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Detection and Monitoring of Aspirin (ASA) Inhibition of Platelet Function Using the Platelet Aggregation Assay (PAA™)

By KA Schwartz*, DE Schwartz*, SL Mantz, G Ens and JM Davis*. * Michigan State University, East Lansing MI and Colorado Coagulation Consultants, Denver, CO. First presented in 1998 at the American Society of Hematology Meeting as a Poster and Abstract.

Abstract: Acetylsalicylic acid (ASA) because of its platelet inhibitory effect is frequently utilized to treat and or prevent atherosclerotic vaso-occlusive disease. We used a platelet aggregation assay (PAA) to evaluate the reliability of Platelet Aggregation Assay (PAA), Analytical Control Systems, Inc. to detect and monitor the amount of platelet inhibition induced by ASA. Platelet rich plasma from 20 normal donors, who had not taken ASA or other known platelet inhibitors for at least 2 weeks, was evaluated before and 24 hours after ingestion of 325mg of ASA. Using 30uM PAA, the slope of the PAA curve completely differentiated aspirinated from normal platelets. ASA platelet slope, mean=18.4+/-3.7 (range = 15.5-40), was significantly decreased. p<0.05 compared to pre-ASA mean = 75.5+/-3.6 (range=50-125). Additionally, time to 50% platelet aggregation with ASA, mean =8.2+/-0.6min (range= 4.7-12.4) was significantly prolonged, p<0.05 compared to pre-ASA mean = 3.8+/-0.2 min. (range = 1.7-6.0). ADP tested at 2.5, 5, 10 & 20 uM failed to discriminate between aspirinated and normal platelets. Colorado Coagulation Consultants independently evaluated the sensitivity of PAA to the long-term platelet inhibitory effects of ASA. 9 normal volunteers were evaluated with 30 uM PAA™ 2, 8, 24, 48 and 96 hours after a single dose of ASA 81 or 325mg. Compared to pre-ASA slope mean = 79.6+/-1.9, the maximal decrease in slope occurred after 2 hours for both 81mg ASA mean = 61.3+/-6.7 and 325mg ASA mean = 12.1+/-1.8. ASA significantly decreased the slope and prolonged the time to 50% aggregation for all time intervals through 48 hours with 81 mg ASA and through 72 hrs with 325mg ASA, p<0.05. The decreased slopes and increased time to 50% aggregation observed at 2, 8 and 24 hours, p<0.05, reflected the greater degree of platelet inhibition with 325 mg as compared to 81mg ASA. We conclude that the PAA detects: 1) the difference between aspirinated and normal platelets with a sensitivity of 100%. 2) the inhibitory effects of a single dose of ASA 72 hours after ingestion, and 3) the greater platelet inhibitory effects of a single dose of 325 mg of ASA when compared to 81 mg ASA. The PAA™ may be useful in more effectively monitoring the anti-platelet activity of ASA (more)

Introduction: The inhibitory effect of acetyl salicylic acid, aspirin, (ASA) on platelet function is used to help prevent occlusive vascular disease. ASA inhibition of platelets is mediated via blockade of the cyclooxygenase enzyme required for production of prostaglandins. Aspirin inhibits platelet production of prostaglandin A2, a strong vasoconstrictor and stimulator of platelet aggregation.

Current clinical tests that measure platelet function include the template bleeding time and the platelet aggregation response to a standard set of platelet agonists as measured by platelet aggregometry. Because these assays do not reliably detect ASA induced platelet inhibition, we investigated a new platelet agonist, Platelet Aggregation Assay to test the hypothesis that the Platelet Aggregometry Assay (PAA™) can detect the effect of ASA on platelet aggregation. Our results show the PAA™ can reliably differentiate normal from ASA treated platelets with a sensitivity of 100 percent.

Methods: After signed informed consent blood from normal donors was drawn into citrate anticoagulant, 3.2%. After centrifugation, 200xG for 10 minutes, platelet rich plasma was harvested from the top layer. Platelet aggregation responses were measured in a Chrono-Log aggregometer. The final concentration of Platelet Aggregation Assay used in the aggregometer cuvette was 30uM.

Summary:

1. One day after ingestion of a single 325mg ASA the slope of the PAA tracing was significantly decreased allowing complete differentiation from normal platelets (100 percent sensitive).
2. The inhibitory effect of a single dose of 325mg ASA was detectable 72 hours after ingestion.
3. The inhibitory effect of a single dose of 81mg of ASA was detectable 48 hours after ingestion.
4. PAA™ detected the stronger inhibition of platelet function produced by a single dose of 325mg ASA as compared to the smaller dose of 81mg ASA.
5. Maximal inhibition of platelet function produced by 14 days of 325mg per day ASA produced a dramatic decrease in the slope of the PAA™ that reflected the stronger inhibition of platelet function produced by 14 days of ASA when compared to a single dose.

Conclusions:

1. Platelet Aggregometry Assay (PAA™) reliably detects the inhibitory effect of ASA on platelet function with a sensitivity of 100 percent.
2. PAA™ is sensitive to the increased inhibition of platelet function by three different regimens of increasing amounts of ASA (single doses of 81mg and 325mg and 14 days of 325mg ASA).

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