1. Effect of Aspirin Dose on Platelet Reactivity in Diabetic Patients Effect of Aspirin Dose on Platelet Reactivity in Diabetic Patients Paul A. Gurbel, MD Director, Sinai Center for Thrombosis Research Sinai Hospital of Baltimore Associate Professor of Medicine Johns Hopkins University School of Medicine Baltimore, MD

2. Background 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.

3. Objectives 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.

4. Methods- Subjects 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 4.0 mg/dL. - Current use of non-steroidal anti-inflammatory drugs, anticoagulants, or antiplatelet drugs other than aspirin.

5. Methods-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-TxB 2 (AspirinWorks (Denver, CO) - Levels were estimated using Enzyme Linked Immunoassay (ELISA) and reported as pg 11-dehydro-TxB 2 /mg creatinine.

6. Methods- 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-TxB 2 /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.

7. Patient Demographics

8. Results 27 14 27 4 17 10 33 23 4 3 0 0 5 10 15 20 25 30 35 40 DiabeticNon-DiabeticDiabeticNon-Diabetic Resistance (%) 81mg ASA 162mg ASA 325mg ASA * + * + * * p  0.05 for within group comparison +p  0.05 for between group comparison 100 80 60 40 20 0 Platelet Aggregation (%) 81 162 325 81 162325 81 162 325 mg mg mg Diabetic Non-Diabetic Diabetic Non-Diabetic 2mM AA-Induced LTA 5mM AA-Induced LTA Resistance Resistant pt #1 Resistant pt #2 100 80 60 40 20 0 Platelet Aggregation (%) 81 162 325 81 162 325 mg mg mg mg mg mg Diabetic Non-Diabetic 2mM AA-Induced LTA5mM AA-Induced LTA Resistance Resistant pt #1 Resistant pt #2 81 162 325 81 162 325 mg mg mg mg mg mg Diabetic Non-Diabetic

9. Results 27 14 27 4 17 10 33 23 4 3 0 0 5 10 15 20 25 30 35 40 DiabeticNon-DiabeticDiabeticNon-Diabetic Resistance (%) 81mg ASA 162mg ASA 325mg ASA * + * + * * p  0.05 for within group comparison +p  0.05 for between group comparison

10. Results 27 14 27 4 17 10 33 23 4 3 0 0 5 10 15 20 25 30 35 40 DiabeticNon-DiabeticDiabeticNon-Diabetic Resistance (%) 81mg ASA 162mg ASA 325mg ASA * + * + * * p  0.05 for within group comparison +p  0.05 for between group comparison

11. Results 27 14 27 4 17 10 33 23 4 3 0 0 5 10 15 20 25 30 35 40 DiabeticNon-DiabeticDiabeticNon-Diabetic Resistance (%) 81mg ASA 162mg ASA 325mg ASA * + * + * * p  0.05 for within group comparison +p  0.05 for between group comparison

12. Results 27 14 27 4 17 10 33 23 4 3 0 0 5 10 15 20 25 30 35 40 DiabeticNon-DiabeticDiabeticNon-Diabetic Resistance (%) 81mg ASA 162mg ASA 325mg ASA * + * + * * p  0.05 for within group comparison +p  0.05 for between group comparison

13. Comparison Between Groups at Specific Dose 27 14 27 4 17 10 33 23 4 3 0 0 5 10 15 20 25 30 35 40 DiabeticNon-DiabeticDiabeticNon-Diabetic Resistance (%) 81mg ASA 162mg ASA 325mg ASA * + * + * * p  0.05 for within group comparison +p  0.05 for between group comparison Diabetic (n=30) Non-Diabetic (n=90) Diabetic vs. Non-Diabetic (p-Value ) 81mg162mg325mg81mg162mg325mg 81 mg 162 mg 325 mg AA Stimulated Assays 2mM AA-LTA (%) 7±184±25±34±1.54±64±1 NS 5mM AA-LTA (%) 8±227±135±45±84±25±4 NS Accumetrics (ARU) 470±66448±57426±45445±48429±38428±430.020.04NS Other Assays 5μm ADP-LTA (%) 64±1360±961±357±12 57±9 0.01NS0.05 2μg/mL Collagen- LTA (%) 54±2333±2129±1631±2327±2027±17 <0.001NS PFA-100 (sec.) 208±69251±67231±73226±74257±63245±65 NS Urinary Thromboxane (pg/mg creatinine) 413±110332±106302±112331±136315±145298±148 0.02NS

14. Evaluation of Dose Effect within Groups 27 14 27 4 17 10 33 23 4 3 0 0 5 10 15 20 25 30 35 40 DiabeticNon-DiabeticDiabeticNon-Diabetic Resistance (%) 81mg ASA 162mg ASA 325mg ASA * + * + * * p  0.05 for within group comparison +p  0.05 for between group comparison Diabetic (n=30) Non-Diabetic (n=90) 81mg162mg325mg81mg162mg325mg AA Stimulated Assays 2mM AA-LTA (%) 7±184±25±34±24±64±1 5mM AA-LTA (%) 8±227±135±45±84±25±4 Accumetrics (ARU) 470±66448±57426±45 + 445±48429±38*428±43 + Other Assays 5μm ADP-LTA (%) 64±1360±961±357±12 57±9 2μg/mL Collagen-LTA (%) 54±2333±21*29±16 + 31±2327±2027±17 PFA-100 (sec,) 208±69251±67*231±73226±74257±63*245±65 Urinary Thromboxane (pg/mg creatinine) 413±110332±106*302±112 + 331±136315±145298±148 * p  0.02 for 81mg vs 162 mg + p  0.02 for 81mg vs 325 mg No dose effect observed between 162mg vs 325mg

15. Conclusion 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.