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Keywords:

  • acute coronary syndrome;
  • aspirin non-responsiveness;
  • C-reactive protein;
  • infection;
  • platelets

Summary.

  1. Top of page
  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References

Background: Epidemiologic studies have shown that there is an association between acute respiratory infection and acute coronary syndrome. The aim of this study was to analyze the thrombotic risk, assessed by platelet aggregation and aspirin non-responsiveness, in patients with an acute coronary syndrome complicated by an infection. Methods: Patients with an acute coronary syndrome who were admitted to the intensive care unit and hospitalized for at least 3 days in 2002 and 2003 were eligible for the study. Three hundred and fifty-eight patients were included, of whom 66 had an infection during their hospital stay. Platelet aggregation was analyzed by an aggregometer using laser light (PA-200, laser light scattering). Aspirin non-responsiveness was defined as a closure time of ≤193 s measured by PFA-100. Results: Platelet aggregation was more pronounced during an infectious complication (P < 0.001). The subgroups of patients with persistent fever, urinary tract infection, and pneumonia all had a higher level of aggregates than the group of patients without an infection (P = 0.007, P = 0.04, and P = 0.01, respectively). Aspirin non-responsiveness was more frequent in the group of subjects with pneumonia compared with those without an infection, 90% vs. 46% (P = 0.006). The CRP levels were independently associated with platelet aggregation and aspirin non-responsiveness (P < 0.001, P < 0.001, respectively). Conclusion: An infectious complication during the course of an acute coronary syndrome leads to more pronounced platelet aggregation. Aspirin non-responsiveness is more frequent in severe infections, such as pneumonia. CRP is an independent predictor of platelet aggregation and aspirin non-responsiveness in the setting of an acute coronary syndrome.


Introduction

  1. Top of page
  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References

Inflammation plays an important role in the pathophysiology of atherosclerosis and plaque rupture [1–3]. A clinical infection may act as a trigger and has been associated with the acute coronary syndrome [4]. Furthermore, acute infection or inflammation impairs endothelial function, which leads to a more prothrombotic and vasospastic state [5]. Platelets may be activated and aggregate, promoting the inflammatory process [6]. Recent studies have shown that activated platelets adhere to circulating leukocytes (heterotypic aggregates) and that these aggregates are associated with ischemic events [7]. The dynamics of platelet aggregation during the course of an ischemic cardiac event are, however, not well known.

Aspirin is standard therapy in the secondary prevention of ischemic stroke and myocardial infarction [8,9]. Recurrent events are, however, common, despite aspirin therapy. In healthy subjects, a low dose of aspirin of 30–75 mg or so will completely block prostaglandin synthesis, but, in the setting of an acute coronary syndrome, the optimal dose of aspirin is not known [10]. The tissue damage leads to an inflammatory response with elevated levels of CRP, cytokines, and fibrinogen, and alternative pathways for thromboxane synthesis may be activated [11,12]. One example is the COX-2 pathway, which may result in thromboxane synthesis that is not inhibited by a low dose of aspirin [13].

In the present study, we evaluated platelet aggregation and inflammatory response during an acute coronary syndrome. We recorded possible and verified infectious complications, such as fever, urinary tract infection (UTI), and pneumonia, and analyzed the impact of these events on platelet aggregation. Finally, we examined the relationship between infectious complications and aspirin responsiveness.

Methods

  1. Top of page
  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References

Study population

Three hundred and fifty-eight consecutive patients with acute coronary syndrome, admitted to the cardiac intensive care unit at Östersund Hospital during the period of January 1, 2002 to October 30, 2003, were included in the study. An in-hospital stay of at least 3 days was required for inclusion. Östersund Hospital is the primary hospital for the County of Jämtland, with a catchment area of approximately 128 000 inhabitants. An acute coronary syndrome was defined as either unstable angina or myocardial infarction with typical chest pain and objective evidence of cardiac ischemia (ECG changes and/or elevated cardiac markers) [14]. Conventional antiplatelet and antithrombotic treatments were used as clinically indicated. The routinely used dose of aspirin was 75 mg. An infection was arbitrarily defined as: (i) the occurrence of fever of >38.0 °C for >2 days; (ii) UTI; or (iii) pneumonia or other significant clinical infections. The diagnosis of infection was made by the physician on duty, who did not participate in the study.

Blood tests

Blood samples were obtained via venipuncture and collected in tubes containing sodium citrate on admission and on the third day in hospital.

PA-200 system testing

The PA-200 aggregometer (Kowa Inc., Tokyo, Japan) has been described in detail by Ozaki et al. [15]. It is a particle counting technique based on laser light scattering (LS). The validity of this method has been confirmed in several studies [16–20]. Briefly, a 20-mW diode laser generates a laser beam measuring 40 μm (wavelength 675 nm) in diameter, which is passed through 300 μL platelet-rich plasma (PRP) stirred in a cylindrical glass cuvette with a 5 mm internal diameter. Light scatter generated by single platelets and platelet aggregates is measured within a region of approximately 30 × 65 × 145 μm by a photocell array. The signal frequency is recorded at 10-s intervals. The LS intensity increases in proportion to the particle size in a suspension and thus gives an estimate of platelet-aggregate size in PRP. Data are expressed as the change over time (s) in the number of aggregates (counts per 10 s) of individual sizes (determined by light intensity, expressed in volts). The LS signals that are obtained are digitized with an A/D converter and processed by a computer. Data are recorded as a two-dimensional graph showing the change over time (s) in the number of aggregates (counts per 10 s) during a period of 10 min. Particles with an intensity of 25–400 mV represent small aggregates (9–25 μm), which are used to study platelet aggregation. To obtain PRP, the blood samples are centrifuged at 150 ×g for 10 min at room temperature. The remaining blood samples are then centrifuged at 300 ×g for 10 min to obtain platelet-poor plasma, which is used as a reference. Adrenalin was used as an agonist in the aggregation analysis (30 μL, 0.1 mg L−1).

PFA-100 system testing

The PFA-100 (Platelet Function Analyzer, Dade Behring, Leiderbach, Germany) measures platelet function as primary hemostasis capacity in citrated whole blood [21]. In the test cartridge, anticoagulated whole blood is aspirated from a sample reservoir through a capillary and an aperture in a membrane, exposing platelets to high shear flow conditions. The membrane is either coated with collagen and epinephrine (CEPI) or collagen and ADP (CAPD), which trigger the platelets to adhere and activate. CEPI, which is sensitive to the aspirin-induced inhibition of platelet function, was used in this study. The time (s) from the start of the test until the platelet plug occludes the aperture is the closure time (CT). The reliability of the PFA-100 in evaluating the effect of aspirin treatment has been previously documented [22,23]. The maximum time recorded is 300 s and, if no occlusion is detected at that time, the measurement is ended. Aspirin non-responsiveness was defined as a CT of ≤193 s, according to previous reports [24,25].

Calculation and statistics

Data were analyzed using spss 13.0 software. We hypothesized that an infectious complication would lead to a standardized effect of 0.40 on platelet aggregation and increase the absolute proportion of patients with aspirin non-responsiveness by 25%. With these assumptions a sample size of approximately 300 participants would be adequate (power 90%, type I error 0.05). Group data are expressed as the mean ± 2 SD for continuous variables and as rates for variables on a nominal scale. Differences between two mean values were assessed with Student's t-test for unpaired data or the Mann–Whitney U-test when appropriate. Differences between multiple groups were assessed with the Kruskal–Wallis test. Differences between proportions were analyzed with the chi-squared test. Independent predictors of platelet aggregation measured by the PA-200 were identified by a multiple linear regression model. Age, CRP, troponin T, the use of low molecular weight heparin (LMWH), and the use of clopidogrel were considered to be of potential importance and included in the models. Some study variables did not show a normal distribution. An approximate normal distribution and improved skewness and kurtosis were achieved by square root transformation of the PA-200 results and logarithmic transformation of the CRP values and the troponin T values. Independent predictors of aspirin non-responsiveness were identified by a multivariate logistic regression model. The variables age, troponin T, the use of low LMWH, and the use of clopidogrel were also included in this model. The null hypothesis was rejected for values of P < 0.05.

Ethics

The ethics committee at Umeå University approved the project. All the patients gave their informed consent to participate in the study.

Results

  1. Top of page
  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References

Two hundred and ninety-two patients had no signs of infection and 66 patients had their hospital stay complicated by an infection. The patient characteristics are shown in Table 1. The total number of patients differs slightly in the tables, as not all inflammatory variables were complete in every patient. Furthermore, four patients with other infections are not shown in the tables (Tables 1–3). Patients with signs of infection had a lower hemoglobin level, a higher white cell count, a higher CRP level, and a lower use of both molecular weight heparin (LMWH) and clopidogrel, compared with patients without signs of infection. The patients with infection were also older than those with no signs of infection. The results of the platelet tests are shown in Table 2. Patients with signs of infection had a higher occurrence of platelet aggregates, while there was no significant difference in CTs between the groups. Aspirin non-responsiveness was examined in the different categories of patients with signs of infection and in those without infection. Aspirin non-responsiveness was more frequent in the group of subjects with pneumonia compared with those without an infection, 90% vs. 46% (P = 0.006) (Table 3). The proportion of patients with aspirin non-responsiveness in the group without infection was similar on admission (42%) and on day 3 (46%). In patients with pneumonia, aspirin non-responsiveness was more common on day 3 (90%) than on admission (44%). There were no differences in aspirin non-responsiveness in patients with fever and UTI as compared with those without infection. The CRP levels were independently associated with platelet aggregation (P < 0.001, R2 = 0.17) as well as with aspirin non-responsiveness (P < 0.001, OR = 1.54; 95% CI: 1.23–1.96) adjusted for age, troponin T-levels, and use of clopidogrel and LMWH. There was no significant difference in treatment with fibrinolysis or aspirin-dose between the groups (P = 0.5, P = 0.8).

Table 1.   Clinical characteristics of the non-infectious group and the different subgroups with infection
 No infection (n = 292)Fever (n = 39)UTI (n = 13)Pneumonia (n = 10)P-value
  1. UTI = urinary tract infection. Fever is defined as a temperature of >38 °C for >2 days. BMI = body mass index; TnT = troponin T. Hemoglobin, platelet count, white cell count, and CRP were measured on day 3.

  2. Values are expressed as the mean ± 2 SD or numbers, unless otherwise indicated.

  3. P-values are calculated by comparing the two main groups: the patients with no signs of infection and those with infection.

Gender, M/F192/10027/127/66/40.7
Age, years70 ± 1274 ± 1081 ± 877 ± 80.001
Smokers61 (21%)10 (26%)1 (8%)2 (20%)0.7
BMI26 ± 4.226 ± 4.425 ± 2.028 ± 5.80.2
Hypertension112 (38%)17 (44%)5 (38%)7 (70%)0.2
Diabetes mellitus50 (17%)10 (26%)3 (23%)1 (10%)0.3
Prior AMI68 (23%)11 (28%)3 (23%)3 (30%)0.4
Aspirin278 (95%)39 (100%)13 (100%)10 (100%)0.7
Clopidogrel116 (40%)8 (21%)5 (38%)2 (20%)<0.001
Statin (on admission)52 (18%)8 (21%)1 (8%)3 (30%)0.7
Enoxaparin252 (86%)29 (74%)13 (100%)7 (70%)0.03
Hemoglobin, g L−1131 ± 15125 ± 13120 ± 17118 ± 17<0.001
Platelet count, ×109218 ± 57207 ± 58217 ± 59201 ± 750.3
White cell count, ×1097.7 ± 2.79.3 ± 3.29.4 ± 3.014.6 ± 8.2<0.001
Cholesterol, mm5.4 ± 1.25.3 ± 1.55.0 ± 1.14.9 ± 1.30.3
CRP, mg L−1, median (interquartile range)11 (28)55 (124)32 (58)134 (80)<0.001
CKMB, mg L−1, median (interquartile range)41 130162 25362 15126 117<0.001
TnT, μg L−1, median (interquartile range)1.09 (2.6)3.18 (5.9)2.96 (5.8)2.04 (6.1)0.03
Table 2.   Comparison of platelet aggregation among patients diagnosed with acute coronary syndrome, with and without clinical signs of infection
 No infection (n = 277)Fever (n = 38)UTI (n = 12)Pneumonia (n = 10)P-value
  1. SPA, small-sized platelet aggregates; PFA, platelet function analyzer; UTI, urinary tract infection.

  2. Data are the median (interquartile range).

  3. P-values calculated using the Kruskall–Wallis test.

SPA (day 1) counts × 1042.8 (6.0)3.6 (5.8)2.4 (8.0)9.5 (5.5)0.08
SPA (day 3) counts × 1045.5 (8.2)9.7 (9.9)10 (9.6)12 (9.7)0.002
PFA-100, s (day 1)234 (167)158 (184)175 (173)194 (176)0.7
PFA-100, s (day 3)205 (156)191 (167)290 (166)167 (43)0.5
Table 3.   Aspirin non-responsiveness among patients diagnosed with acute coronary syndrome, with pneumonia and patients without clinical signs of infection
 Aspirin non-responsivenessAspirin responsivenessTotal
  1. Aspirin non-responsiveness defined as CT ≤193 s. P-value on day 3 = 0.006.

No infection, day 1110 (42%)155 (58%)265
Pneumonia, day 14 (44%)5 (56%)9
Total114160274
No infection, day 3128 (46%)149 (54%)277
Pneumonia, day 39 (90%)1 (10%)10
Total137150287

Discussion

  1. Top of page
  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References

This study shows that platelet aggregation increases in patients with signs of infection during the course of an acute coronary syndrome. The increased aggregation of platelets occurs despite standard treatment with aspirin and clopidogrel.

Furthermore, it is shown that a more serious infectious complication, pneumonia in the present study, is associated with aspirin non-responsiveness. Patients with milder infectious signs, such as fever and UTI, had more small-sized platelet aggregates (P = 0.007, P = 0.04), but the frequency of aspirin non-responsiveness did not differ compared with patients without infection. Finally, this study shows that platelet aggregation and aspirin non-responsiveness are related to the inflammatory response measured by CRP.

Aspirin non-responsiveness is a poorly defined condition and several possible mechanisms have been described, ranging from lack of compliance to thromboxane-independent platelet aggregation [26]. The present body of evidence shows that an aspirin dose that effectively inhibits platelet activation in healthy volunteers may not be effective in patients with an acute disease. Borna et al. showed that aspirin non-responsiveness was increased by an ST-elevation myocardial infarction and correlated with adenosine diphosphate levels [25]. It may have been the more pronounced inflammatory response in ST-elevation myocardial infarction that caused the difference in aspirin non-responsiveness. Whether an increased dose of aspirin can improve platelet inhibition in subsets of patients with aspirin non-responsiveness has, however, not been conclusively studied. A recently published study found that the residual arachidonic acid-induced platelet activation is COX-1 and COX-2 independent and could partly be inhibited by clopidogrel. Speculatively, the inflammatory response with an increase in CRP levels could be linked to the ADP-induced platelet aggregation [27]. In the setting of an acute infection, enhanced shear-induced platelet aggregation may be important but has not been explored in the present study.

Previous studies with the PA-200 aggregometer have shown an increased formation of small platelet aggregates in smokers, in diabetics, and in patients with stable or acute coronary syndromes. It has also been shown in small studies that this correlates with an increase in the incidence of cardiovascular events.

Our study adds to previous knowledge. It shows that platelet aggregation in an acute coronary syndrome can be more pronounced by different infectious complications. Moreover, our results make it clear that platelet aggregation and aspirin non-responsiveness are dynamic processes associated with the systemic inflammatory response.

The systemic inflammatory response was assessed by the CRP levels in the present study. It is well known that elevated CRP is not only a marker of a future risk of developing cardiovascular disease but also predicts poorer outcome in patients with an acute coronary syndrome. There are in vitro data linking CRP to platelet aggregation. CRP exists in two different conformations; the native pentamer (nCRP) and the modified CRP (mCRP), where the last of these appears to have pro-inflammatory actions not seen with nCRP, which instead appears to have vasculoprotective effects [28]. Modified CRP may promote platelet aggregation, while nCRP appears to inhibit aggregation. The former CRP is bound to the intima and is not measured in blood samples. However, native CRP may undergo conformational changes into mCRP and promote platelet aggregation, but knowledge at this time is limited.

Conclusion

  1. Top of page
  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References

Our study shows that a systemic infection during the course of an acute coronary syndrome leads to more pronounced platelet aggregation. Furthermore, aspirin non-responsiveness is more frequent in severe infections. CRP is an independent predictor of platelet aggregation and aspirin non-responsiveness in the setting of an acute coronary syndrome. Further studies are needed to clarify the prognostic consequences as well as the treatment aspects of these findings.

Disclosure of Conflict of Interests

  1. Top of page
  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References

This study was supported by grants from the Research and Development Unit at Jämtland County Council, the Heart Foundation of Northern Sweden and the Joint Committee of the Northern Sweden Health Care Region.

References

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  2. Summary.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Conflict of Interests
  9. References
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