Multi-Analyte, Genetic, and Thrombogenic Markers of Atherosclerosis

Cardiovascular diseases (CVD), primarily coronary artery disease are the leading cause of
death and disability in the United States and Europe. The cost of cardiovascular disease in
the United States in 2009 is estimated to be $475.3 billion, according to the American Heart
Association and the National Heart, Lung, and Blood Institute. Although there have been
significant accomplishments in reducing cardiovascular events over the past decade, too many
people still die of heart and vascular diseases. Therefore, the improvement of risk
stratification of CVD by identification of new biomarkers has been extensively investigated
in both primary and secondary clinical settings in the past decade. A substantial number of
biomarkers, representing various stages of atherogenesis and impaired cardiac function, have
been evaluated against modifiable traditional risk factors, such as cholesterol, blood
pressure, smoking status, and diabetes. However, little is known about the true extent that
these identified multi-analyte, genetic, and thrombogenic markers contribute to the presence
and degree of atherosclerosis.

Patients with severe stenosis of coronary arteries may have a different profile of
biochemical and genetic markers than patients with "clear" coronary vessels. Therefore, more
research is required to improve the predictability and specificity of these known and novel
factors before physicians fully implement these tests into their routine clinical practice.

At present, physicians rely on conventional cardiovascular risk factors to try to identify
at-risk patients. A number of risk factors stem from genetic or biologic conditions such as
gender, age, ethnicity and family history of heart disease. While many risk factors cannot
be changed, risk factors such as high cholesterol, high blood pressure, obesity, tobacco
smoking, stress, physical inactivity can be modified. One of the most significant risk
factor for the development of CVD is diabetes mellitus, whereby both heredity and lifestyle
play a major role. Nevertheless, there are patients without these known classical risk
factors who develop severe CVD. Conversely, there are patients with these classical risk
factors without relevant coronary artery disease. "The CVD Risk Factor Paradox" may be
explained by a combination of biological, environmental, and genetic factors that are under
investigation.

Animal and human studies have established the role of cholesterol in the development and
progression of atherosclerosis. Epidemiological studies directly implicated LDL-C to the
development of atherosclerosis and CVD. Furthermore, LDL-C level appears to be directly
related to the development and recurrence of CVD. While LDL-C is the primary lipid marker
for assessing risk, evidence has demonstrated the important role of other lipoproteins
components in atherogenesis. These include lipoprotein (a), LDL pattern density, HDL
subtypes, VLDL, and intermediate-density lipoprotein. A substantial body of evidence has
also demonstrated Lp-PLA2 as a cardiovascular risk marker in both primary and secondary
prevention that provides new information, over and above new traditional risk factors. Most
recently oxidized low-density lipoprotein (oxLDL)/ β2-glycoprotein I (β2GPI) complexes have
been implicated in atherogenesis. More accurate and expanded depiction of the lipid profile
compared to the standard lipid profile may identify important emerging risk factors and
secondary targets of therapy for cardiovascular disease.

It is also been established that heightened plaque metabolism together with increased blood
vulnerability characterized by hypercoagulability, heightened platelet reactivity and
inflammation, are important processes responsible for plaque rupture and subsequent
occlusive ischemic events occurrence during ACS. Recent developments in catheter-based
near-infrared spectroscopy may help to identify vulnerable plaques by characterizing
chemical components. However, information regarding blood vulnerability based on a specific
biomarker profile is lacking.

Multiple lines of evidence suggest the critical role of platelets in the development of
atherosclerosis and thrombosis. By expressing adhesion molecules platelets facilitate the
diapedesis of leukocytes into the vascular wall during atherosclerotic development.
Additionally, P-selectin and CD40L trigger release of RANTES from platelets and subsequently
augment monocyte recruitment and secretion of inflammatory cytokines from monocytes.
Previous studies have demonstrated the roles of MCP-1 and IL-8 during plaque development and
cardiovascular clinical outcomes. Gurbel et. al. demonstrated that incremental changes in
platelet function, demonstrated by increases in GPIIb/IIIa expression and increased release
of RANTES, IL-8 and MCP-1, as clinical disease progressed from the asymptomatic state to
stable angina and finally to unstable angina.These findings further reinforce the critical
role of platelets in plaque destabilization.

The role of inflammation during atherosclerotic plaque rupture and subsequent development of
thrombus generation at the site of plaque rupture by enhancing tissue factor expression and
thrombin generation is well recognized. Several studies have demonstrated the association of
high IL-6, -8, and -18, elevated CD40 ligand (CD40L), myeloperoxidase, tumor necrosis factor
and CRP levels with ischemic events.

Epidemiological evidence in addition to experimental and clinical data supports the
hypothesis that CRP may be a "marker" as well as an active participant in the development of
atherothrombotic complications. More interestingly, recent in vitro studies suggest that
there is a direct role of CRP on endothelial and platelet function. In autopsy studies, CRP
immune reactivity was detected in atherosclerotic arteries but not in normal arteries. In
addition, the levels of CRP in fibrous tissue and atheroma of atherectomy specimens were
higher in patients with unstable angina and myocardial infarction compared to patients with
stable angina. Gurbel et al. demonstrated a statistically significant increase in specific
inflammation markers, importantly CRP, IL-8, RANTES and MCP-1 in patients with progressive
CAD compared to patients with asymptomatic disease. Although serial changes in markers were
not studied in the same patient, the incremental changes among various markers across the
study population, clearly suggests the transition between disease states.

Linking vulnerable blood characterized by elevated inflammation markers, hypercoagulability,
and highly reactive platelets to the vulnerable patient who is at risk for thrombotic
complications has been the subject of much discussion in recent years.

Platelet activation and aggregation are the most critical factors in the generation of
ischemic events, including stent thrombosis and myocardial infarction (MI). Unlike the
routine measurement of blood glucose, cholesterol and C-reactive protein performed during
the management of patients with atherosclerosis, the measurement of platelet function is
largely ignored during the management of cardiovascular patients, even in those at the
highest risk. Multiple laboratory and translational research studies performed at our
research center as well as others have demonstrated the importance of platelet reactivity as
a new emerging risk factor. At our center we primarily utilize two methods to determine
coagulation and platelet reactivity, thrombelastography (TEG) and platelet aggregation. The
indications for TEG testing include assessing bleeding of unclear etiology, and assessing
hypercoagulable states. In addition, TEG platelet mapping has been utilized to monitor
antiplatelet therapy. It has been hypothesized that high platelet-fibrin clot strength
measured by TEG predicts robust clot formation at the site of plaque rupture. In support of
this premise, Gurbel et al. demonstrated that high thrombin-induced platelet-fibrin clot
strength and high platelet reactivity has been associated with ischemic events in patients
with CVD. In fact, all of the current data support that uniform measurement of on-treatment
platelet reactivity may be a major diagnostic strategy, not only in the treatment of
patients who have undergone PCI, but also in all patients with cardiovascular disease at
risk for thrombotic events.

Presently, the gold standard to diagnose patients with CVD is coronary angiography. If
significant lesions are detected, coronary intervention with angioplasty or coronary artery
bypass grafting can be performed to reestablish flow in the blocked coronary arteries.
Modification of cardiovascular risk factors and pharmacological management is subsequently
implemented to prevent recurrent cardiovascular events including myocardial infarction and
stent thrombosis. The importance of the ADP-P2Y12 receptor interaction and COX-inhibition
has been demonstrated by the clinical benefits associated with the addition of clopidogrel
to aspirin therapy in patients with acute coronary syndromes and patients treated with
stents. However, the" one size fits all" antiplatelet management strategy has proven to be
flawed due to clopidogrel and aspirin response variability.

Incomplete inhibition of platelet thromboxane generation has been associated with an
increased risk of cardiovascular events. In the CHARISMA study, Eikelboom et. al.
demonstrated that urinary concentrations of 11-dehydro thromboxane B2 potentially was a
modifiable determinant of stroke, myocardial infarction, or cardiovascular death in patients
at risk for atherothrombotic events. In addition, variability in clopidogrel response is
well established with multiple translational research studies demonstrating a relationship
between antiplatelet nonresponsiveness, high on-treatment platelet reactivity, and the
occurrence of ischemic events in percutaneous coronary intervention patients.

Recently, the loss-of-function CYP2C19*2 allele has been shown to be associated with
decreased activation of clopidogrel and antiplatelet effect and with increased
cardiovascular events in patients receiving clopidogrel. Individuals with this genotype have
reduced protection from thrombotic events as outlined in the FDA black box warning for
clopidogrel. Despite the black box warning, there has been no large scale study performed to
personalize antiplatelet treatment of P2Y12 inhibitors with CYP2C19 genotyping primarily
because the technology for point-of-care genotyping is not commercially available. From a
clinical perspective, reporting 2C19 test results in a rapid fashion will help guide
therapeutic decisions while patients are in the inpatient setting prior to discharge.

Large-scale genome-wide association studies using high-density, single nucleotide
polymorphism genotyping arrays have revealed genetic variants that are robustly associated
with CAD and CAD-related traits such as type 2 diabetes and obesity. Also, evidence has been
obtained that multiple rare alleles with fairly strong phenotypic effects may contribute to
the genetic heritability of CAD Although, the involvement of specific genes and their level
of contribution to CAD have not been established by research, it is known that CAD often
results from the blended effects of multiple genes. These so-called polygenic effects mean
that the genetics of CAD are extremely complicated, with many different genes influencing
person's risk. In most cases, CAD is not inherited in a clearly dominant or recessive
manner. Instead, a person may have mutations in some genes that increase risk and mutations
in other genes that decrease risk, and their combined effect plays a role in the development
of atherosclerosis. At present the replication of results in the reported studies is poor,
probably because of the lack of high-quality environmental data and not counting for the
gene-environment interactions.

Many patients have undetected coronary artery disease that, if accurately identified, would
lead to more aggressive early treatment strategies, including lifestyle modification and
targeted pharmacologic therapy. A goal of current study is to determine potential
biochemical and genetic markers associated with the presence and progression of CVD.

The current studies hypothesis is that specific biomarkers and genetic profile will
precisely identify with the severity of angiographically-defined coronary lesions,
independently of the classic risk factors for atherosclerosis. The investigators believe
that this will enhance the treatment of patients with cardiovascular disease by implementing
personalized treatment strategies.

3. STUDY DESIGN 3.1. Outline A total of 1300 subject's ≥18 years undergoing coronary
angiography (inpatient cohort)or who have undergone coronary angiography within 5 years
(outpatient cohort, not to exceed 50 subjects) will be enrolled. In addition, 300 healthy
controls free from any pharmacologic therapy will also be enrolled. After giving informed
consent, a lifestyle questionnaire on topics of diet, physical activity, history of high
blood pressure, hyperlipidemia, diabetes, smoking and family history of early heart disease
will be completed. A blood (approximately 35 ml) and urine sample will be obtained prior to
angiogram for the inpatient cohort, or on the day of enrollment for the outpatient cohort
for laboratory assessments. The presence and severity of CAD will be determined according to
the following three categories: category 1 - no disease or minimal stenosis (<25%) of major
branches without need of any medical therapy; category 2 - intermediate stenosis (25%-75%)
of major branches and/or patient needs only conservative treatment (no need for PTCA);
category 3 - severe stenosis (>75%) of major branch and/or patient needs PTCA or CABG.
Statistical tools will be used to detect any correlation between the studied markers and the
extent of atherosclerotic plaques. Subgroup analysis will be performed to detect the similar
correlation in certain risk groups like smokers, obese subjects, diabetes, or subjects with
hypercholesterolemia. Subjects will be contacted once a year for up to 5 years by telephone
and information regarding antiplatelet therapy and cardiovascular events (death, myocardial
infarction, stent thrombosis, stroke, revascularization, major bleeding) will be collected.

Inclusion Criteria:

- Age ≥18 years

- Subjects scheduled for coronary angiography

- Subjects who have undergone coronary angiography within 5 years

Exclusion Criteria:

- Female subjects who are pregnant

- Subjects who suffer currently from an acute infection

- Subjects, who have received an experimental drug or who gave a blood donation of ≥ 1
pint within 8 weeks prior to screening

- Subjects with any coagulation, bleeding or blood disorders

- Subjects who are undergoing treatment for neoplastic diseases

- Subjects with autoimmune disease or connective tissue disease

- Subjects with HIV or hepatitis C.

- Subjects with any abnormal laboratory value or physical finding that according to the
investigator may interfere with the interpretation of the study results, be
indicative of an underlying disease state, or compromise the safety of a potential
subject