Multi-Analyte, Genetic, and Thrombogenic Markers of Atherosclerosis
Multi-Analyte, Genetic, and Thrombogenic Markers of Atherosclerosis
About 13 million people in the United States have coronary artery disease (CAD). It is the
 leading cause of death in both men and women.

 Coronary artery disease (CAD) occurs when the blood vessels that supply blood to the heart
 muscle (the coronary arteries) become hardened and narrowed. The arteries harden and narrow
 due to buildup of fatty and calcified material called plaque on their inner walls. The
 buildup of plaque is also called atherosclerosis. This is a process which starts early in
 life, but can be influenced by multiple factors.

 Several factors increase the risk of developing atherosclerosis. They include high blood
 pressure, smoking, diabetes, high cholesterol and being related to someone who had a heart
 attack or a stroke. The more risk factors you have, the greater the chance that you have
 severe atherosclerosis. Some of the risk factors cannot be modified, like age and family
 history of early heart disease. The influenceable factors include high blood pressure, high
 blood cholesterol, high blood sugar, cigarette smoking, overweight or obesity, and lack of
 physical activity.

 Nevertheless, there are patients without any above mentioned risk factors who develop
 atherosclerosis. In addition to that, there are also patients with several risk factors who
 do not develop severe coronary artery disease.

 According to research studies high blood levels of some substances in the blood (biochemical
 markers) as well as some genes in the DNA of our cells may be associated with an increased
 risk of developing CAD and faster progression of the disease.

 The purpose of this study is to find a correlation between certain blood markers and growth
 of the plaques, regardless of the presence of the classic risk factors for atherosclerosis.
 If we prove our hypothesis we will be one step closer to predicting the extent of
 atherosclerosis by performing certain blood tests.
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