Novel Biomarkers of Thrombotic Risk
Conditions: Myocardial Infarction
Sponsor: University of Vermont

Novel Markers of Thrombotic Risk 
 
 
 
 
Clinical Trial Registration:  NCT02505217 
 
 
 
 
Document Prepared July 6, 2015 
 
 

 
 
Objectives: 
1. To determine whether four novel biomarkers identified in blood early after myocardial 
infarction will be evident in the same subjects 6 months later.  The biomarkers are 
increased platelet expression of FcγRIIa, the ratio TF:TFPI, and the reduction in 
coagulation factors II and X, and thrombin generation.  
2. To determine whether the four biomarkers defined above are associated with a greater 
risk of cardiovascular events (stroke, myocardial infarction and death) and a greater risk 
of bleeding. 
 
Protocol 
After informed consent is obtained, the patients will be interviewed and their medical records will 
be reviewed.  We will ask about previous medical problems and medications.  Clinical 
characteristics will be recorded in a manner that protects confidentiality.  Blood (15 ml, 1 
tablespoon) will be taken by peripheral venipuncture.  The following biochemical assays will be 
performed 
1. Platelet expression of FcγRIIa with the use of fluorochrome labelled antibodies and flow 
cytometry 
2. The concentration of coagulation factors in blood will be quantified. 
3. A thrombin generation assay will be performed.  In this assay, thrombin production will 
be initiated by lipidated tissue factor in both platelet rich and platelet poor plasma.  
Thrombin formed will hydrolyze a specific substrate to give a fluorescent signal that is 
continuously recorded.  This analysis will assess the entire process of thrombin 
generation with respect to the initiation, propagation, and termination phases of the 
reaction. 
Patients will have an ambulatory visit 6 months later.  This visit will be a research visit but we 
will attempt to perform the research visit in the context of a clinical visit.  During this visit, the 
patient will be interviewed (focusing on current medications and clinical events that occurred 
during the previous 6 months), medical records will be reviewed, and clinical characteristics will 
be recorded in a manner that protects confidentiality.  Blood (15 ml, 1 tablespoon) will be taken 
by peripheral venipuncture.  The biochemical assays above will be performed a second time. 
 
Patients will be contacted by telephone 1 year after study initiation and a final time after the last 
patient enrolled completes 1 year of follow-up.  The patient will be interviewed and, if 
appropriate, medical records will be reviewed. We will ask about medical conditions focusing on 
events that occurred during the previous year. Clinical characteristics will be recorded in a 
manner that protects confidentiality. 
 
Clinical care of patients will not be altered by participation in this study.  Participation will end 
after telephone interview performed 1 year after the last patient is enrolled. 
 
Statistical Plan 
Sample Size Calculations:  We propose to enroll 200 subjects.  We have not yet established a 
specific thresholds to identify high platelet reactivity, high TF:TFPI, depletion of coagulation 
factors, or altered thrombin generation.  For the purposes of this study we will define the 

upper/lower 33% of the group as a whole as expressing ‘high’ values or depletion of coagulation 
factors.  Power calculations are based on the association of the novel biomarkers with 
subsequent clinical events.  Based on recent trial results, we anticipate that at least 10-12% of 
the entire group will have a cardiovascular event during the subsequent year.  Based on our 
previous retrospective results we hypothesize that high expression of FcγRIIa will be ≥11,000 
molecules/platelet. For the hypotheses above to have clinical utility, they must identify 
substantial differences.  Accordingly we powered the study to identify groups that exhibit a 2-
fold increase in risk.  An event rate of 14% in the high risk group and 7% in the control group 
can be identified with a power of 95% (α<0.05) when 200 subjects are enrolled.   
 
Statistical Rationale:  Our primary analysis will test each biomarker and associated hypothesis 
independently.  Because an acute event such as a myocardial infarction may amplify the extent 
of alteration (i.e. enhance the elevation or depletion) the initial analysis will determine whether 
the initially identified groups are significantly different at the time of the second analysis.  In 
addition we will determine the extent of change for each of the biomarkers over time, assessing 
all subjects (the total group) and the originally defined groups.  Secondary analysis will assess 
the extent of overlap between the originally defined groups and the relative predictive value of 
each parameter as well as correlating each of the parameters with subsequent risk of 
cardiovascular events.  Sensitivity analyses will be performed to assess additional thresholds to 
identify high and low expression of biomarkers. 
 
Primary Endpoint Analysis:  We will compare results between the 2 groups with analysis of 
variance (ANOVA).  Multiple analysis of variance with time as a covarate will be used to assess 
changes over time in all subjects and each of the originally defined groups. 
 
Secondary Endpoint Analysis:  The incidence of clinical events in the originally defined groups 
will be compared with the use of chi squared analysis.  A logistic regression model will be 
implemented to examine the joint relationship of clinical endpoint incidence relative to baseline 
group classifications.  Potential confounding measures will be included to adjust for confounding 
group differences.  A full time-to-event analysis will start with the estimation of Kaplan-Meier 
plots to characterize the subject data for the full follow-up time period.  This analysis will be 
followed by primary survival analysis modeling performed with the use of Cox regression 
methods.