Levene’s test yielded a P value <0.05 for the F value, then equal variances were not assumed and data were analyzed using the Mann-Whitney test. The independent samples t test was used to analyze data with equality of variance. The level of significance for P values was adjusted according to the number of group comparisons using Bonferroni correction, such that significance was attributed to P values <0.05, 0.025, or 0.01 according to the experiment. Further details of the statistical analysis methods and detailed results are available in the online-only Data Supplement.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Platelet Function
Comparison of the aggregation profiles of P2Y12–/– mice and P2Y12+/+ mice revealed that significantly fewer P2Y12–/– platelets aggregated in response to 30 µmol/L ADP (14.78±2.626% versus 55.89±4.89%; n=6; P<0.01). To determine the role of P2Y12 receptor activation in platelet P-selectin expression in whole blood, we investigated the responses of platelets from P2Y12–/– and P2Y12+/+ mice to PAR-4 TRAP (Figure 1). In whole blood from wild-type mice, P-selectin expression was observed in response to 1, 3, and 10 mmol/L TRAP and was significantly higher than that observed in blood from P2Y12–/– mice in response to 3 mmol/L TRAP (n=6; P<0.01). In P2Y12-deficient mice, only stimulation with the highest concentration of TRAP (10 mmol/L) caused levels of P-selectin expression that were more than nonspecific interactions of the antibody.
|
; n=6) and P2Y12–/– (
n=6) mice. *P<0.01; (Mann–Whitney test with correction of level of significance for multiple group comparison).
FeCl3 Injury
Vascular injury in the presence of flow produced significantly less thrombus in P2Y12-deficient animals after 30 minutes (Figures 2 and 3
). The following data (Figures 3 and 4) are generated with the average morphometric values from 5 sequential sections per artery. A comparison of FeCl3-injured arteries from P2Y12–/– and P2Y12+/+ animals, at 30 minutes after injury, revealed significantly less thrombus (0.003±0.001 versus 0.039±0.006 mm2 respectively; n=5; P<0.025) and a correspondingly greater lumen area in P2Y12–/– arteries (0.058±0.004 versus 0.024±0.006 mm2; n=5; P<0.025) (Figure 3A). At 21 days after injury, significantly less neointima (0.011±0.001 versus 0.061±0.006 mm2; n=4; P<0.01) and a correspondingly greater lumen area (0.094±0.006 versus 0.037±0.008 mm2, respectively; n=4; P<0.01) were observed in P2Y12–/– arteries (Figure 3B and 3C).
|
|
|
Arteries from P2Y12+/+ animals administered clopidogrel for the 21-day period after injury exhibited less neointimal area to arteries from P2Y12+/+ animals administered water only by gavage, and a similar effect was seen when clopidogrel was administered 4 hours before, and only 1 additional dose 24 hours after, FeCl3 injury (Figure 4). Although nonsignificant trends were noted between the control groups and the separate clopidogrel-treated groups (P=0.032 and 0.056 for prolonged or short-duration clopidogrel, respectively, versus control), the overall effect of any clopidogrel treatment was significantly inhibitory on neointima formation (P<0.025 for both groups combined versus control).
No significant differences in media or total cross-sectional areas were observed between groups at either time point. Representative artery sections after FeCl3 injury are shown in Figure 2.
Bone marrow transplantation was used to examine the importance of vessel wall P2Y12 versus platelet P2Y12 in the response to arterial injury. Two types of chimeric mice, P2Y12+/+ mice with P2Y12–/– bone marrow (–/– to +/+) and P2Y12 –/– mice with P2Y12+/+ bone marrow (+/+ to –/–), were generated. Control groups of P2Y12+/+ to P2Y12+/+ and P2Y12–/– to P2Y12–/– transfers were also performed. Chimeric and control mice were subjected to FeCl3-induced injury 6 weeks after bone marrow transplantation. Analysis of arterial sections from these animals 21 days after injury (Figure 5) revealed less neointima formation in –/– to +/+ animals compared with the positive (+/+ to +/+) control group (0.034±0.011; n=8 versus 0.925±0.298; n=9; P<0.01) or compared with the +/+ to –/– animals (P<0.01). No significant difference was found between –/– to +/+ animals and the negative control (–/– to –/–) group.
|
Wire Injury
To assess the influence of P2Y12 with a weaker stimulus than FeCl3 injury, P2Y12–/– and P2Y12+/+ animals were also subjected to femoral wire injury or sham operation. The intima-media ratio was significantly greater in arteries proximal to the muscular branch from P2Y12+/+ compared with P2Y12–/– animals (P<0.05; Figure 6). In the sham-operated arteries from animals of either strain, neointima was only observed distal to, but not proximal to, the muscular branch artery.
|
, –/–) animals. P<0.05 (Mann–Whitney test). |
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
To investigate the activation status of murine platelets in response to PAR-4 TRAP, we determined the levels of binding of fluorochrome-conjugated P-selectin antibody to P-selectin on the surface of platelets. P-selectin expression is a marker of
-granule secretion and hence release of inflammatory mediators.20,21 Deficiency of the P2Y12 receptor abolishes P-selectin expression at lower concentrations of TRAP; however, P-selectin expression occurs independently of P2Y12 receptor activation at high concentrations of TRAP. This finding provides further evidence that the P2Y12 receptor plays a major role in P-selectin expression in response to PAR-4–mediated platelet activation in mice and illustrates the important role of P2Y12 in amplification of -granule release. This supports the conclusions of a study by Hollinstat et al,22 who described a synergistic interaction of P2Y12 and PAR-4 in human platelets. Application of FeCl3 to the carotid artery represents an established rodent model of arterial injury and thrombosis.16,17 FeCl3 injury induces oxidative damage to the endothelium, resulting in widespread deendothelialization as a result of transendothelial migration of ferric ion and subsequent oxidation in the arterial lumen.23 This particular model was chosen because of the 2-stage nature of the response to injury: an initial thrombus consisting of platelets, erythrocytes, and fibrin24 replaced with a cellular lesion at 21 days after injury.23 Despite the presence of a smooth muscle cell–rich neointima 21 days after FeCl3 injury, this model is standardly used to examine thrombus formation. We observed an 80% reduction in neointima formation and a 60% increase in lumen area in arteries from P2Y12 receptor–deficient animals at 21 days after injury. This mirrors findings at 30 minutes after injury in which we observed substantial thrombus formation in the lumen of control animal arteries yet little thrombus accumulation in arteries from P2Y12-deficient animals. These data agree with a previous study that demonstrated that P2Y12 deficiency was associated with a delayed onset of cyclic flow reductions and lack of occlusion in injured mesenteric arteries.14 Our findings further contribute to this area by highlighting the relationship between early thrombus formation and late neointima development, demonstrating a role for the P2Y12 receptor in later stages of the vessel wall response to injury. We also used a less thrombotic femoral wire-injury model to further investigate the role of P2Y12 and demonstrated a reduced vessel wall response to injury in P2Y12-deficient mice using this model. Smooth muscle cells are a consistent feature in the neointima at 21 days after FeCl3 injury. The P2Y12 receptor is also expressed by vascular smooth muscle cells,15 raising the question of whether the effect of loss of the P2Y12 receptor reported here is attributable to platelet or vessel wall (vascular smooth muscle cell) P2Y12. To address this question, we used a bone marrow transplantation technique to create 2 types of chimeric mice: 1 with bone marrow cells and circulating platelets expressing P2Y12 with the remaining tissues including the vessel wall being deficient in P2Y12, and a second with bone marrow cells and platelets deficient in P2Y12 with the remaining tissues expressing P2Y12 normally. Chimeric mice and controls were injured by the FeCl3 method. At 21 days after injury, we observed a significantly reduced intima-media ratio (a corrected measure of neointima) and neointimal area in mice from the group with P2Y12-deficient platelets, whereas the neointima response in mice with P2Y12+/+ platelets but P2Y12-deficient vessel wall was not statistically different from the positive control. This finding supports the notion that neointima formation after FeCl3 injury in this murine model is predominantly dependent on platelet-mediated thrombus formation, with the platelet P2Y12 receptor playing a profound role in this process. However, additional studies are required to further characterize the potential roles of vascular smooth muscle cell P2Y12 in the vascular response to injury, such as through mediation of vasoconstriction. The effects on intima-media ratio and neointimal area are smaller than those seen in nontransplanted +/+ mice, and this reflects an effect of the bone marrow transplantation procedure on neointimal formation. We have observed this phenomenon previously in response to vascular injury (carotid ligation) performed after bone marrow transplantation and reconstitution.19
Clopidogrel has been found previously to inhibit thrombus formation in a murine FeCl3 injury model.25 Wild-type mice were administered clopidogrel, the active metabolite of which acts as an antagonist at the P2Y12 receptor. After FeCl3 injury, a significantly reduced neointimal response was observed at 21 days in treated wild-type animals compared with wild-type mice that were not administered clopidogrel. This observation remained significant whether antagonism of P2Y12 was achieved via clopidogrel administration before and 24 hours after injury or for the duration of the 21 days after injury. This suggests that early inhibition of thrombus formation via P2Y12 is sufficient to prevent neointima formation at later time points.
Given the importance of the platelet in the vessel response to injury, it seems possible that inhibition of platelet activation via P2Y12 inhibition could prevent the pathological response to injury after PCI that culminates in restenosis. Small-scale clinical trials of thienopyridine compounds have hinted at their ability to reduce restenosis after PCI26; however, no adequately powered, randomized controlled trials to date have examined the effects of clopidogrel on restenosis. The Clopidogrel for Reduction of Events During Observation (CREDO) study27 compared the strategies of starting clopidogrel either before or after PCI but was not powered or designed to assess the impact of an effective loading regimen of clopidogrel on restenosis. A study by Momi et al28 compared the effect of nitroaspirin, clopidogrel, and aspirin alone or in combination on intimal thickening induced by photochemical injury of the femoral artery in mice. A reduction in intimal thickening 21 days after injury was observed in clopidogrel-treated animals, but this was not significant, possibly related to the low dose of clopidogrel employed (0.5 mg/kg) and/or differences in the models of injury between this study and our own. Although clopidogrel is used routinely in patients undergoing PCI, it only achieves partial blockade of the P2Y12 receptor population,29 and studies of newer, more efficacious antagonists of P2Y12 may provide direct evidence for the role of the receptor in the pathogenesis of restenosis.30
In conclusion, these data demonstrate a role for platelet P2Y12 in the vessel wall response to arterial injury and thrombosis and highlight the relationship between early thrombotic response and later neointima formation after arterial injury. This illustrates the manner in which platelets may contribute to atherogenesis and restenosis and provides a rationale for the further investigation into the therapeutic use of P2Y12 antagonists for their prevention.
|
Acknowledgments |
|---|
We are grateful to Schering-Plough Research Institute and Dr Madhu Chintala for providing the P2Y12 knockout mice.
Sources of Funding
This work was supported by a grant to the University of Sheffield from the British Heart Foundation (PG/03/124/16118).
Disclosures
Dr Storey has received research grants and/or honoraria from pharmaceutical companies including AstraZeneca, Eli Lilly, Daiichi Sankyo, and The Medicines Company. No pharmaceutical company influenced the design, analysis, interpretation, or manuscript construction for the present study.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Kroll MH, Harris TS, Moake JL, Handin RI, Schafer AI. von Willebrand factor binding to platelet GpIb initiates signals for platelet activation. J Clin Invest. 1991; 88: 1568–1573.[Medline] [Order article via Infotrieve]
2. Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989; 86: 2839–2843.
3. Day SM, Reeve JL, Pedersen B, Farris DM, Myers DD, Im M, Wakefield TW, Mackman N, Fay WP. Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood. 2005; 105: 192–198.
4. Hermann A, Rauch BH, Braun M, Schror K, Weber AA. Platelet CD40 ligand (CD40L)–subcellular localization, regulation of expression, and inhibition by clopidogrel. Platelets. 2001; 12: 74–82.[CrossRef][Medline] [Order article via Infotrieve]
5. Scheuerer B, Ernst M, Durrbaum-Landmann I, Fleischer J, Grage-Griebenow E, Brandt E, Flad HD, Petersen F. The CXC-chemokine platelet factor 4 promotes monocyte survival and induces monocyte differentiation into macrophages. Blood. 2000; 95: 1158–1166.
6. Schober A, Manka D, von Hundelshausen P, Huo Y, Hanrath P, Sarembock IJ, Ley K, Weber C. Deposition of platelet RANTES triggering monocyte recruitment requires P-selectin and is involved in neointima formation after arterial injury. Circulation. 2002; 106: 1523–1529.
7. Assoian RK, Sporn MB. Type beta transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells. J Cell Biol. 1986; 102: 1217–1223.
8. Lindemann S, Tolley ND, Dixon DA, McIntyre TM, Prescott SM, Zimmerman GA, Weyrich AS. Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis. J Cell Biol. 2001; 154: 485–490.
9. Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A. 1998; 95: 8070–8074.
10. Leon C, Freund M, Ravanat C, Baurand A, Cazenave JP, Gachet C. Key role of the P2Y1 receptor in tissue factor-induced thrombin-dependent acute thromboembolism: studies in P2Y1-knockout mice and mice treated with a P2Y1 antagonist. Circulation. 2001; 103: 718–723.
11. Foster CJ, Prosser DM, Agans JM, Zhai Y, Smith MD, Lachowicz JE, Zhang FL, Gustafson E, Monsma FJ Jr, Wiekowski MT, Abbondanzo SJ, Cook DN, Bayne ML, Lira SA, Chintala MS. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs. J Clin Invest. 2001; 107: 1591–1598.[Medline] [Order article via Infotrieve]
12. Wang K, Zhou X, Zhou Z, Tarakji K, Carneiro M, Penn MS, Murray D, Klein A, Humphries RG, Turner J, Thomas JD, Topol EJ, Lincoff AM. Blockade of the platelet P2Y12 receptor by AR-C69931MX sustains coronary artery recanalization and improves the myocardial tissue perfusion in a canine thrombosis model. Arterioscler Thromb Vasc Biol. 2003; 23: 357–362.
13. 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: 925–934.[CrossRef][Medline] [Order article via Infotrieve]
14. Andre P, Delaney SM, LaRocca T, Vincent D, DeGuzman F, Jurek M, Koller B, Phillips DR, Conley PB. P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries. J Clin Invest. 2003; 112: 398–406.[CrossRef][Medline] [Order article via Infotrieve]
15. Wihlborg AK, Wang L, Braun OO, Eyjolfsson A, Gustafsson R, Gudbjartsson T, Erlinge D. ADP receptor P2Y12 is expressed in vascular smooth muscle cells and stimulates contraction in human blood vessels. Arterioscler Thromb Vasc Biol. 2004; 24: 1810–1815.
16. Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res. 1990; 60: 269–280.[CrossRef][Medline] [Order article via Infotrieve]
17. Farrehi PM, Ozaki CK, Carmeliet P, Fay WP. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice. Circulation. 1998; 97: 1002–1008.
18. Sata M, Takahashi A, Tanaka K, Washida M, Ishizaka N, Ako J, Yoshizumi M, Ouchi Y, Taniguchi T, Hirata Y, Yokoyama M, Nagai R, Walsh K. Mouse genetic evidence that tranilast reduces smooth muscle cell hyperplasia via a p21(WAF1)-dependent pathway. Arterioscler Thromb Vasc Biol. 2002; 22: 1305–1309.
19. Chamberlain J, Evans D, King A, Dewberry R, Dower S, Crossman D, Francis S. Interleukin-1β and signaling of interleukin-1 in vascular wall and circulating cells modulates the extent of neointima formation in mice. Am J Pathol. 2006; 168: 1396–1403.
20. Yokoyama S, Ikeda H, Haramaki N, Yasukawa H, Murohara T, Imaizumi T. Platelet P-selectin plays an important role in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates. J Am Coll Cardiol. 2005; 45: 1280–1286.
21. Leytin V, Mody M, Semple JW, Garvey B, Freedman J. Flow cytometric parameters for characterizing platelet activation by measuring P-selectin (CD62) expression: theoretical consideration and evaluation in thrombin-treated platelet populations. Biochem Biophys Res Commun. 2000; 269: 85–90.[CrossRef][Medline] [Order article via Infotrieve]
22. Holinstat M, Voss B, Bilodeau ML, McLaughlin JN, Cleator J, Hamm HE. PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation. J Biol Chem. 2006; 281: 26665–26674.
23. Tseng MT, Dozier A, Haribabu B, Graham UM. Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery. Thromb Res. 2006; 118: 275–280.[CrossRef][Medline] [Order article via Infotrieve]
24. Lockyer S, Kambayashi J. Demonstration of flow and platelet dependency in a ferric chloride-induced model of thrombosis. J Cardiovasc Pharmacol. 1999; 33: 718–725.[CrossRef][Medline] [Order article via Infotrieve]
25. Wang X, Smith PL, Hsu MY, Ogletree ML, Schumacher WA. Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor. J Thromb Haemost. 2006; 4: 403–410.[CrossRef][Medline] [Order article via Infotrieve]
26. Nagaoka N, Matsubara T, Okazaki K, Masuda N, Shikaura K, Hotta A. Comparison of ticlopidine and cilostazol for the prevention of restenosis after percutaneous transluminal coronary angioplasty. Jpn Heart J. 2001; 42: 43–54.[CrossRef][Medline] [Order article via Infotrieve]
27. Steinhubl SR, Berger PB, Mann JT III, Fry ET, DeLago A, Wilmer C, Topol EJ; CREDO Investigators. Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial. JAMA. 2002; 288: 2411–2420.
28. Momi S, Pitchford SC, Alberti PF, Minuz P, Del Soldato P, Gresele P. Nitroaspirin plus clopidogrel versus aspirin plus clopidogrel against platelet thromboembolism and intimal thickening in mice. Thromb Haemost. 2005; 93: 535–543.[Medline] [Order article via Infotrieve]
29. 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: 407–413.[CrossRef][Medline] [Order article via Infotrieve]
30. Greenbaum AB, Grines CL, Bittl JA, Becker RC, Kereiakes DJ, Gilchrist IC, Clegg J, Stankowski JE, Grogan DR, Harrington RA, Emanuelsson H, Weaver WD. Initial experience with an intravenous P2Y12 platelet receptor antagonist in patients undergoing percutaneous coronary intervention: results from a 2-part, phase II, multicenter, randomized, placebo- and active-controlled trial. Am Heart J. 2006; 151: 689.e1–689.e10.[CrossRef][Medline] [Order article via Infotrieve]
|
Footnotes |
|---|
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.762690/DC1.
Related Article:
Circulation 2009 119: 1-4.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||