A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy

TR-06020; No of Pages 7 Thrombosis Research xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Thrombosis Research journal homepage: www.elsevier.com/locate/thromres

A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy Alessia Fabbri a,⁎, Rossella Marcucci a, Anna Maria Gori a,b, Betti Giusti a, Rita Paniccia a, Daniela Balzi c, Alessandro Barchielli c, Serafina Valente d, Cristina Giglioli d, Rosanna Abbate a, Gian Franco Gensini a,b a

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy Don Carlo Gnocchi Foundation, Florence, Italy Epidemiology Unit, Local Health Unit 10, Florence, Italy d Heart and Vessel Department, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy b c

a r t i c l e

i n f o

Article history: Received 19 December 2014 Received in revised form 26 May 2015 Accepted 30 June 2015 Available online xxxx Keywords: Antiplatelet agents Atherothrombosis Acute Coronary Syndrome (ACS) High on-treatment platelet reactivity (HPR)

a b s t r a c t Introduction: Limited data are available on the natural history of high on treatment platelet reactivity (HPR) by arachidonic acid and ADP - markers of unfavorable prognosis in acute coronary syndrome patients -. Material and methods: In a cohort of acute coronary syndrome male patients (n = 101), we evaluated the timecourse of HPR by ADP (platelet aggregation by 10 μM ADP ≥ 70%) and arachidonic acid (platelet aggregation by 1 mmol arachidonic acid ≥ 20%) measuring platelet function in the acute phase (T0), at 6 months (T1) and 1 year (T2). Results: We identified persistent (HPR at T0,T1 and T2), acute non persistent (HPR only at T0), and late (HPR only at T1 or T2). Patients with persistent HPR by ADP were more frequently with higher values of BMI. Patients carrying CYP2C19*2 variant were more prevalent in the group of persistent HPR (33%). Significant higher values of immature platelet fraction and high immature platelet fraction at 6 and 12 months and mean platelet volume were present in patients with late HPR. Immature platelet fraction was the only variable significantly associated with late HPR by ADP at multivariate analysis (OR = 1.6 (1.08-2.3), p = 0.016). Patients with persistent HPR by arachidonic acid were more frequently diabetics. Immature platelet fraction at 6 months and high immature platelet fraction at 6 and 12 months were the parameters associated with late HPR by AA (OR = 1.4 (1.0-1.9), p = 0.036; OR = 1.5 (1.08-2.4), p = 0.05; OR = 4.9 (1.3-18.8), p = 0.018, respectively). Conclusions: About 25% of 101 patients has persistent HPR; they are more frequently diabetics, overweight or carriers of CYP2C19*2. The occurrence of an inflammatory state, indicated by the increase of immature platelet fraction, is associated with the occurrence of late HPR. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction During the last years, a number of studies described the crucial role of platelet activation and aggregation in thrombus formation in patients with acute coronary syndromes (ACS) [1]. In large clinical trials, adverse vascular events remain a serious clinical problem that occurs in a significant proportion of ACS patients despite of, the wide use of the antiplatelet therapy and the significant benefits reported with combined antiplatelet treatment (clopidogrel and aspirin) [2–4]. During the acute phase of coronary artery disease, a growing body of evidence has demonstrated a higher prevalence of high on-treatment ⁎ Corresponding author at: Department of Experimental and Clinical Medicine, University of Florence, Azienda Ospedaliero-Universitaria Careggi, Largo Brambilla 3, 50134 Florence, Italy. E-mail address: [email protected] (A. Fabbri).

platelet reactivity (HPR) on antiplatelet therapy and several mechanisms leading to HPR involve genetic, clinical and cellular factors [5]. It is well documented that HPR, in ACS patients on dual antiplatelet treatment, has a relevant clinical role because of it is significantly associated with an increased risk of cardiovascular events [6]. Limited data, in unstable and stable patients are available in the literature, on the prevalence of HPR by ADP - which reflects the socalled clopidogrel non –responsiveness, during the follow-up period after percutaneous coronary intervention (PCI) [7,8]. Whereas no data are available on the prevalence of HPR by arachidonic acid (AA) in both stable and unstable patients after PCI and on the factors associated with the HPR throughout the late phase of ACS. To provide this lack of information we evaluated the time-course of HPR by ADP and AA and its association with clinical and laboratory characteristics during 1 year of follow-up from the acute event of a cohort of ACS male patients receiving drug-eluting stent or bare-metal stent on dual antiplatelet treatment.

http://dx.doi.org/10.1016/j.thromres.2015.06.040 0049-3848/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: A. Fabbri, et al., A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.040

2

A. Fabbri et al. / Thrombosis Research xxx (2015) xxx–xxx

2. Methods

2.3. Light transmission aggregometry measurement

2.1. Study population

Turbidimetric platelet aggregation (PA) was used to measure agonist-induced PA. Citrated whole blood samples were centrifuged for 10 minutes (min) at 250 x g to obtain platelet-rich plasma (PRP). Platelet-rich plasma was stimulated with 10 μM ADP (Mascia Brunelli, Milan,Italy) and with 1 mmol AA (Sigma-Aldrich,Milan, Italy) using an APACT 4 aggregometer (Helena Laboratories Italia S.P.A, Milan, Italy). Platelet aggregation was evaluated, according to Born’s method, considering the maximal percentage of platelet aggregation in response to different stimuli (ADP-PA and AA-PA) after 10 min. High on-treatment platelet reactivity by ADP or AA was diagnosed in the presence of ADP-PA ≥ 70% and AA-PA ≥ 20% respectively. We defined patients with dual HPR those patients with PA induced by AA ≥ 20% and by ADP ≥ 70% according to the literature [14] and studies from our group [15,16].

The study population consisted of a group of 157 male patients discharged from the Coronary Unit of Careggi University Hospital, Florence, Italy from April 2008 to April 2009 and enrolled in the frame of the Florence Acute Myocardial Infarction-2 (AMI-Florence 2) registry. Twenty-two patients were excluded as they interrupted dual antiplatelet therapy for major surgery. Thirty-four patients dropped out from the program. Therefore the final study population included 101 patients. The AMI-Florence 2 registry is a second-wave survey of the AMIFlorence registry; it included all patients who arrived alive, between April 2008 and April 2009, to the emergency departments of one of the six participating hospitals in the Florence health district with a suspected ACS [9]. Acute coronary syndromes was diagnosed according to criteria established by the European Society of Cardiology [10]. Briefly: acute myocardial infarction (MI) was defined as typical rise and gradual fall of troponin, or more rapid rise and fall of CK-MB, defined as N 99% of normal levels (troponin T N 0.05 ng/ml; CK-MB N10 ng/ml), with at least one of the following: acute onset of typical ischaemic chest pain; some Q waves in V1-V3, 30 ms Q waves ≥1 mm in two contiguous leads; ST- segment elevation or depression in ≥ 2 leads, ≥ 0.2 mV in V1-V3, N 0.1 mV in other leads. Unstable angina was defined as a history of new-onset, more frequent, more persistent or rest episode of chest pain, without typical changes of myocardial enzymes and with ECG evidence of myocardial ischaemia (transient ST segment displacement N 0.1 mV during chest pain). All patients underwent coronary angiography performed by the Judkins’ technique and percutaneous coronary intervention (PCI). Before PCI, all patients received a loading dose of 500 mg of acetylsalicylic acid (ASA) and 300 mg of clopidogrel, followed by 100 (15.9%) or 325 mg (84.1%) of ASA daily and 75 mg of clopidogrel daily. Unfractioned heparin 70 IU/kg was used in all patients during PCI as anticoagulant. A clinical follow-up was performed in all patients at 6 and 12 months. Current smoking status was determined at the time of blood collection. The subjects were classified as having hypertension according to the guidelines of European Society of Hypertension/European Society of Cardiology [11] or if they reported taking antihypertensive medications, as verified by the physician. Diabetic subjects were defined in agreement with the American Diabetes Association [12] or on the basis of self-report data if confirmed by medication or chart review. Dyslipidaemia was defined according to the Third report of the National Cholesterol Education Program (NCEP-III) [13] or if they reported taking antidyslipidaemic drugs, as verified by the physician. A positive family history was defined as the presence of at least one first-degree relative who had developed coronary artery disease (CAD) before the age of 55 years. On the day of examination patients took their clopidogrel medication in the morning before blood sampling and their acetylsalicylic acid medication in the afternoon. Antiplatelet therapy with aspirin and clopidogrel remained unmodified during the entire follow-up for all patients. All subjects gave informed consent; the study complies with the Declaration of Helsinki and was approved by the local ethic committee.

2.2. Blood collection Venous blood samples anticoagulated with 0.109 M sodium citrate for platelet aggregometry were taken at baseline within 24–48 hours (h) from clopidogrel and acetylsalicylic acid loading dose (T0), and after 6 (T1) and 12 (T2) months. To minimize the effect of anticoagulant on platelets, all measurements were performed within 2 h after blood collection.

2.4. Reticulated platelets A fully automated method has been developed that uses blood cell counter for the quantification of reticulated platelets (RP) [17]. Reticulated platelets were measured by using the Sysmex XE-2100 haematology analyser (Sysmex, Kobe, Japan). Briefly, the flow cytometric determination of RP uses a proprietary fluorescent dye containing polymethine and oxazine. These two dyes penetrate the cell membrane staining the mRNA in RP. The stained cells were passed through a semiconductor diode laser and the resulting forward scatter light and fluorescent intensity were measured. A computer algorithm (Sysmex IPF Master) applies a pre-set gate to separate mature platelets (blue dots) and RP (green dots). Reticulated platelets were expressed as a percentage (%) of the total optical platelet count (immature platelet fraction; IPF). The immature platelet fraction indicates the rate of platelet production. Reticulated platelets were also expressed as the percentage of platelets, within the immature platelet fraction, with a major amount of highly fluorescent m-RNA (highly fluorescent immature platelet fraction; H-IPF). The average coefficients of variation (CV) for IPF and H-IPF were 10.6% and 18.8%, respectively.

Table 1 Characteristics of the Study Population. Baseline (T0) Male, (n = 101) Age, median and IQR (y) BMI, median and IQR (Kg/m2) Diabetes, n (%) Hypertension, n (%) Hypercholesterolemia, n (%) Hypertriglyceridemia, n (%) Cigarette smoking, n (%) Familiary history for CAD, n (%) Prior PCI, n (%) Prior CABG, n (%) Prior ACS, n (%) Admission for NSTEMI/UA, n (%) Admission for STEMI, n (%) PCI, n (%) DES, n (%) BMS, n (%) CABG, n (%) CYP2C19*2 +/−, n (%) PA-ADP 10 μM, median and IQR (%) PA-AA 1 mmol, median and IQR (%)

67 (58–74) 26 (24–26) 21 (21) 64 (63) 59 (58) 33 (33) 41 (41) 48 (48) 17 (17) 8 (8) 24 (24) 45 (45) 56 (55) 87 (86) 63 (62) 21 (21) 9 (9) 25 (25) 52 (36–64) 13 (9–16)

IQR = interquartile range, BMI = body mass index, CAD = coronary artery disease, PCI = percutaneous coronary intervention, DES = drug eluting stent, BMS = bare metal stent, CABG = coronary artery bypass graft, ACS = acute coronary syndrome, NSTEMI = non-ST-segment elevation myocardial infarction, STEMI = ST-segment elevation myocardial infarction, UA = unstable angina, CYP2C19*2 = Cytochrome P450 2C19, PA-ADP 10 μM = platelet aggregation by ADP 10 μM, PA-AA 1 mM = platelet aggregation by arachidonic acid.

Please cite this article as: A. Fabbri, et al., A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.040

A. Fabbri et al. / Thrombosis Research xxx (2015) xxx–xxx

3

Fig. 1. High platelet reactivity by ADP or by AA at baseline vs 6 months and at baseline vs 12 months.

Immature platelet fraction and H-IPF evaluation was performed after 6 (T1) and 12 (T2) months. 2.5. Genotyping Genomic DNA was isolated from venous peripheral blood using Tecan, Freedom EVO liquid handler (Tecan Group, Männedorf, Switzerland) and with the magnetic bead using the GeneCatchergDNA Blood kit (Invitrogen, Carlsbad, California, U.S.A.). DNA purity and concentration were determined by NanoDrop spectrophotometer (Thermo Scientific, Wilmington, Delaware, U.S.A.). Genotyping of the CYP2C19*2 loss-of-function polymorphism (681G N A, rs4244285) was performed using TaqMan validated Drug Metabolism Genotyping assay with the 7900HT Sequence Detection System (Applied Biosystems, Foster City, California, U.S.A.) [18]. 2.6. Statistical analysis Statistical analysis was performed using the SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) software for Windows (Version 20.0). For intraindividual correlation analyses, were included all patients with a drug compliance N 90% (assessed by a questionnaire). Values are presented as median and interquartile range (IQR). The Kruskal-Wallis test for non-parametric data was used to analyze differences among different groups. Post-hoc comparison between two

groups was performed by using Bonferroni correction. The Mann– Whitney test, for unpaired data, was used for comparison between two groups. Dichotomous variables were compared by Chi2 test. To test the independent association between several confounders and HPR by ADP or by AA, we performed a multiple logistic regression analysis adjusted for: age, diabetes, proton pump inhibitor (PPI), 6- and 12month IPF and H-IPF, statin use, Cytochrome P450 2C19 (CYP 2C*19) and body mass index (BMI). All odds ratios (OR) are given with their 95% confidence interval (CI). All p values b 0.05 were considered to be statistically significant. 3. Results Demographic and clinical characteristics of the study population are shown in Table 1. Age was the parameter associated with HPR by AA at baseline (Table 3). No recurrent adverse events (acute myocardial infarction, stent thrombosis, ischemic stroke and cardiovascular death) were observed during follow-up. The median values of PA-ADP and PA-AA at T0 were reported in Table 1. The median values of PA-ADP and PA-AA at T1 were 60% (IQR: 45–72) and 14% (IQR: 11–16.5), respectively; the median values of PA-ADP and PA-AA at T2 were 65% (IQR: 49–76) and 15% (IQR: 11–18.5), respectively. The occurrence of HPR by ADP was found in 12 patients at T0, in 18 patients at T1 and in 25 patients at T2 (Fig. 1).

Table 2 Time course of HPR by ADP.

Age median and IQR (y) Familiary history n, (%) BMI median and IQR, (Kg/m2) Diabetes n, (%) Hypertension n, (%) Hypercholesterolemia n, (%) Hypertriglyceridemia n, (%) Cigarette smoking n, (%) CYP219*2 +/− n, (%)

HPR by ADP ≥ 70% at baseline n = 12

HPR by ADP b 70% at baseline n = 89

P Value

HPR by ADP ≥ 70% at 6 months n = 18

HPR by ADP b 70% at 6 months n = 83

P Value

HPR by ADP ≥ 70% at 12 months n = 25

HPR by ADP b 70% at 12 months n = 76

P Value

68 (57–74)

67 (58–75)

0.725

76 (61–78)

66 (56–72)

0.008

69 (57–76)

67 (58–74)

0.568

45 (51)

0.096

39 (47)

0.817

13 (52)

35 (46)

0.605

26 (25–28)

0.791

25 (24–28)

0.040

28 (24–29)

26 (24–28)

0.267

2 (17)

19 (21)

0.708

7 (39)

14 (17)

0.037

9 (36)

14 (18)

0.069

8 (67)

56 (63)

0.800

13 (72)

57 (69)

0.749

20 (80)

56 (74)

0.302

6 (50)

53 (60)

0.529

9 (50)

50 (60)

0.424

17 (68)

51 (67)

0.778

3 (25)

32 (36)

0.454

7 (39)

29 (35)

0.897

16 (64)

30 (39)

0.517

5 (42)

36 (40)

0.936

3 (17)

15 (18)

0.871

5 (20)

11 (14)

0.385

4 (33)

21 (24)

0.463

3 (17)

22 (27)

0.381

5 (20)

20 (26)

0.526

3 (25) 25 (24–30)

9 (50) 27 (25–30)

IQR = interquartile range, BMI = body mass index, CYP2C19*2 = Cytochrome P450 2C19.

Please cite this article as: A. Fabbri, et al., A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.040

4

A. Fabbri et al. / Thrombosis Research xxx (2015) xxx–xxx

Table 3 Time course of HPR by AA.

Age median and IQR (y) Familiary history n, (%) BMI median and IQR (Kg/m2) Diabetes n, (%) Hypertension n, (%) Hypercholesterolemia n, (%) Hypertriglyceridemia n, (%) Cigarette smoking n, (%)

HPR by AA ≥20% at baseline n = 18

HPR by AA b 20% at baseline n = 83

P Value

HPR by AA ≥20% at 6 months n = 10

HPR by AA b20% at 6 months n = 91

P Value

HPR by AA ≥20% at 12 months n = 16

HPR by AA b20% at 12 months n = 85

P Value

71 (64–77)

66 (56–74)

0.042

75 (58–78)

67 (57–74)

0.101

63 (55–74)

68 (58–75)

0.615

10 (56)

38 (46)

0.452

43 (47)

0.869

40 (47)

0.829

26 (24–27)

26 (25–28)

0.207

26 (24–28)

0.657

26 (24–28)

0.429

6 (33)

15 (18)

0.148

5 (50)

16 (18)

0.016

6 (38)

17 (20)

0.126

13 (72)

51 (61)

0.390

9 (90)

61 (67)

0.135

9 (56)

67 (79)

0.055

10 (56)

49 (59)

0.786

3 (30)

56 (62)

0.055

9 (56)

59 (69)

0.272

8 (44)

27 (33)

0.336

2 (20)

33 (36)

0.357

7 (44)

39 (46)

0.812

7 (39)

34 (41)

0.871

3 (30)

15 (17)

0.289

6 (38)

10 (12)

0.010

5 (50) 26 (23–28)

8 (50) 28 (24–29)

IQR = interquartile range, BMI = body mass index.

The occurrence of HPR by AA was found in 18 patients at T0, in 10 patients at T1 (vs T0, p = 0.054) and in 16 patients at T2 (vs T0, p = 0.025) (Fig. 1). The occurrence of dual HPR (HPR by ADP and by AA) was found in 4 patients (3.9%) at T0, in 6 patients (5.9 %) at T1 and in 10 patients (9.9 %) at T2. The prevalence of CYP2C19*2 variant +/− was 25 % (Table 1). The prevalence of CYP2C19*2 variant −/− was 75%. At baseline high on-treatment platelet reactivity by AA was associated with older age (Table 3). No differences were detected in the other traditional cardiovascular risk factor according to HPR by ADP or HPR by AA (Tables 2 and 3). At 6 months, patients with HPR by ADP or by AA were older, with a higher prevalence of diabetes and with a higher BMI than the patients without HPR (Tables 2 and 3). At 12 months, we observed a higher prevalence of cigarette smoking in patients with HPR by AA than patients without it (Tables 2 and 3). Interesting, in patients with dual HPR (defined as HPR by ADP and by AA) we found a higher prevalence of diabetes at 6 and 12 months and of cigarette smoking at 12 months (Table 4). Fig. 1 shows the time-course of platelet reactivity during 12 monthfollow up.

According to these results we identified four groups of patients: 1- PERSISTENT HPR by ADP or by AA: patients with HPR by ADP or by AA at T0, T1 and T2. 2- ACUTE NON PERSISTENT HPR by ADP or by AA : patients with HPR by ADP or AA only at T0. 3- LATE HPR by ADP and AA: patients with HPR by ADP or AA only at T1 or T2. 4- NO HPR: patients without HPR by ADP or AA at T0, T1 and T2. Patients with persistent HPR by ADP were more frequently overweight (Table 5). Proton pump inhibitors and statin use were not significant different among the four groups of HPR by ADP. Patients carrier CYP2C19*2 variant were more prevalent in the group of persistent and acute non persistent HPR (33%) with respect to the other groups, even if the difference does not reach the statistical significance due to the low number of patients. Significant higher values of IPF, high IPF at 6 and 12 months and mean platelet volume were present in patients with late HPR than in patients without HPR by ADP (Table 5). Patients with persistent HPR by AA were more frequently diabetics and older than the other groups.

Table 4 Time course of HPR by ADP and AA.

Age median and IQR (y) Familiary history n, (%) BMI median and IQR (Kg/m2) Diabetes n, (%) Hypertension n, (%) Hypercholesterolemia n, (%) Hypertriglyceridemia n, (%) Cigarette smoking n, (%)

HPR by AA ≥20% and ADP ≥ 70% at baseline n=4

HPR by AA b 20% and ADP b 70% at baseline n = 75

P Value

HPR by AA ≥20% and ADP ≥ 70% at 6 months n=6

HPR by AA b20% and ADP b 70% at 6 months n = 79

P Value

HPR by AA ≥20% and ADP ≥ 70% at 12 months n = 10

HPR by AA b20% and ADP b 70% at 12 months n = 70

P Value

69 (62–80)

66 (56–74)

0.359

76 (59–78)

65 (57–72)

0.108

66 (58–65)

68 (60–74)

0.959

36 (48)

0.531

37 (47)

0.999

33 (47)

0.447

26 (25–28)

0.118

25 (24–28)

0.381

26 (24–28)

0.089

1 (25)

14 (19)

0.577

4 (67)

13 (16)

0.003

5 (50)

13 (19)

0.026

3 (75)

46 (61)

0.583

6 (100)

54 (68)

0.101

7 (70)

54 (77)

0.620

7(64)

55 (61)

0.667

4 (67)

51 (65)

0.917

6 (60)

48 (69)

0.543

2 (50)

26 (35)

0.493

1 (17)

25 (32)

0.432

6 (60)

29 (41)

0.330

1 (25)

30 (40)

0.091

2 (33)

14 (18)

0.315

4 (40)

9 (13)

0.030

1 (25) 25 (24)

3 (50) 26 (24–31)

6 (60) 28 (26–29)

IQR = interquartile range, BMI = body mass index.

Please cite this article as: A. Fabbri, et al., A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.040

A. Fabbri et al. / Thrombosis Research xxx (2015) xxx–xxx

5

Table 5 Characteristics of the Study Population according to persistent, acute non persistent, late and no HPR by ADP. PERSISTENT HPR by ADP, n = 3

ACUTE NON PERSISTENT HPR by ADP, n = 9

Age median and IQR (y) Diabetes n, (%) PPI n, (%) IPF at 6 months follow-up median and IQR, (%) H-IPF at 6 months follow-up median and IQR, (%)

56 (54.0) 0 3 (100) 2.8 (1.9)

69 (62.5-74.5) 2 (22.2), 4 (44.4) 2.6 (1.8-3.6)

0.8 (0.6)

0.6 (0.4-0.7)

IPF at 12 months follow-up median and IQR, (%)

2.8 (1.9)

2.55 (1.1-5.7)

H-IPF at 12 months follow-up median and IQR, (%)

0.7 (0.5)

0.55 (0.18-1.48)

Reticulated platelets median and IQR

5600 (3400)

4600 (2600–10600)

Mean platelets volume median and IQR, (f/l)

11,1 (10,7)

12,55 (9,95-16,25)

Statins n, (%) CYP 2C19 +/− n, (%) BMI median and IQR, (Kg/m2)

3 (100) 1 (33.3) 29.7 (28.5) *p = 0.011

9 (100) 3 (33.3) 25.2 (23.1-25.7)

LATE HPR by ADP, n = 25 70 (57.5-76.5) 8 (32.0), 18 (72.0) 3.6 (2.5.-5.75), *p = 0.011 1.15 (0.5-1.7), *p = 0.038 4 (2.5-4.9), *p = 0.009 1.1 (0.7-1.4), *p = 0.036 8100 (5000–11600) *p = 0.011 12 (11–17,2) *p = 0.021 22 (88.0) 3 (12.0) 26.9 (25.5-28.6)

NO HPR by ADP, n = 64

P for trend

67 (56.5-73.0) 11 (17.2) 44 (68.8) 2.1 (1.5-3.85)

0.462 0.365 0.274 0.077

0.6 (0.4-1.15)

0.161

2.1 (1.5-3.8)

0.055

0.5 (0.3-1.1)

0.104

4700 (3200–7600)

0.061

11,25 (10,20-12,05)

0.126

61 (95.3) 18 (28.1) 26 .0 (24.62-28.0)

0.465 0.389 0.010

PPI = Proton Pump Inhibitor, IQR = InterQuartile Range, IPF = Immature Platelet Fraction, H-IPF = High Immature Platelet Fraction, CYP 2C19 = Cytochrome P450 2C19, BMI = Body Mass Index, *p vs NO HPR.

Immature platelet fraction and high IPF at 6 and 12 months were higher in patients with late HPR by AA (Table 6). Significant higher values of BMI, IPF at 6 and 12 months, H-IPF at 12 months and mean platelet volume were present in patients with late HPR than without HPR by ADP after the exclusion of diabetic patients (Table 7). Immature platelet fraction and H-IPF at 6 and 12 months and mean platelet volume were higher in patients with late HPR than without HPR by AA (Table 8). Due to the low number of subjects included, logistic regression analysis was not performed in patients with persistent HPR by both ADP and AA. Immature platelet fraction at 12 months (OR = 1.6 (1.08-2.3), p = 0.016) was the only variable significantly associated with late HPR by ADP at multivariate backward logistic regression analysis. At multivariate logistic regression analysis - adjusted for age, familiary history, BMI, diabetes, hypertension, hypercholesterolemia, hypertriglyceridemia, cigarette smoking-, IPF at 6 months and H-IPF at 6 and 12 months were the parameters associated with late HPR by AA (OR = 1.4 (1.0-1.9), p = 0.036; OR = 1.5 (1.08-2.4), p = 0.05; OR = 4.9 (1.3-18.8), p = 0.018, respectively). 4. Discussion This study shows that it is possible to identify four different patterns in the natural history of platelet reactivity in ACS patients on dual antiplatelet therapy. The main finding is that a low number of patients with HPR at baseline maintains this phenotype at the follow-up, i.e. 4/12 (33%) for HPR

by ADP and 4/18 (22%) for HPR by AA. This means that about 25% of patients identified as HPR at the time of acute event will persist in this condition thereafter. These results might be ascribed to the effect of the acute phase and of the activation of inflammation which naturally decreases within 6 months. We do not know whether only patients with persistent HPR will be those at high risk of thrombotic events or not. Nevertheless HPR is a marker of thrombotic risk with high sensitivity and low specificity, meaning that only a minority of patients identified as HPR at the time of the acute event will develop a thrombotic complication. The question whether this group is represented by patients with persistent HPR is not answered by this study. Interesting, the phenotype persistent HPR by ADP is associated with the highest prevalence, even if not statistically significant due to the low number of patients, of *2 variant carriers and with the higher prevalence of elevated BMI; this indirectly confirms the presence of a non-modifiable marker of platelet hyper-reactivity. Differently from Hochholzer et al., our results, obtained in a population of ACS patients followed up for 12 months, indicate that HPR associated with genetic polymorphism is a phenotype stable over time [7]. Also regarding HPR by AA, the group of persistent HPR was associated with diabetes (p = 0.004). Furthermore, regarding diabetes, it is well known that elevated admission of glucose is a powerful risk factor for increased mortality and in-hospital complications in patients with acute myocardial infarction. Our data strongly suggest that in the acute phase of ACS, platelet reactivity might be impaired by the stress hyperglycemia [19]. The group of acute non persistent HPR identifies patients with HPR documented only at the time of the acute event.

Table 6 Characteristics of the Study Population according to persistent, acute non persistent, late and no HPR by AA.

Age median and IQR, (y) Diabetes n, (%) IPF at 6 months follow-up median and IQR, (%) H-IPF at 6 months follow-up median and IQR, (%) IPF at 12 months follow-up median and IQR, (%) H-IPF at 12 months follow-up median and IQR, (%) Reticulated platelets median and IQR Mean platelets volume median and IQR, (%) BMI median and IQR, (Kg/m2)

PERSISTENT HPR by AA, n = 2

ACUTE NON PERSISTENT HPR by AA, n = 16

LATE HPR by AA, n = 13

NO HPR by AA, n = 70

P for trend

75.5 (75.0), 2 (100), *p = 0.004 5.3 (3–5) 1.3 (0.9) 4.55 (3.5) 1.1 (0.9) 11800 (7800) 16,35 (11,5) 26.3 (25.9)

69 (61.3-79.0) 4 (25.0) 2.6 (2.1-4.4) 0.7 (0.5-1.1) 2.5 (1.7-6.48) 0.65 (0.5-2.23) 6150 (2650–12950) 11,8 (10,23-12,38) 25.8 (22.9-27.5)

60 (52.5-74.0) 3 (23.1) 4.1 (2.67-5.93), *p = 0.031 1.25 (0.825-1.85), *p = 0.033 4.1 (3.3-4.7), *p = 0.030 1.3 (0.8-1.5), *p = 0.019 8100 (5600–9700) 11,9 (11,5-13,2) 28 (25.3-29.4)

67 (56–73.3) 12 (17.1) 2.1 (1.5-3.73) 0.6 (0.4-1.2) 2.3 (1.5-3.6) 0.55 (0.3-0.93) 4800 (3275–7825) 11,10 (10,2-12,6) 26.0 (24.7-28.0)

0.157 0.040 0.051 0.093 0.113 0.077 0.125 0.245 0.183

PPI = Proton Pump Inhibitor, IQR = InterQuartile Range, IPF = Immature Platelet Fraction, H-IPF = High Immature Platelet Fraction, BMI = Body Mass Index,*p vs NO HPR.

Please cite this article as: A. Fabbri, et al., A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.040

6

A. Fabbri et al. / Thrombosis Research xxx (2015) xxx–xxx

Table 7 Characteristics of the Study Population according to persistent, acute non persistent, late and no HPR by ADP without diabetic patients. PERSISTENT HPR by ADP, n = 3

ACUTE NON PERSISTENT HPR by ADP, n = 7

Age median and IQR, (y) PPI n, (%) IPF at 6 months follow-up median and IQR, (%)

56 (54) 3 (100) 2.8 (1.9)

67 (60–72) 3 (42.9) 2.6 (1.4-3.2)

H-IPF at 6 months follow-up median and IQR, (%)

0.8 (0.6)

0.4 (0.4-0.7)

IPF at 12 months follow-up median and IQR, (%)

2.8 (1.9)

2.6 (0.9-3.5)

H-IPF at 12 months follow-up median and IQR, (%)

0.7 (0.5)

0.6 (0.2-0.6)

Reticulated platelets median and IQR, (%)

5600 (3400)

Mean platelets volume median and IQR, (f/l)

11.1 (10.7)

11 (9.9-15.9)

Statins n, (%) CYP 2C19 +/− n, (%) BMI median and IQR, (Kg/m2)

3 (100) 1 (33.3) 29.7 (28.5) p = 0.014

7 (100) 2 (28.6) 25.3 (24.4-27.9)

4600 (1900–6475)

LATE HPR by ADP, n = 17 60 (52.5-76.5) 13 (76.5) 4.1 (2.4-5.9) *p = 0.015 1.2 (0.5-1.8) *p = 0.060 4.2 (3.8-4.9), *p = 0.004 1.2 (0.8-1.4), *p = 0.034 8400 (6375–11150) *p = 0.006 12 (11.1-18.5) *p = 0.011 15 (88.2) 1 (5.9) 27.7 (26.0-28.5) *p = 0.023

NO HPR by ADP, n = 53

P for trend

65 (54–72) 38 (71.7) 2.1 (1.5-3.6)

0.857 0.246 0.075

0.5 (0.4-1.1)

0.134

2.2 (1.5-3.7)

0.018

0.6 (0.4-1.1)

0.044

5000 (3300–7375)

0-020

11.1 (10.2-11.7)

0.084*

50 (94.3) 15 (28.3) 26 (24–28)

0.668 0.284 0.010

PPI = Proton Pump Inhibitor, IQR = InterQuartile Range, IPF = Immature Platelet Fraction, H-IPF = High Immature Platelet Fraction, CYP 2C19 = Cytochrome P450 2C19, BMI = Body Mass Index, *p vs NO HPR.

Finally, we have identified a group of late HPR, meaning that patients with HPR only at 6 or 12 months. From a biological point of view, it is difficult to explain the occurrence of platelet reactivity so far from the acute event. The first and more plausible explanation is related to a lack of compliance from patients, meaning that a number of patients interrupted antiplatelet drugs. The patient not following the medications are associated with poor therapeutic outcomes, progression of disease, and an estimated burden of billions per year in avoidable direct health care costs [20]. Approximately 50% of patients with cardiovascular disease are not following their prescribed medications [21]. Recent studies have shown a discrepancy between the recommended therapy and that which is actually received despite of the strong evidence supporting the use of aspirin, clopidogrel, beta-blockers, statins, and ACE inhibitors as secondary preventive medicine following acute myocardial infarction (AMI) [22]. In a study, analyzing 30,028 AMI patients, during outpatient care, 82% of these patients received a beta-blocker, 73% a statin, 69% an ACE inhibitor, 66% aspirin, and 61% clopidogrel after 5 years with the largest decline observed in the first year following the infarction [23]. Nevertheless, an analysis of the parameters associated with this phenotype has shown us that the number of reticulated platelets at 6 and 12 months was significantly associated with late and acute non persistent HPR. Hence, we documented the concomitant presence of a higher number of reticulated platelets with late HPR. We also documented significant higher values of mean platelet volume in patients with late HPR. As the entity of reticulated platelets mirrors an increased turn-over from bone-marrow associated with an inflammatory response, we might speculate that, at least in part, in these patients the occurrence of late HPR is linked to the occurrence of an inflammatory state at 12

months. Based on literature data [24], we know that an inflammatory state may precede or be concomitant with a plaque destabilization leading to an acute vascular event. Our novel finding of an association between HPR and the number of reticulated platelets supports this link between inflammation, enhanced platelet turn over and enhanced platelet reactivity. Excluding diabetic patients, the analysis showed that the association between IPF and high-IPF with platelet hyper-reactivity not only persisted but became stronger than that found in the whole population, suggesting that the increased platelet activation was not only a characteristic of diabetic patients. A limitation of this study is the low number of patients enrolled which does not allow us to achieve, in many cases, statistically significant results. On the other hand, as far as we know, no study is found in the literature documenting the natural history of platelet reactivity by both ADP and AA with a time course of 12 months, in high risk vascular patients such as ACS patients. A second limitation is related to the lack of data on the clinical outcome. Our next step will be to evaluate the possible correlation between these different groups of patients with clinical events, both ischemic and hemorrhagic. 5. Conclusion We have documented the natural history of platelet reactivity by evaluating platelet function on dual antiplatelets by both ADP and AA. We found persistent, acute non persistent and late HPR patients, in relation to the presence of HPR at baseline, 6 or 12 month follow-up. About 25% of patients has persistent HPR by ADP or AA; they were

Table 8 Characteristics of the Study Population according to persistent, acute non persistent, late and no HPR by AA without diabetic patients. PERSISTENT HPR by AA n = 0 Age median and IQR, (y) IPF at 6 months follow-up median and IQR, (%) H-IPF at 6 months follow-up median and IQR, (%) IPF at 12 months follow-up median and IQR, (%) H-IPF at 12 months follow-up median and IQR, (%) Reticulated platelets median and IQR Mean platelets volume median and IQR, (f/l) BMI median and IQR, (Kg/m2)

ACUTE NON PERSISTENT HPR by AA n = 12

LATE HPR by AA n = 10

NO HPR by AA n = 58

P for trend

67.5 (61.3-80.5) 2.5 (2.1-3.6) 0.5 (0.4-0.8) 1.8 (1.7-4.1) 0.6 (0.5-0.95) 5300 (3100–7950) 11.6 (10.0-12.0) 25.9 (24.3-27.2)

59 (50.3-74.8) 4.5 (3.2-5.95), *p = 0.033 1.3 (0.9-1.9), *p = 0.029 4.3 (3.9-4.8), *p = 0.006 1.4 (0.95-1.7), *p = 0.005 8600 (6950–9750) *p = 0.014 11.9 (11.6-15.20) *p = 0.034 27.0 (24.4-29.1)

63 (54.8-72.0) 2.1 (1.5-3.6) 0.6 (0.4-1.2) 2.5 (1.55-3.6) 0.6 (0.45-0.95) 5000 (3300–8050) 11.05 (10.23-12.20) 26.0 (24.9-28.8)

0.279 0.063 0.050 0.020 0.014 0.038 0.107 0.590

PPI = Proton Pump Inhibitor, IQR = InterQuartile Range, IPF = Immature Platelet Fraction, H-IPF = High Immature Platelet Fraction, BMI = Body Mass Index,*p vs NO HPR.

Please cite this article as: A. Fabbri, et al., A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.040

A. Fabbri et al. / Thrombosis Research xxx (2015) xxx–xxx

more frequently diabetics, with elevated BMI or carriers of genetic variants related to altered clopidogrel metabolism. Finally, in addition to non adherence to medication, the occurrence of an inflammatory state – indicated by a higher number of reticulated platelets – is associated with the occurrence of late HPR. Concluding, diabetes, BMI, CYP2C19*2 variant and the number of reticulated platelets are the markers of HPR by both ADP and AA. From a clinical point of view, this implies that in these patients – diabetics, overweight, carriers of *2 variant– platelet function could be taken into account to identify patients who could benefit from a careful clinical follow-up. Finally, an intercurrent inflammatory process, documented by a higher number of reticulated platelets and by a higher values of mean platelet volume, might be associated with an increased platelet reactivity. Hence, a strict surveillance to compliance and antiplatelet treatment in these conditions would be useful to avoid thrombotic complications. Conflict of interest Dr Marcucci reported receiving honoraria for lectures from Daiichi Sankyo/Eli Lilly and Merck Sharp & Dohme. Dr Gensini reported receiving consulting fees from Bayer, Boehringer Ingelheim, and Eli Lilly; lecture fees from AstraZeneca, GlaxoSmithKline, Instrumentation Laboratory, Menarini, and Sigma Tau; and research grant funding from Novo Nordisk, Merck Sharp & Dohme, Pfizer, Pierrel, sanofi-aventis, and Servier. Dr Abbate reported receiving consulting fees from Eli Lilly; lecture fees from Instrumentation Laboratory and Sigma Tau; and research grant funding from Bayer, Boehringer Ingelheim, and Pfizer. No other disclosures were declared. References [1] J.F. Bentzon, F. Otsuka, R. Virmani, E. Falk, Mechanisms of plaque formation and rupture, Circ. Res. 114 (2014) 1852–1866. [2] R. Marcucci, C. Cenci, G. Cioni, A. Lombardi, B. Giusti, G.F. Gensini, Antiplatelets in acute coronary syndrome: personal perspectives, Expert. Rev. Cardiovasc. Ther. 10 (2012) 1487–1496. [3] S.R. Steinhubl, P.B. Berger, J.T. Mann III, E.T. Fry, A. DeLago, C. Wilmer, et al., Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial, JAMA 288 (2002) 2411–2420. [4] S.R. Mehta, S. Yusuf, R.J. Peters, M.E. Bertrand, B.S. Lewis, M.K. Natarajan, 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 358 (2001) 527–533. [5] R. Marcucci, A.M. Gori, R. Paniccia, C. Giglioli, P. Buonamici, D. Antoniucci, et al., Residual platelet reactivity is associated with clinical and laboratory characteristics in patients with ischemic heart disease undergoing PCI on dual antiplatelet therapy, Atherosclerosis 195 (2007) 217–223.

7

[6] R. Marcucci, A.M. Gori, R. Paniccia, B. Giusti, S. Valente, C. Giglioli, et al., High ontreatment platelet reactivity by more than one agonist predicts 12-month followup cardiovascular death and non-fatal myocardial infarction in acute coronary syndrome patients receiving coronary stenting, Thromb. Haemost. 104 (2010) 279–286. [7] W. Hochholzer, C.T. Ruff, R.A. Mesa, J.F. Mattimore, J.F. Cyr, L. Lei, et al., Variability of Individual Platelet Reactivity Over Time in Patients Treated With Clopidogrel: Insights From the ELEVATE–TIMI 56 Trial, J. Am. Coll. Cardiol. 64 (2014) 361–368. [8] G. Campo, G. Parrinello, P. Ferraresi, B. Lunghi, M. Tebaldi, M. Miccoli, et al., Prospective Evaluation of On-Clopidogrel Platelet Reactivity Over Time in Patients Treated With Percutaneous Coronary Intervention: Relationship With Gene Polymorphisms and Clinical Outcome, J. Am. Coll. Cardiol. 57 (2011) 2474–2483. [9] F. Cesari, R. Marcucci, A.M. Gori, R. Caporale, A. Fanelli, G. Casola, et al., Reticulated platelets predict cardiovascular death in acute coronary syndrome patients. Insights from the AMI-Florence 2 Study, Thromb. Haemost. 109 (2013) 846–853. [10] C.W. Hamm, J.P. Bassand, S. Agewall, J. Bax, E. Boersma, H. Bueno, et al., ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation, Eur. Heart J. 32 (2011) 2999–3054. [11] R. Cifkova, S. Erdine, R. Fagard, C. Farsang, A.M. Heagerty, W. Kiowsky, et al., ESH/ESC Hypertension Guidelines Committee Practice guidelines for primary care physicians: 2003 ESH/ESC Hypertension guidelines, J. Hypertens. 21 (2003) 1779–1786. [12] Report of the expert committee on the diagnosis and classification of diabetes mellitus, Diabetes Care 26 (2003) 5–20. [13] Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adult (Adult Treatment Panel III) final report, Circulation 106 (2002) 3143–3421. [14] P.A. Gum, K. Kottke-Marchant, P.A. Welsh, J. White, E.J. Topol, A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease, J. Am. Coll. Cardiol. 41 (2003) 961–965. [15] G. Parodi, R. Marcucci, R. Valenti, A.M. Gori, A. Migliorini, B. Giusti, et al., High residual platelet reactivity after clopidogrel loading and long-term cardiovascular events among patients with acute coronary syndromes undergoing PCI, JAMA 306 (2011) 1215–1223. [16] P. Buonamici, R. Marcucci, A. Migliorini, G.F. Gensini, A. Santini, R. Paniccia, et al., Impact of platelet reactivity after clopidogrel administration on drug-eluting stent thrombosis, J. Am. Coll. Cardiol. 49 (2007) 2312–2317. [17] C. Briggs, S. Kunka, D. Hart, S. Oguni, S.J. Machin, Assessment of an immature platelet fraction(IPF) in peripheral thrombocytopenia, Br. J. Haematol. 126 (2004) 93–99. [18] C. Saracini, A. Vestrini, S. Galora, A. Armillis, R. Abbate, B. Giusti, Pharmacogenetics of clopidogrel: comparison between a standard and a rapid genetic testing, Genet. Test. Mol. Biomarkers 16 (2012) 500–503. [19] M. Kosiborod, S.E. Inzucchi, H.M. Krumholz, L. Xiao, P.G. Jones, S. Fiske, et al., Glucometrics in patients hospitalized with acute myocardial infarction: defining the optimal outcomes-based measure of risk, Circulation 117 (8) (2008) 1018–1027. [20] S.H. Naderi, J.P. Bestwick, D.S. Wald, Adherence to drugs that prevent cardiovascular disease: meta-analysis on 376,162 patients, Am. J. Med. 125 (2012) 882–887. [21] I.M. Kronish, S. Ye, Adherence to cardiovascular medications: lessons learned and future directions, Prog. Cardiovasc. Dis. 55 (2013) 590–600. [22] F. Sofi, A. Fabbri, R. Marcucci, A.M. Gori, D. Balzi, A. Barchielli, et al., Lifestyle modifications after acute coronary syndromes in a subset of the AMI-Florence 2 Registry, Acta Cardiol. 66 (2011) 791–796. [23] S. Mangiapane, R. Busse, Prescription prevalence and continuing medication use for secondary prevention after myocardial infarction: the reality of carerevealed by claims data analysis, Dtsch. Arztebl. Int. 108 (2011) 856–862. [24] M.I. Tomey, J. Narula, J.C. Kovacic, Advances in the understanding of plaque composition and treatment options: year in review, J. Am. Coll. Cardiol. 63 (2014) 1604–1616.

Please cite this article as: A. Fabbri, et al., A time course study of high on treatment platelet reactivity in acute coronary syndrome male patients on dual antiplatelet therapy, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.040