Incomplete Inhibition of Thromboxane Biosynthesis by Acetylsalicylic Acid

Determinants and Effect on Cardiovascular Risk

CHARISMA OCTOBER 2008CIRCULATION

John W. Eikelboom, FRACP, FRCPA; Graeme J. Hankey, MD, FRACP, FRCP; Jim Thom, MSc; Deepak L. Bhatt, MD; P. Gabriel Steg, MD; Gilles Montalescot, MD, PhD; S. Claiborne Johnston, MD, PhD; Steven R. Steinhubl, MD; Koon-Hou Mak, MD, FRCP; J. Donald Easton, MD; Christian Hamm, MD; Tingfei Hu, MS; Keith A.A. Fox, MB, ChB, FRCP, FESC; Eric J. Topol, MD, on behalf of the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management

and Avoidance (CHARISMA) Investigators

Background

• Incomplete inhibition of platelet thromboxane

generation, as measured by elevated urinary 11-dehydro thromboxane B2 concentrations, has been associated with an increased risk of cardiovascular events.

• We aimed to determine the external validity of this association in aspirin-treated patients enrolled in the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance (CHARISMA)

Background

• trial and to determine whether there are any modifiable factors or interventions that lower urinary 11-dehydro thromboxane B2 concentrations that could thereby reduce cardiovascular risk.

• Acetylsalicylic acid (ASA) reduces the risk of serious cardiovascular events by 20% in a broad range of high-risk patients.The primary effect of ASA on hemostasis is to acetylate platelet cyclooxygenase (COX)-1 and thereby inhibit the synthesis of thromboxane A2, a powerful platelet agonist.

• Acetylation of platelet COX-1 by ASA is rapid, irreversible, and permanent (for the life of the platelet), because platelets lack the biosynthetic machinery necessary to synthesize new protein, and it is believed to be saturable at low doses.

• doses. Acetylation of platelet COX-1 does not attain functional relevance until the maximal capacity to generate thromboxane A2 is reduced by at least 95%.

• However, very small amounts of residual COX-1 activity can generate sufficient amounts of thromboxane to support thromboxane-dependent platelet function. Thus, as much as 99% inhibition of serum thromboxane may be necessary to optimally inhibit platelets.

• Some patients treated with ASA continue to generate thromboxane A2 and thereby activate platelets.

• Possible mechanisms of continued thromboxane generation despite ASA treatment include

• poor compliance with ASA treatment,• inadequate ASA dose,• concomitant use of other COX inhibitors that interfere

with the antiplatelet effects of ASA, • increased rate of platelet turnover, transcellular

metabolism of prostaglandin precursors, and true "resistance" of COX-1 to the inhibitory effects of ASA (eg, due to a genetic polymorphism of the COX-1 gene).

• Continued thromboxane production despite ASA therapy, as manifested by elevated concentrations of 11-dehydro thromboxane B2 (a stable metabolite of thromboxane A2) in the urine, has been associated with an increased risk of serious cardiovascular events in 1

study of high-vascular-risk patients.16 However, this finding has not been validated externally in an independent data set. If it were externally validated, knowledge of the determinants of elevated concentrations of 11-dehydro thromboxane B2 could

generate new interventions to lower concentrations of 11-dehydro thromboxane B2 and thereby reduce cardiovascular risk.

• n this prespecified substudy of the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance (CHARISMA) trial, we aimed to prospectively verify the hypothesis that incomplete suppression of thromboxane generation with usual doses of ASA, as measured by elevated concentrations of 11-dehydro thromboxane B2 in the urine, is associated with an increased risk of subsequent serious vascular events.

• We also aimed to identify the independent and significant determinants of urinary 11-dehydro thromboxane B2 concentrations, as well as whether the addition of an ADP receptor antagonist, clopidogrel, to ASA would reduce ASA-insensitive thromboxane biosynthesis and

thereby improve survival free of cardiovascular events.

Methods

• CHARISMA was a multinational, multicenter,

randomized, parallel-group, double-blind trial of clopidogrel versus placebo in high-risk patients at risk of atherothrombotic events.

• The institutional review committee at each participating center approved the study, and all subjects gave informed consent. A total of 15 603 patients with either clinically established

cardiovascular disease or multiple risk factors were randomly assigned to receive clopidogrel (75 mg/d) or placebo.

• All patients received ASA (75 to 162 mg/d). Patients were followed up for a median of 28 months.

• The primary efficacy outcome for the

CHARISMA trial was a composite of stroke, myocardial infarction (MI), and cardiovascular death.

Patients

• A total of 3261 patients from 224 sites in 12 countries complied with a request to provide a first-morning-urine specimen at least 1 month after randomization.

• A minimum 1-month interval between randomization and urine collection ensured steady state suppression of thromboxane generation and reduced the likelihood of recruiting a patient soon after an acute atherothrombotic event, which might be a cause of platelet activation.

Follow-Up and Ascertainment of Clinical Outcomes

• Patients were followed up prospectively at 1, 3, and 6 months after randomization and every 6 months thereafter until the trial was completed. At each follow-up, we recorded use of medications, including ASA dose, and clinical outcomes.

• The primary outcome was the composite of stroke, MI, and cardiovascular death.

• The urine samples were thawed and assayed for 11-dehydro thromboxane B2 with a commercially available enzyme immunoassay (Cayman

Chemical, Ann Arbor, Mich). • This assay has interassay and intra-assay

coefficients of variation of 12.1% and 10%, respectively. Control urine samples with assigned values for 11-dehydro thromboxane B2 were run with each batch (kindly supplied by AspirinWorks,

Broomfield, Colo).

Baseline Characteristics

All Patients* (n=15 603)

Stroke, MI, or CV Death (n=144)

No Stroke, MI, or CV Death (n=3117)

Age, median, y 64.0 69.0 64.0

Female sex, n (%) 4644 (29.8) 39 (27.1) 870 (27.9)

BMI, kg/m2

Mean (SD) 27.9 (5.1) 27.6 (4.8) 28.7 (5.2)

Median 27.1 27.9

Inclusion subgroup, n (%) Documented vascular disease

12 153 (77.9) 117 (81.3) 2349 (75.4)

Multiple risk factors

3284 (21.0) 26 (18.1) 747 (24.0)

Smoking status, n (%)

Baseline Characteristics

All Patients* (n=15 603)

Stroke, MI, or CV Death (n=144)

No Stroke, MI, or CV Death (n=3117)

Age, median, y 64.0 69.0 64.0

Female sex, n (%) 4644 (29.8) 39 (27.1) 870 (27.9)

BMI, kg/m2

Mean (SD) 27.9 (5.1) 27.6 (4.8) 28.7 (5.2)

Median 27.1 27.9

Inclusion subgroup, n (%)

Documented vascular disease

12 153 (77.9) 117 (81.3) 2349 (75.4)

Multiple risk factors

3284 (21.0) 26 (18.1) 747 (24.0)

Smoking status, n (%)

Current 3155 (20.2) 26 (18.1) 632 (20.3)

Former 7613 (48.8) 76 (52.8) 1547 (49.6)

MI 5397 (34.6) 52 (36.1) 1082 (34.7)

TIA 1864 (11.9) 17 (11.8) 303 (9.7)

Stroke 3837 (24.6) 57 (39.6) 749 (24.0)

PAD 3531 (22.6) 38 (26.4) 670 (21.5)

PCI 3554 (22.8) 30 (20.8) 748 (24.0)

CABG surgery 3079 (19.7) 34 (23.6) 568 (18.2)

Carotid endarterectomy

825 (5.3) 12 (8.3) 140 (4.5)

Angioplasty / bypass

1737 (11.1) 20 (13.9) 322 (10.3)

MedicationsAll Patients* (n=15

603)Stroke, MI, or CV

Death (n=144)No Stroke, MI, or CV

Death (n=3117)ASA

ASA treatment, n (%)

15 552 (99.7) 144 (100) 3114 (99.9)

ASA dose in mg, median

100.0 81.0 81.0

ASA <100 mg/d, n (%)

7269 (46.7) 90 (62.5) 1,928 (61.9)

ASA 100–149 mg/d, n (%)

4984 (32.0) 28 (19.4) 737 (23.7)

ASA 150 mg/d, n (%)

3299 (21.2) 26 (18.1) 449 (14.4)

Clopidogrel, n (%)

Study clopidogrel 7802 (50.0) 64 (44.4) 1536 (49.3)

Any clopidogrel 8574 (55.0) 105 (72.9) 1683 (54.0)

NSAIDS, n (%) 3378 (21.6) 42 (29.2) 749 (24.0)

Treated Not Treated PRandomized clopidogrel treatment, n

1600 1661

11-Dehydro thromboxane B2, ng/mmol creatinine Median (Q1, Q3) 57.1 (40, 90) 58.5 (40, 92) 0.50

Minimum, maximum

0.5, 3509 7.1, 3741

Log-transformed mean (SD)

4.2 (0.76) 4.2 (0.72)

Geometric mean 63.6 64.1

NSAID, n 791 2470

11-Dehydro thromboxane B2, ng/mmol creatinine

ASA Dose

P<100 mg/d 100–149 mg/d 150 mg/dNo. of subjects 2018 765 475

11-Dehydro thromboxane B2, ng/mmol creatinine Median (Q1, Q3)

58.7 (40, 92) 59.8 (42, 93) 50.3 (36, 80) <0.001

Minimum, maximum

0.5, 3509 2.8, 3741 12.5, 694

Log-transformed mean (SD)

4.2 (0.75) 4.2 (0.75) 4.0 (0.66)

Geometric mean

64.8 66.5 56.2

11-Dehydro Thromboxane B2

Stroke, MI, or CV Death (n=144)

No Stroke, MI, or CV Death (n=3117)

Median (Q1, Q3) 72.7 (41, 135) 57.4 (40, 90)

Minimum, maximum 12, 1235 0.5, 3741

Log-transformed mean (SD) 4.4 (0.96) 4.1 (0.73)

Geometric mean 84.1 63.1

CV indicates cardiovascular; Q, quartile.

Baseline Characteristic OR 95% CIConcomitant statin use

0.72 0.61–0.87

Hypercholesterolemia 0.74 0.63–0.88

Concomitant NSAID use

0.78 0.67–0.90

Average ASA dose (per 50 mg/d) until urine sample collected

0.78 0.71–0.87

Age, in 10-year increments

1.19 1.11–1.27

Concomitant ACE inhibitor use

1.22 1.07–1.38

Concomitant use of oral hypoglycemic drugs

1.42 1.24–1.62

Discussion

• The principal findings were as follows: (1) values of urinary 11-dehydro thromboxane B2 concentration in the upper quartile in a broad population of high-risk patients treated with usual doses of ASA (75 to 162 mg/d) were independently associated with an increased risk of serious cardiovascular events; (2) increasing age, female sex, history of peripheral artery disease, current smoking, and oral hypoglycemic or angiotensin-converting enzyme inhibitor therapy were independently associated with higher concentrations of 11-dehydro thromboxane B2 in the urine, whereas ASA dose 150 mg/d, history of treatment with NSAIDs, history of hypercholesterolemia, and statin treatment were associated with lower concentrations; and (3) randomization to clopidogrel (versus placebo) did not reduce urinary 11-dehydro thromboxane B2 levels or reduce the hazard of cardiovascular events in patients in the highest quartile of urinary 11-dehydro thromboxane B2

levels.