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European Heart Journal Advance Access originally published online on February 12, 2008
European Heart Journal 2008 29(14):1729-1738; doi:10.1093/eurheartj/ehn027
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Determinants of thrombin generation, fibrinolytic activity, and endothelial dysfunction in patients on dual antiplatelet therapy: involvement of factors other than platelet aggregability in Virchow's triad

Yuichiro Yano1, Tsukasa Ohmori2,*, Satoshi Hoshide1, Seiji Madoiwa2, Keiji Yamamoto1, Takaaki Katsuki1, Takeshi Mitsuhashi1, Jun Mimuro2, Kazuyuki Shimada1, Kazuomi Kario1 and Yoichi Sakata2

1 Division of Cardiovascular Medicine, Department of Medicine, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
2 Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan

Received 4 October 2007; revised 27 December 2007; accepted 10 January 2008; online publish-ahead-of-print 12 February 2008.

* Corresponding author. Tel: +81 285 58 7397, Fax: +81 285 44 7817, Email: tohmori{at}jichi.ac.jp

See page 1699 for the editorial comment on this article (doi:10.1093/eurheartj/ehn257)


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Aims: The aim of the study was to assess mechanisms and clinical backgrounds in order to determine residual platelet aggregability in dual antiplatelet therapy and to ascertain whether platelet aggregability is involved in systemic thrombogenicity.

Methods and results: A cross-sectional study was conducted in 85 consecutive patients who underwent dual antiplatelet therapy (aspirin and thienopyridine/cilostazol) after percutaneous coronary intervention (PCI). Although serum thromboxane B2 and dephosphorylation of vasodilator-stimulated phosphoprotein were significantly abolished, the platelet aggregation tests showed inter-individual differences that could be partly explained by plasma glucose levels. Platelet aggregability was not related to other factors involved in thrombogenicity. Thrombin generation assessed by soluble fibrin was independently associated with total cholesterol (β = 0.349, P < 0.001), brain natriuretic peptide (β = 0.222, P = 0.018), and ankle-brachial index (β = –0.330, P = 0.001). Plasminogen activator inhibitor-1 was associated with the apnea–hypopnea index (β = 0.300, P = 0.006). E-selectin was correlated with diabetes mellitus (β = 0.279, P = 0.008) and body mass index (β = 0.323, P = 0.002).

Conclusion: Although dual antiplatelet therapy effectively inhibited its pharmacological targets, thrombin generation, inhibition of fibrinolytic activity, and endothelial dysfunction were determined by other clinical backgrounds. Our data suggested that some patients remain at risk of thrombotic complications after PCI and that these may benefit from anticoagulant treatment despite adequate dual antiplatelet therapy.

Key Words: Percutaneous coronary intervention • Aspirin • Thienopyridine • Antiplatelet drug resistance • Thrombin generation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Platelet aggregation plays a central role in the development of thrombotic complications after percutaneous coronary intervention (PCI).1–3 The role of aspirin in secondary prevention of ischaemic cardiovascular diseases is universally accepted. Furthermore, dual antiplatelet therapy of aspirin combined with thienopyridine (and/or cilostazol) including clopidogrel or ticlopidine is the gold standard for preventing major cardiovascular events in patients undergoing PCI, especially since the beginning of the balloon-expandable stent era.4–6 In contrast, nearly 20% of patients continue to have further cardiovascular events after PCI, despite the superior protection conferred by dual antiplatelet therapy, as shown in a number of clinical trials.7

The mechanism by which antiplatelet therapy fails in certain patients after PCI, in part, thought to be attributed to the fact that some individuals have impaired antiplatelet responses, is referred to as ‘aspirin resistance’ or ‘clopidogrel resistance’.8–10 There is evidence that not all patients respond comparably to antiplatelet drugs, as evaluated by non-specific laboratory test such as aggregometry, and hence the concept of drug ‘resistance’ has arisen.11–14 However, recent evidence suggest that when the definition of resistance is limited to situations in which the drugs fail to hit their pharmacological targets, resistance against antiplatelet drug appears to be rare.15–18 Many published studies of antiplatelet resistance have been carried out using nonspecific platelet aggregation tests, which merely identify patients on antiplatelet therapy with high residual platelet activation.7,18 Despite this drawback, identification of patients with high residual platelet reactivity may be useful for predicting individuals risks of atherothrombotic events.7–10,13,17

The results of clinical trials on the use of anticoagulant agents and the involvement of fibrin fibrils and inflammatory cells in the formation of occlusive thrombi suggest that not only platelets but also the coagulation cascade, fibrinolytic system, inflammation, and endothelial dysfunction may orchestrate in vivo thrombus formation, thereby leading to clinical treatment failure under dual antiplatelet therapy.19–21 Indeed, the clinical outcomes of patients undergoing PCI were reported to be associated with the levels of D-dimer, plasminogen activator inhibitor-1 (PAI-1), E-selectin, and markers for thrombin generation.22–25 However, there is no sufficient data that correlate heightened platelet reactivity during dual antiplatelet therapy with other markers for coagulation, fibrinolysis, and endothelial dysfunction. The aims of the present study were to assess the various clinical backgrounds associated with high residual platelet aggregability under dual antiplatelet therapy and to clarify any association with thrombin generation, fibrinolytic activity, and endothelial dysfunction that might lead to clinical failure against antiplatelet therapy.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Patients and study protocol
The institutional review board at the Jichi Medical University approved the study protocols, and written informed consent was obtained from all participants. We enrolled consecutive hospitalized patients from July 2006 to April 2007 who were treated by PCI because of symptomatic coronary artery disease, including unstable angina, and non-ST-elevation or ST-elevation myocardial infarction. We estimated the sample size required using a general formula for the correlation coefficient.26 We set {alpha} = 0.05, β = 0.20, and expected a correlation coefficient, r = 0.30–0.35. Using the formula, at least 62–85 participants would be required for the study. All patients had taken dual antiplatelet therapy, consisting of 100 mg/day of aspirin and 200 mg/day of ticlopidine, 75 mg/day of clopidogrel, or 200 mg/day of cilostazol (Table 1). The exclusion criteria were as follows: acute coronary syndrome within 10 days; New York Heart Association Class III or IV heart failure; ingestion of other drugs affecting platelet function or coagulation; platelet counts of <10x107 or >40x107 ml–1; myeloproliferative disorders; autoimmune diseases; malignant diseases; and atrial fibrillation. Compliance with antiplatelet drugs was determined by nursing staff during hospitalization. After normalization of cardiac enzymes (just before discharge), patients underwent blood sampling, ambulatory blood pressure monitoring (ABPM; TM-2425; A&D Co., Inc., Tokyo, Japan), ankle-brachial index (ABI) monitoring (FORM/ABI; Colin Co. Ltd., Ehime, Japan), and cardiorespiratory monitoring (Somte; Compumedics, Melbourne, Australia). To assess the effects of antiplatelet therapy, 20 healthy individuals who were not taking any antiplatelet drugs were enrolled as controls.


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  		Table 1 Characteristics of the study population

 
Platelet aggregation
A fasting venous sample was carefully collected via a 21-gauge needle into a syringe containing 1/10 volume of sodium citrate between 07:30 and 08:00 h. Platelet-rich plasma (PRP) was obtained by centrifuging whole blood at 200 g for 12 min. The time from blood collection to measurement was standardized to 1 h. The aggregation response was measured based on the light scattering intensities obtained with a PA-200 Platelet Aggregation Analyzer (Kowa Co. Ltd., Tokyo, Japan).27 This device is particularly sensitive for detecting the sizes of small platelet aggregates.27,28 Platelet aggregation was performed without any agonists, or with collagen (Hormon-Chemie, Munich, Germany), adenosine diphosphate (ADP) (MC Medical Co., Tokyo, Japan), and thrombin receptor-activating peptide (TRAP; Invitrogen Co., Carlsbad, CA, USA), a specific agonist for protease-activating receptor-1. Spontaneous small platelet aggregation was defined by small aggregate formation by stirring without agonist.

Phosphorylation of vasodilator-stimulated phosphoprotein in platelets
Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) is regulated by the cAMP level, which is thus believed to be a marker of P2Y12 receptor reactivity.29 To determine the VASP phosphorylation state of whole blood, we used a standardized flow cytometric assay (PLT VASP/P2Y12; Biocytex, Marseille, France) with some modifications. We found that the commercially available VASP phosphorylation assay appeared to contain an extremely high concentration of ADP. In our protocol, cAMP elevation by 1 µM PGI2 increased the VASP phosphorylation level by stimulation of adenylate cyclase. When simultaneously stimulated with 2 µM ADP, the signaling from Gi activation mediated via P2Y12 reduced the phosphorylation of VASP induced by PGI2. However, when the P2Y12 receptor was successfully inhibited by active metabolites of thienopyridines, or phosphodiesterase that was inhibited by cilostazol, ADP was unable to reduce PGI2-induced VASP phosphorylation. The phosphorylation of VASP was quantified by flow cytometry according to the manufacturer's instructions. The reduction of VASP phosphorylation induced by ADP was expressed as the % of PGI2; the mean fluorescence intensity of PGI2 plus ADP was devided by that of PGI2.

Laboratory testing, ambulatory blood pressure monitoring, ankle-brachial index, and cardiorespiratory monitoring
Methods are described in detail in the supplementary materials. The intraassay and interassay coefficients of laboratory tests were all <10%. The data obtained from patients are shown in Table 2.


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  		Table 2 Physiological and biochemical characteristics of the study population

 
Statistical analysis
All statistical analyses were performed with SPSS version 11 software (SPSS, Inc., Chicago, IL, USA). The Mann–Whitney U-test was used to compare measurements of platelet activation between patients and healthy volunteers. The associations between the individual parameters were calculated using Spearman's correlation method. To identify independent factors, we used a step-wise multivariable linear regression analysis in which a P-value of 0.05 or less in a simple regression analysis was used as the criterion for entry into the model. We validated independent explanatory variables by Mann–Whitney U-test after categorization into two groups. All reported P-values are two-sided; a P-value of less than 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Patients
Of the 94 patients recruited, two were not included because of advanced gastric cancer or spastic angina, and three did not take dual antiplatelet drugs at the time blood was collected. An additional four patients were excluded from the analysis because of incomplete blood collection or failure of polysomnography or ABPM. Thus, 85 patients were finally included in the analysis (Table 1).

Dual antiplatelet therapy effectively inhibits its pharmacological targets
To precisely assess the effects of aspirin, we measured serum thromboxane B2 (TxB2) concentration, which reflects platelet-cyclooxygenase (COX)-dependent TxA2 production. As has been described,15,17 the serum TxB2 concentration was uniformly abolished in all patients compared with control patients (Figure 1A). We also simultaneously evaluated VASP dephosphorylation after ADP stimulation, which reflects Gi-dependent cAMP reduction. As shown in Figure 1B, cAMP reduction by ADP was effectively inhibited by dual antiplatelet therapy. These data suggested that dual antiplatelet therapy efficiently inhibits its pharmacological targets in patients undergoing PCI.


Figure 1
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  		Figure 1 Serum thromboxane B2 (TxB2) concentration and vasodilator-stimulated phosphoprotein index in patients taking dual antiplatelet therapy. (A) The serum concentration of TxB2 was measured by EIA. (B) The vasodilator-stimulated phosphoprotein phosphorylation was assessed by flow cytometry. ADP-induced vasodilator-stimulated phosphoprotein dephosphorylation was expressed as % of PGI2. Data are expressed as box-and-whisker plots.

 
Inter-individual differences in platelet reactivity under dual antiplatelet therapy
Next, we examined the effects of dual antiplatelet therapy on platelet aggregation patterns using an aggregometry method that simultaneously measures both light transmission and light scattering. Although platelet aggregation assessed by light transmission was significantly decreased in the patients, the results of platelet aggregation tests induced by different agonists showed some inter-individual differences compared with serum TxB2 and VASP phosphorylation (Figure 2A). We compared the changes of VASP phosphorylation and all platelet aggregations in the cilostazol group (n = 10) with those in the thienopyridine group (n = 75). We did not find any significant differences in platelet activation status, suggesting that drug differences could not explain the heterogeneity of platelet aggregation. Use of a laser-light scattering method to quantitatively evaluate the aggregate sizes and numbers revealed that the number of small aggregates increased after stimulation with all agonists, except for the lower concentration of ADP (Figure 2B). The inhibition of medium and large aggregates was clearer for low-dose agonist stimulation (data not shown), indicating that the platelet reactivity generating large platelet aggregates from small aggregates after agonist stimulation was highly concentration-dependent. Furthermore, the degrees of platelet aggregation induced by different agonists within a given subject significantly correlated with each other (Table 3). The number of small platelet aggregates spontaneously formed without agonist stimulation was significantly correlated with the collagen-induced platelet aggregation assessed by light transmission (R = 0.398, P < 0.001). We also found that small aggregate formation induced by a lower dose of agonist (1 µg/mL of collagen or 2 µM ADP) strongly correlated with light transmission induced by all higher concentrations of agonist (R = 0.563–0.815, P < 0.001). These data suggested that platelet aggregability under dual antiplatelet therapy may be determined by differences in the thresholds of each patient's platelets, rather than by differences in antiplatelet drug efficacies.


Figure 2
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  		Figure 2 Platelet aggregation patterns in patients taking dual antiplatelet therapy. Platelets in platelet-rich plasma obtained from control subjects (C) or patients taking dual antiplatelet therapy (P) were stimulated with the indicated agonists for 5 min. (A) Changes in the maximum light transmission were monitored using conventional methods. (B) Light scattering intensities that represent small aggregate formation were measured simultaneously. Data are expressed as box-and-whisker plots.

 

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  		Table 3 Spearman's correlation coefficients among platelet aggregation (light transmission), PAI-1, D-dimer, SF, and E-selectin

 
As activated platelets offer the scaffold of a coagulation cascade in arterial thrombus formation, we supposed that residual platelet activation under dual antiplatelet therapy may be involved in a systemic thrombin generation. To determine whether in vitro platelet aggregation is related to blood thrombogenicity, we compared the results of platelet aggregation tests with the plasma levels of SF (a marker for thrombin generation), D-dimer (a marker for fibrinolysis), PAI-1 (an inhibitor of fibrinolysis), and E-selectin (a marker for endothelial dysfunction). None of these variables was associated with the results of platelet aggregation (Table 3). Next, we attempted to determine factors influencing platelet aggregability by comparing the clinical backgrounds and other laboratory tests. Interestingly, we found that only the fasting glucose level was significantly correlated with the number of spontaneously formed small platelet aggregates and collagen-induced platelet aggregates (R = 0.498, P < 0.001 and R = 0.243, P = 0.025, respectively), regardless of the presence of diabetes mellitus (Table 4). Although many drugs including angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and statin can influence platelet activation and blood coagulation, the use of these drugs did not affect the results of platelet aggregation tests, or the levels of PAI-1, D-dimer, SF, or E-selectin (data not shown).


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  		Table 4 Spearman's correlation coefficients between patient characteristics and thrombogenetic factors in patients taking dual antiplatelet therapy

 
Determinants of thrombin generation, fibrinolytic activity, and endothelial dysfunction
Finally, we examined the clinical characteristics that determine thrombin generation, fibrinolytic activity, and endothelial dysfunction. SF was significantly correlated with total cholesterol, BNP, ABI, and the number of coronary vessels affected Table 4. By multivariable regression analysis including these significant covariates, total cholesterol, BNP, and ABI remained independently correlated with the SF level (Table 5). BNP was also an independent predictor of the D-dimer level in a multivariable regression analysis (Table 5). On the other hand, PAI-1 was significantly correlated with body mass index (BMI) and AHI Table 4. By multivariable analysis, only AHI remained independently correlated with the PAI-1 level (Table 5). E-selectin was significantly associated with age, BMI, diabetes mellitus, 24 h DBP, and AHI (Table 4). By multivariable regression analysis, BMI and diabetes mellitus remained independently correlated with the E-selectin level (Table 5). The significance of these explanatory variables was confirmed by Mann–Whitney U-test after categorization into two groups (see Supplementary material online, Figure S1). These results suggested that total thrombogenicity under antiplatelet therapy may be orchestrated by a variety of patient backgrounds that affect platelet reactivity, thrombin generation, fibrinolysis, and endothelial dysfunction.


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  		Table 5 Multivariate analyses for determination of thrombogenetic factors in patients taking dual antiplatelet therapy

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary material
 Funding
 Acknowledgements
 References
 
Activated platelets are critically involved in thrombotic complications after PCI and in acute coronary syndrome.8–10 The issue of resistance to antiplatelet agents has been emphasized in the literature, leading to growing concern about the efficacy of antiplatelet therapy and about possible unfavorable clinical outcomes.10–12 However, the term ‘resistance’ is frequently misleading when it refers to individuals who develop cardiovascular events despite antiplatelet therapy.10–12 More accurately, we should properly distinguish patients who develop cardiovascular events despite antiplatelet therapy as ‘treatment failure’.30 From the viewpoint of Virchow's triad, arterial thrombosis may occur through complex interactions of a variety of components, including platelet activation, coagulation/fibrinolytic activity, endothelial dysfunction, and blood flow.31,32

On the basis of the results of our study, true antiplatelet drug resistance as defined by a specific test appears rare. This observation is consistent with recent studies, reporting that aspirin resistance other than non-compliance appears to be exceptional.15–18,33 Although studies that used specific tests to measure the pharmacological effects of thienopyridines showed a wide variability in the responses to these drugs,12 VASP dephosphorylation was significantly inhibited by dual antiplatelet therapy, and was not associated with ADP-induced platelet aggregation (data not shown). This discrepancy may be because of differences in the concentrations of ADP used; the commercially available VASP phosphorylation kit appears to use a high concentration of ADP (see Materials and Methods). As well, it is possible that pharmacokinetic differences related to race exist in the metabolism of thienopyridine antiplatelet drugs.

Although antiplatelet resistance has been defined by in vitro platelet function, there appears a widespread misunderstanding that in vitro platelet function directly represents inhibition of a drug target.30 Here, we found that platelet aggregation elicited by different agonists were significantly correlated with each other and associated with small aggregate formation without or with lower agonist stimulation. These data suggest that the platelet aggregability under dual antiplatelet therapy may be determined by differences in the thresholds of each patient's platelets, rather than by differences in antiplatelet drug efficacies. Our finding is supported by recent reports that a 150 mg maintenance dose of clopidogrel is associated with enhanced antiplatelet effects compared with a 75 mg dose, although suboptimal responses were still present in 60% of patients.34 Furthermore, Michelson et al.35 reported that pre-existing variability in platelet responses to ADP accounts for clopidogrel resistance assessed by aggregometory.

We previously showed that an unknown factor, other than COX-1, determines inter-individual differences in platelet aggregation in aspirin-treated patients.17 In this study, only fasting glucose level was significantly correlated with platelet aggregability, regardless of diabetes mellitus. Acute hyperglycemia during oral glucose tolerance tests was correlated with the number of small platelet aggregates.28 Angiolillo et al.34 reported that patients with hyperglycemia exhibit increased platelet reactivity, despite dual antiplatelet therapy, that continues to persist even after administration of a higher maintenance dose of clopidogrel. These findings indicate the importance of suppressing transient hyperglycemia by tight glucose control to prevent thrombotic complications after PCI. Indeed, elevated plasma glucose, with or without a diabetic status, was reportedly an independent predictor of outcomes in acute coronary syndrome patients.36,37

Treatment failure under antiplatelet drug therapy may be influenced by many factors. The coagulation cascade and its regulation are important contributors to clinical events after PCI.19–21 Activated platelets provide phosphatidylserine exposure on their surface that provokes the coagulation cascade, thereby amplifying thrombin generation.38,39 However, residual platelet activation was not correlated with systemic thrombin generation assessed by plasma SF and resultant fibrinolytic activation assessed by the D-dimer level. The major determinant of thrombin generation was found to be independently associated with total cholesterol, BNP, and ABI, suggesting that thrombin generation in PCI subjects under dual antiplatelet therapy is mainly determined by the degree of impaired cardiac function and/or arteriosclerosis. Plasma PAI-1 was also associated with the presence of sleep apnea syndrome. Although circulating platelets account for increases in plasma PAI-1 and release it following activation,40 platelet aggregability was not associated with PAI-1. Taken together, these data suggested that many factors may be involved in systemic thrombogenicity, independent of platelet aggregability.

Our data suggested that some patients may benefit from the addition of anticoagulant treatment after PCI. The American College of Cardiology/American Heart Association guidelines recommend anticoagulant therapy in patients with an acute ST-elevation myocardial infarction with extensive regional wall motion abnormalities. However, the routine use of anticoagulant drugs without thienopyridine should be avoided in patients who have undergone PCI because treatment with aspirin and ticlopidine results in a lower rate of stent thrombosis as compared with a combination of aspirin plus warfarin.41 No trial has closely evaluated the safety and efficacy of anticoagulant therapy in combination with dual antiplatelet therapy in patients undergoing PCI. Large-scale trials are thus needed to confirm any recommendations. Our study should be interpreted in light of its limitations; for ethical reasons we could not obtain proper control patients who had not taken any antiplatelet drug after PCI. This was because dual antiplatelet therapy is the gold standard to reduce clinical events in patients who have undergone PCI.

In conclusion, the current study has demonstrated that dual antiplatelet therapy effectively inhibited its pharmacological targets, although we found inter-individual variability in platelet aggregation, which was at least partly explained by hyperglycemia. On the other hand, thrombin generation, inhibition of fibrinolytic activity, and endothelial dysfunction were not determined by platelet aggregability, but by other aspects of the patients' backgrounds, such as obesity, sleep apnea, diabetes mellitus, cardiac dysfunction, and/or atherosclerotic burden. Our findings indicated that some patients remain at risk of subsequent thrombotic complications after PCI despite adequate dual antiplatelet therapy. Large-scale prospective studies are required to determine which markers are associated with the risk of further cardiovascular events after PCI and to examine interventions such as tight plasma glucose control, anticoagulation, and continuous positive air way pressure therapy.


   Supplementary material
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
Funding
 Acknowledgements
 References
 
Supplementary material is available at European Heart Journal online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
Acknowledgements
 References
 
This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education and Science, Health and Labour Science Research Grants for Research from the Ministry of Health, Labour and Welfare and Grants for ‘High-Tech Center Research’ Projects for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2002–2006.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
References
 
The authors are grateful for the hard work of the Coronary Care Unit staff in patient recruitment and management. We also thank N. Matsumoto, H. Taguchi, and M. Ito for their excellent technical assistance.

Conflict of interest: none declared.


   References
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 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 

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