Serum Thromboxane B2 Assay as a Measure of Platelet Production in Healthy Volunteers Taking Aspirin
Phase: NA
Conditions: Aspirin
Sponsor: Hamilton Health Sciences Corporation

Proposal 
Serum thromboxane B2 Assay as a Measure of Platelet Production in Healthy Volunteers 
Taking Aspirin 
 
Short study name: COX-1 and TxB2 
 
Principal Investigator: Dr Noel Chan1 
Laboratory Investigator: Dr Geoffrey Werstuck1,  
Co-investigators: Dr Jack Hirsh2, Dr John Eikelboom1,2,3, Dr Jeffrey Ginsberg1,2, Dr Ke Xu3, Dr 
Vinai Bhagirath2,3, Dr Paul Kruger3. 
 
 
Affiliations: 
1 Thrombosis and Atherosclerosis Research Institute (TaARI),  
2 Department of Medicine, McMaster University  
3 Population Health Research Institute, Hamilton Health Sciences McMaster University  
 
 
 

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Overall objective:  
To develop a valid method to quantify platelet production (without the use of radioactive 
isotopes) in order to examine the hypothesis that enhanced platelet production is a common 
cause of poor aspirin responsiveness in patients with atherothrombosis.  
 
Specific objective:  
To validate the Cayman Chemical serum thromboxane immunoassay as a measure of platelet 
production in healthy subjects who are treated with aspirin by demonstrating that the recovery 
of their serum thromboxane B2 levels occurs at a rate of about 10% per day after aspirin 
cessation.  
 
Background and Rationale:  
Cardiovascular disease is the most common cause of preventable death globally. Given once-
daily, low dose aspirin reduces the risk of major cardiovascular events (MACE) by 25% in patients 
with atherothrombosis1. By irreversibly acetylating cyclooxygenase-1 (COX-1) in platelets, aspirin 
blocks the production of thromboxane (TX) A2, a potent stimulator of platelet aggregation, 
thereby mediating its anti-thrombotic effect. Despite its rapid clearance (30 minutes) from the 
circulation, the effect of aspirin on the inhibition of TXA2 synthesis lasts for the lifespan of the 
platelet, which is about 10 days2. In healthy subjects, a single 100-mg dose of aspirin inhibits 
almost 100 percent of TXA2 synthesis within 30 to 60 minutes of its administration. TXA2 synthesis 
then recovers slowly as the aspirin is cleared and new platelets with non-acetylated COX-1 
(‘unacetylated’ platelets) are released from the bone marrow. Since only about 10% of platelets 

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are replaced daily by new platelets entering the circulation, at 24 hours after a dose of aspirin, 
TXA2 synthesis is still suppressed by > 90%. At this level of suppression, TXA2 mediated platelet 
aggregation remains maximally inhibited. So, provided that no more than 10% of ‘unacetylated’ 
platelets enter the circulation per day, once daily aspirin maintains an optimal antithrombotic 
effect. 
 
Soon after aspirin was shown to be effective as an antithrombotic agent, several investigators 
reported that unlike healthy subjects, about a third of patients treated with aspirin showed 
incomplete suppression of TX synthesis or arachidonate-induced platelet aggregation3. These 
patients were called poor responders and subsequent studies showed that poor response to once 
daily aspirin, identified by either TX or by platelet aggregation assays, was associated with 
reduced antithrombotic effectiveness4. Several groups, including ours, proposed that, in certain 
conditions 
(e.g. 
post-coronary 
artery 
bypass 
grafting5, 
diabetes6, 
and 
essential 
thrombocythemia7), poor response to aspirin was caused by an increased rate of platelet 
production. Such a mechanism is a plausible cause of poor response to aspirin because an 
increased rate of platelet production would result in an increased proportion of ‘unacetylated’ 
platelets entering the circulation after aspirin is cleared, which would shorten the duration of 
platelet COX-1 suppression to <24 h. Because full suppression of TXA2-dependent platelet 
function requires >90% inhibition of COX-1 activity, even a modest recovery of this activity can 
sustain a substantial platelet response8. Consistent with this proposed mechanism, we and others 
showed that a poor response to aspirin was reversed by giving aspirin twice daily (instead of 
once)6,7. It is possible that an increased rate of platelet production is responsible for most cases 

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of poor response, in which case, the response could be restored by twice daily aspirin 
administration. Our ultimate aim is to test this hypothesis, but to achieve this aim we need to 
develop an accurate test of platelet production to identify patient groups that would benefit from 
twice daily aspirin. 
 
How can we identify patients in whom an increased rate of platelet production is responsible for 
poor response to aspirin? Previous methods to measure platelet production in healthy subjects, 
which were based on survival of radiolabeled platelets, have shown that platelets survive in the 
circulation for about 10 days. The specific objective of this study is to demonstrate that in healthy 
subjects in whom production of new platelets occurs at rate of 10% per day, the recovery of 
serum thromboxane B2 per day would be about 10%. Instead of labeling with radioactive 
isotopes, we will use the acetylation of platelet COX-1 by aspirin to identify the rate of platelet 
production. Our method is based on the observation that in normal subjects, after a single dose 
of aspirin, unacetylated COX-1 recovers at a rate of about 10 percent per day as new platelets 
enter the circulation9. However, measuring acetylation status of protein requires specialized 
proteomics assays. Instead, we intend to use an assay to quantify serum TXB2 as an indirect 
measure of the effect of unacetylated COX-1 from new platelets.  If true, then the daily recovery 
of serum TXB2 would provide a readout of new platelet production. 
 
The serum thromboxane B2 method has the additional advantage of providing information on 
both the rate of platelet production and the degree of platelet inhibition by aspirin. Such an 
advantage would facilitate the identification of non-responders whose reduced response is due 

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to an increased rate of platelet production, as well as the assessment of response following 
aspirin dose modification in future studies. 
 
 
Implications of this study:  
Successful validation of serum thromboxane B2 assay performed in-house as a measure of 
platelet production will be critical to pursuing our multistep research program to:  1) investigate 
the frequency with which increased platelet production results in poor response to aspirin; 2) 
identify patient populations in whom increased production is a major cause of poor response to 
aspirin; 3) test the hypothesis that poor response in those populations can be overcome by twice 
daily aspirin; and 4) evaluate the effect of twice daily aspirin in those populations on reducing 
the risk of cardiovascular events. 
 
Methods: 
Study design: Healthy volunteers will take aspirin 81 mg daily for 5 days. Serum thromboxane 
B2 (sTxB2) will be measured before taking aspirin (baseline), at 4h after last dosing (daily peak 
level), and then daily for 5 days after aspirin cessation.  
Population: 20 healthy volunteers. Enrollment criteria are listed below: 
 Inclusion criteria:  
o Healthy non-smoking volunteers;  
o Age ≥ 18 years; 
 Exclusion criteria:  

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o Allergy or intolerance to aspirin; 
o Current pregnancy; 
o Use of aspirin or drugs interfering with platelet function (NSAIDs, anticoagulants) 
within one week of study enrolment. 
 
Assays:  
 Serum Thromboxane B2: serum TXB2 concentrations were measured using an enzyme 
immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the instructions 
provided by the manufacturer. In-house controls were tested with each batch. 
 
Blood collection: 2.5 ml of blood in a serum tube for sTxB2 assay, and 3 ml of blood in EDTA for 
platelet count at baseline.  
 
Statistical considerations: 
 Sample size: As a pilot study, we plan to enroll 20 patients as recruitment of this number 
is feasible. This study will provide estimates of mean/median and variability (e.g. 
standard deviation and range) of serum thromboxane B2 and recovery in healthy 
volunteers and will inform on sample size calculation of future studies with this assay. 
 Proposed analyses:  
■ Biological variability of serum TXB2 will be represented as mean/median and standard 
deviation/range, at each time point of sampling.  

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■ Residual serum TXB2 will be plotted against time and recovery rate of serum TXB2will be 
estimated, and compared with 10% rate of new platelet production reported in historic 
studies using radiolabeling method for detection.  
 
Anticipated result: 
In healthy subjects in whom production of new platelets occurs at rate of 10% per day, we 
expect a 10% increase in serum thromboxane B2 per day; 
 
Study coordination, recruitment and logistics 
This study will be performed at PHRI & TaARI. Healthy volunteers will be screened and recruited 
by the investigators or research assistants. Informed consent will be obtained from participants 
before we conduct our study. Samples will be collected by our research assistant or 
investigators and then transferred to TaARI for testing. 
 
Role of the principal investigator 
Dr Chan is the principal investigator, and will have lead roles in study oversight, protocol 
development, Research Ethics Board submission, creation of database, develop the detailed 
statistical analysis plan, and draft the manuscript for reporting study results.  
 
Ethical considerations:  
We will ensure that each participant fully understands the purpose and procedures of this 
study. Written informed consent will be obtained from each participant. The risk of aspirin is 

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well documented, and unforeseeable risk limited. We will keep in close contact with the 
participants for the 2 weeks of study. Participant data will only be used for the purpose of the 
study. We will ensure confidentiality by storing data in a keyword-secured computer in an ID 
badge-secured building and restricting data access to research personnel only. 
 
Figure 1. Study procedure 
 
 
* ASA, acetylsalicylic acid; sTxB2, serum thromboxane B2; ICF, informed consent form. 
References: 
1 
Antithrombotic Trialists, C. Collaborative meta-analysis of randomised trials of 
antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high 
risk patients. BMJ 324, 71-86 (2002). 
2 
Patrono, C., Garcia Rodriguez, L. A., Landolfi, R. & Baigent, C. Low-dose aspirin for the 
prevention of atherothrombosis. N Engl J Med 353, 2373-2383, 
doi:10.1056/NEJMra052717 (2005). 
3 
Campbell, C. L. & Steinhubl, S. R. Variability in response to aspirin: do we understand the 
clinical relevance? J Thromb Haemost 3, 665-669, doi:10.1111/j.1538-
7836.2005.01119.x (2005). 

Serum thromboxane B2 and platelet production 
Protocol v2.0 
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4 
Hankey, G. J. & Eikelboom, J. W. Aspirin resistance. Lancet 367, 606-617, 
doi:10.1016/S0140-6736(06)68040-9 (2006). 
5 
Paikin, J. S. et al. Multiple daily doses of acetyl-salicylic acid (ASA) overcome reduced 
platelet response to once-daily ASA after coronary artery bypass graft surgery: a pilot 
randomized controlled trial. J Thromb Haemost 13, 448-456, doi:10.1111/jth.12832 
(2015). 
6 
Rocca, B. et al. The recovery of platelet cyclooxygenase activity explains interindividual 
variability in responsiveness to low-dose aspirin in patients with and without diabetes. J 
Thromb Haemost 10, 1220-1230, doi:10.1111/j.1538-7836.2012.04723.x (2012). 
7 
Pascale, S. et al. Aspirin-insensitive thromboxane biosynthesis in essential 
thrombocythemia is explained by accelerated renewal of the drug target. Blood 119, 
3595-3603, doi:10.1182/blood-2011-06-359224 (2012). 
8 
Eikelboom, J. W., Hirsh, J., Spencer, F. A., Baglin, T. P. & Weitz, J. I. Antiplatelet drugs: 
Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of 
Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 141, e89S-119S, 
doi:10.1378/chest.11-2293 (2012). 
9 
Burch, J. W., Stanford, N. & Majerus, P. W. Inhibition of platelet prostaglandin 
synthetase by oral aspirin. J Clin Invest 61, 314-319, doi:10.1172/JCI108941 (1978). 
10 
Santilli, F. et al. Platelet cyclooxygenase inhibition by low-dose aspirin is not reflected 
consistently by platelet function assays: implications for aspirin "resistance". J Am Coll 
Cardiol 53, 667-677, doi:10.1016/j.jacc.2008.10.047 (2009). 

Serum thromboxane B2 and platelet production 
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11 
Patrignani, P. et al. Reappraisal of the clinical pharmacology of low-dose aspirin by 
comparing novel direct and traditional indirect biomarkers of drug action. J Thromb 
Haemost 12, 1320-1330, doi:10.1111/jth.12637 (2014).