Effect of Aspirin Dose on Effect of Aspirin Dose on Platelet Reactivity in Platelet Reactivity in

Diabetic Patients Diabetic Patients

Paul A. Gurbel, MDDirector, Sinai Center for Thrombosis Research

Sinai Hospital of Baltimore Associate Professor of Medicine

Johns Hopkins University School of Medicine Baltimore, MD

BackgroundBackground• Diabetes is a “prothrombotic state” characterized by accelerated atherosclerosis and inflammation.

• Diabetic patients with acute coronary syndromes (ACS) are at higher risk for recurrent ischemic events compared to non-diabetic ACS patients.1

• Aspirin treatment in diabetic patients is less effective in inhibiting platelet thromboxane synthesis compared to aspirin treatment in non-diabetics.2

• In the primary prevention project (PPP), a higher cardiovascular risk was observed in diabetic patients on low dose aspirin compared to non-diabetic patients, suggesting that the benefits of aspirin therapy may be outweighed by aspirin insensitive mechanisms of platelet activation.3

• There are limited data quantifying the prevalence of platelet aspirin resistance in diabetic patients.

• No study has prospectively analyzed the effect of varying aspirin doses on platelet function in both diabetics and non-diabetics using multiple methods.

• Antithrombotic Trialists' Collaboration. Collaborative meta-analysis of randomized trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002;324:71-86

• Watala C. Blood platelet reactivity and its pharmacological modulation in people with diabetes mellitus. Current Pharmaceutical Design. 2005;11:2331-2365.

3. Sacco M et al. Primary prevention of cardiovascular events with low-dose aspirin and vitamin E in type 2 diabetic patients: results of the Primary Prevention Project (PPP) trial. Diabetes Care. 2003;12:3264-72.

ObjectivesObjectives

• To determine whether diabetic patients with coronary artery disease during therapy with 81mg daily aspirin will exhibit a higher prevalence of aspirin resistance and a state of high platelet reactivity to multiple agonists compared to non- diabetics.

• To determine whether higher daily aspirin doses (162 or 325mg/day) will reduce platelet reactivity to multiple agonists and decrease the prevalence of aspirin resistance in diabetic patients.

MethodsMethods- - SubjectsSubjects

• Stable CAD outpatients (n=120) from the ASpirin-Induced Platelet EffeCT (ASPECT) study were examined.

- Random double blind drug treatment sequence of 81,162, and 325mg/day for 4 weeks each over a 12 week period (Williams design). - Diabetes (n=30) was defined as fasting glucose 126mg/dL or treatment with oral hypoglycemic agents insulin.

• Exclusion Criteria:

- Hx of gastrointestinal bleeding, hemorrhagic stroke, illicit drug or alcohol abuse, coagulopathy or major surgery within 6 weeks prior to randomization.

- Plt < 100,000/mm3, Hct< 30%, Cr >4.0 mg/dL.

- Current use of non-steroidal anti-inflammatory drugs, anticoagulants, or antiplatelet drugs other than aspirin.

• Stable CAD outpatients (n=120) from the ASpirin-Induced Platelet EffeCT (ASPECT) study were examined.

- Random double blind drug treatment sequence of 81,162, and 325mg/day for 4 weeks each over a 12 week period (Williams design). - Diabetes (n=30) was defined as fasting glucose 126mg/dL or treatment with oral hypoglycemic agents insulin.

• Exclusion Criteria:

- Hx of gastrointestinal bleeding, hemorrhagic stroke, illicit drug or alcohol abuse, coagulopathy or major surgery within 6 weeks prior to randomization.

- Plt < 100,000/mm3, Hct< 30%, Cr >4.0 mg/dL.

- Current use of non-steroidal anti-inflammatory drugs, anticoagulants, or antiplatelet drugs other than aspirin.

MethodsMethods-Platelet Testing-Platelet Testing• Light Transmittance Aggregometry - Maximum % change in light transmittance using platelet poor plasma as a reference. - Platelets were stimulated with 2 and 5mM arachidonic acid (AA), 5uM adenosine diphosphate (ADP) and 2ug/mL collagen.

• VerifyNow™ (Accumetrics, San Diego, CA) - Measures platelet aggregation to fibrinogen-coated beads after stimulation with arachidonic acid and reports this value in aspirin reaction units (ARU).

• PFA-100 (Dade-Behring, West Sacramento, CA)

- Uses a test cartridge containing a collagen/epinephrine coated

membrane to measure the closure time (seconds) required for platelets

to aggregate and arrest blood flow through an aperture.

• Urinary 11-dehydro-TxB2 (AspirinWorks (Denver, CO)

- Levels were estimated using Enzyme Linked Immunoassay (ELISA)

and reported as pg 11-dehydro-TxB2/mg creatinine.

MethodsMethods- Definitions- Definitions

Aspirin Resistance

> 20% AA-, > 70% ADP- and > 70% collagen-induced platelet aggregation.1

550 ARU (VerifyNow).

< 193 seconds to closure (PFA-100).

Upper quartile (>420 pg 11-dehydro-TxB2/mg creatinine) during treatment with 81mg.2

Aspirin Resistance

> 20% AA-, > 70% ADP- and > 70% collagen-induced platelet aggregation.1

550 ARU (VerifyNow).

< 193 seconds to closure (PFA-100).

Upper quartile (>420 pg 11-dehydro-TxB2/mg creatinine) during treatment with 81mg.2

1. Gum PA et al. J Am Coll Cardiol. 2003;41:961-965. 2. Eikelboom JW et al. Circulation 2002;105:1650-1655.

Patient DemographicsPatient Demographics

Total

Group (n=120)

Diabetic Group (n=30)

Non-Diabetic Group (n=90)

Diabetic vs. Non-

Diabetic (p-value)

Age (years) 6510 649 66±11 0.38 Gender and Ethnicity n, (%) Male 65 (65) 18 (60) 47 (67) 0.33 Caucasian 73 (73) 22 (73) 51 (73) 1.0 Weight (lbs) 195±52 210±55 192±50 0.11 Risk Factors/Past Medical History n,(%) Current Smoking 8 (8) 2 (7) 6 (9) 0.74 Previous Smoking 30 (30) 10 (33) 20 (29) 0.69 Family History of Coronary Artery Disease 38 (38) 13 (43) 25 (36) 0.51 Hypertension 65 (65) 22 (73) 43 (61) 0.25

Prior Myocardial Infarction 18 (18) 6 (20) 12 (17) 0.37 Prior Coronary Artery Bypass Grafting 31 (31) 9 (30) 22 (31) 0.13

Prior Coronary Angioplasty 33 (33) 10 (33) 23 (33) 1.0 Prior Cerebrovascular Accident 6 (6) 5 (17) 1 (1) 0.002 Baseline Medications n, (%) Beta Blockers 56 (56) 18 (60) 38 (54) 0.58 ACE Inhibitors 52 (52) 19 (63) 33 (47) 0.15 Calcium Channel Blockers 19 (19) 5 (17) 14 (20) 0.72 Lipid Lowering Therapy 76 (76) 23 (77) 53 (76) 0.91 Laboratory Data

White Blood Cell Count (x 1000/mm3) 6.4±2.2 6.3±2.5 6.4±1.9 0.82 Platelets (x 1000/mm3) 225±67 208±71 229±63 0.14 Hemoglobin (g/dL) 13.3±2.0 12.8±3.6 13.6±2.2 0.17 Hematocrit (%) 40.8±5.1 38.2±11.1 41.2±5.2 0.09 Creatinine (g/dL) 1.1±0.6 1.1±0.2 1.2±0.7 0.45 Total Cholesterol 165±41 155±43 170±44 0.12 Low Density Lipoprotein 95±36 91±37 98±38 0.40 High Density Lipoprotein 50±12 45±13 51±11 0.02

ResultsResults

100

80

60

40

20

0

Pla

tele

t A

gg

reg

atio

n (

%)

81 162 325 81 162 325 81 162 325 81 162 325mg mg mg mg mg mg mg mg mg mg mg mg

Diabetic Non -Diabetic Diabetic Non -Diabetic

2mM AA -Induced LTA 5mM AA -Induced LTA

Resistance

Resistant pt #1Resistant pt #2

100

80

60

40

20

0

Pla

tele

t A

gg

reg

atio

n (

%)

81 162 325 81 162 325 mg mg mg mg mg mg Diabetic Non-Diabetic

2mM AA-Induced LTA 5mM AA-Induced LTA

Resistance

Resistant pt #1Resistant pt #2

81 162 325 81 162 325 mg mg mg mg mg mg Diabetic Non-Diabetic

ResultsResults

27

14

27

4

17

10

3 3

23

4 3

00

5

10

15

20

25

30

35

40

Diabetic Non-Diabetic Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA

162mg ASA

325mg ASA

*+

* +*

*p0.05 for within group comparison+p0.05 for between group comparison

5uM ADP-Induced LTA 2ug/mL Collagen-Induced LTA

27

14

27

4

17

10

3 3

23

4 3

00

5

10

15

20

25

30

35

40

Diabetic Non-Diabetic Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA

162mg ASA

325mg ASA

*+

* +*

*p0.05 for within group comparison+p0.05 for between group comparison

27

14

27

4

17

10

3 3

23

4 3

00

5

10

15

20

25

30

35

40

Diabetic Non-Diabetic Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA

162mg ASA

325mg ASA

*+

* +*

*p0.05 for within group comparison+p0.05 for between group comparison

5uM ADP-Induced LTA 2ug/mL Collagen-Induced LTA

ResultsResults

13

33 334

0

2

4

6

8

10

12

14

16

18

20

Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA162mg ASA325mg ASA

VerifyNow Aspirin Assay

+

+p0.05 for between group comparison

13

33 334

0

2

4

6

8

10

12

14

16

18

20

Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA162mg ASA325mg ASA

VerifyNow Aspirin Assay

+

+p0.05 for between group comparison

ResultsResults

41 40

1714

27 29

0

10

20

30

40

50

60

70

Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA

162mg ASA

325mg ASA

PFA-100

**

*p0.05 for within group comparison

41 40

1714

27 29

0

10

20

30

40

50

60

70

Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA

162mg ASA

325mg ASA

PFA-100

**

*p0.05 for within group comparison

ResultsResults

37

17

23

1316 16

0

10

20

30

40

50

60

Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA162mg ASA

325mg ASA

*p0.05 for within group comparison+p0.05 for between group comparison

Urinary Thromboxane

* +

37

17

23

1316 16

0

10

20

30

40

50

60

Diabetic Non-Diabetic

Re

sis

tan

ce

(%

)

81mg ASA162mg ASA

325mg ASA

*p0.05 for within group comparison+p0.05 for between group comparison

Urinary Thromboxane

* +

Comparison Between Groups at Specific Dose

Diabetic (n=30)

Non-Diabetic (n=90)

Diabetic vs.

Non-Diabetic

(p-Value)

81mg 162mg 325mg 81mg 162mg 325mg 81 mg

162mg

325mg

AA Stimulated Assays

2mM AA-LTA (%) 7±18 4±2 5±3 4±1.5 4±6 4±1 NS NS NS

5mM AA-LTA (%) 8±22 7±13 5±4 5±8 4±2 5±4 NS NS NS

Accumetrics (ARU) 470±66 448±57 426±45 445±48 429±38 428±43 0.02 0.04 NS

Other Assays

5μm ADP-LTA (%) 64±13 60±9 61±3 57±12 57±12 57±9 0.01 NS 0.05

2μg/mL Collagen-LTA (%)

54±23 33±21 29±16 31±23 27±20 27±17 <0.001 NS NS

PFA-100 (sec.) 208±69 251±67 231±73 226±74 257±63 245±65 NS NS NS

Urinary Thromboxane

(pg/mg creatinine) 413±110 332±106 302±112 331±136 315±145 298±148 0.02 NS NS

Evaluation of Dose Effect within Groups

Diabetic (n=30)

Non-Diabetic (n=90)

81mg 162mg 325mg 81mg 162mg 325mg

AA Stimulated Assays

2mM AA-LTA (%) 7±18 4±2 5±3 4±2 4±6 4±1

5mM AA-LTA (%) 8±22 7±13 5±4 5±8 4±2 5±4

Accumetrics (ARU) 470±66 448±57 426±45+ 445±48 429±38* 428±43+

Other Assays

5μm ADP-LTA (%) 64±13 60±9 61±3 57±12 57±12 57±9

2μg/mL Collagen-LTA (%) 54±23 33±21* 29±16+ 31±23 27±20 27±17

PFA-100 (sec,) 208±69 251±67* 231±73 226±74 257±63* 245±65

Urinary Thromboxane(pg/mg creatinine) 413±110 332±106* 302±112+ 331±136 315±145 298±148

*p0.02 for 81mg vs 162 mg

+p0.02 for 81mg vs 325 mg

No dose effect observed between 162mg vs 325mg

ConclusionConclusion

• Low dose aspirin may not provide adequate platelet inhibition in diabetic patients.

• In general, increasing the dose of aspirin in the diabetic patient reduces the prevalence of resistance to a level observed in the non- diabetic patient.

• However, we did not observe a dose dependent effect of aspirin on resistance measured by ADP-induced aggregation in diabetic patients suggesting a potential benefit of using ADP receptor blockers in addition to higher dose aspirin in selected diabetic patients.

• Future large scale studies are needed to evaluate the clinical efficacy of higher dose aspirin in diabetic patients.

• The dose dependent effects of aspirin suggest that the antithrombotic properties of the drug are not all explained by COX-1 inhibition.