PAR Regulation of Platelet Function in Diabetic Patients
PAR Regulation of Platelet Function in Diabetic Patients
Thrombin is the most potent activator of platelets, and platelet activation is a hallmark of
 thrombosis. Coronary artery disease (CAD) is the major cause of mortality and morbidity in
 the United States and other industrialized countries, and thrombotic sequelae are the key
 cause of death in diabetes. The accumulation of thrombin at sites of vascular injury provides
 one of the major mechanisms of recruiting platelets into a hemostatic plug. Thrombin works by
 activation of the G protein-coupled protease activated receptors PAR1 and PAR4 on human
 platelets to initiate signaling cascades leading to increases in [Ca]i, secretion of
 autocrine activators, trafficking of adhesion molecules to the plasma membrane, and shape
 change, which all promote platelet aggregation. The thrombin receptors work in a progressive
 manner, with PAR1 activated at low thrombin concentrations, and PAR4 recruited at higher
 thrombin concentrations. As direct thrombin inhibitors become widely used in clinical
 practice, it is important to assess their effects on vascular function. Our hypothesis is
 that PAR1 and PAR4 do not signal through the same G protein pathways, and that PAR4 is not a
 strong platelet agonist. To investigate this hypothesis, the investigators will study the G
 protein pathways downstream of PAR4, and assess ex-vivo platelet responsiveness to thrombin,
 PAR1, and PAR4 agonist peptides, both in normal human subjects, and along the stages of
 pathology, from patients with stable angina as well as unstable angina who are undergoing
 angioplasty. Similarly, the investigators will examine platelet function in patients with
 metabolic syndrome as well as diabetes, along the continuum from insulin resistance to
 full-blown disease. These studies will provide deeper insight into the G protein pathways
 used by PARs. They will elucidate the contribution of PAR receptors to normal platelet
 function as well as the abnormal platelet activation in thrombotic states. The long term goal
 is to understand the implications for PAR receptors as therapeutic targets for anti-platelet
 therapies that may carry less bleeding risk.
Given the known roles of proteases and PARs in coagulation, inflammation, pain, healing and
 protection, the need for development of a PAR antagonist as a therapeutic agent for treatment
 of thrombosis, atherosclerosis and inflammation is well-recognized. Thus, blocking PAR action
 by inhibiting the PAR-G protein interface is an alternative target for blocking downstream
 consequences of thrombin-mediated cellular activation. Since there are two PARs on human
 platelets, PAR1 and PAR4, it is critical to define the roles of both receptors in several
 likely clinical settings where PAR antagonists would be used. In this proposal both the G
 protein pathways underlying PAR signaling mechanisms as well as their roles in pathologies
 characterized by activated platelets will be studied in detail. We propose in our grant to
 investigate the specific roles of individual PARs in mediating the events leading to the
 multi-stage process of platelet activation and clot formation. The long term goals of these
 studies are to determine novel PAR-specific anti-platelet therapies. The 2002 estimates of
 the DHHS suggest that 6.3% of the population of the United States (18.2 Million people) have
 diabetes, with estimated annual costs of $132 Billion (Centers for Disease Control and
 Prevention). The complications of coronary artery and of cerebrovascular disease account for
 up to 65% of deaths in patients with diabetes. The prevalence, complexity and complications
 of cardiovascular disease are increased in patients with diabetes. This appears related to
 the frequent association of diabetes and of insulin resistance with traditional risk factors
 for atherosclerosis. Recent studies by Haffner and other investigators have established the
 concept of diabetes as a coronary risk equivalent; this is reflected in the guidelines for
 therapeutic targets set by the ADA, AHA and ACC.

 The clinical importance of platelet activation is reflected by the benefits of aspirin and
 clopidogrel and GPIIbIIIa inhibitors in diabetics. Epidemiologic data demonstrates that the
 metabolic syndrome is associated with an increased incidence of atherosclerotic events. Type
 2 diabetes shares many features in common with the metabolic syndrome, and may represent a
 precursor condition, Stern described this in the "common soil hypothesis". Hsueh and Law have
 more recently proposed that the progression of insulin resistance to type 2 diabetes
 parallels the progression of endothelial dysfunction to atherosclerosis.

 These studies were designed to study PAR signaling and G protein activation states in
 patients with platelet activation in the setting of the continuum of the metabolic syndrome
 and diabetes mellitus. The degree of platelet activation as determined by assays of platelet
 reactivity and by the expression of markers of platelet activation will be correlated with
 the changes in PAR signaling. These studies will provide a comprehensive assessment of
 platelet activation in the setting of the metabolic syndrome, and will compare the extent of
 activation in this setting with that in a group of contemporaneously studied patients with
 diabetes mellitus. Finally, the influence of the direct thrombin inhibitor, bivalirudin on
 platelet activation and signaling will be assessed.
Inclusion Criteria:

 -  Age: over 18, Sex: male and female.

 -  Patients who undergo clinically indicated coronary angiography and/or PCI. Patients in
 Group 1 (elective PCI) include those presented with stable angina (ACC definition for
 stable angina).

 -  Coronary angiography reveals severe stenosis (>70%) that requires PCI.

 -  Patients in Group 2 (elective PCI in subjects with diabetes) include those who present
 with stable angina, or with findings on non-invasive testing (exercise or
 pharmacologic stimulation with imaging by nuclear perfusion imaging or stress
 echocardiography) in whom coronary angiography reveals severe stenosis (>70%) that
 requires PCI.

 -  Patients in Group 2 (ACS) include those presented with unstable angina or non-ST
 elevation myocardial infarction (as defined by the ACC).

 -  Coronary angiography reveals severe stenosis (>70%) that requires PCI.

 Exclusion Criteria:

 -  Significant left main coronary artery disease.

 -  Severely impaired left ventricular systolic function (EF<35%).

 -  Prior treatment with enoxaparin, Bivalirudin (or other thrombin inhibitors), Warfarin,
 or thrombolytic agents <48 hours. Prior history of myocardial infarction (<6 weeks).

 -  Prior history of stroke (<6 weeks).

 -  Prior history of coronary intervention (<6 weeks).

 -  History of HIV/AIDS.

 -  The patients will be identified in the following manner:

 -  All subjects will be picked from a pool of patients diagnosed with stable angina and
 diabetes from the Vanderbilt Page-Campbell Heart Institute at Vanderbilt University
 Medical Center and undergo a complete history and physical examination. Patients with
 acute coronary syndrome will be referred from the acute cardiology patient service at
 Vanderbilt University Medical Center.

 -  Subjects with hematologic, renal (creatinine > 2.0 mg/dl), hepatic, inflammatory, and
 neoplastic disorders, and those who sustained a recent (< 1 month) myocardial
 infarction, ACS, or stroke will be excluded.

 -  Patients who used nonsteroidal anti-inflammatory drugs, corticosteroids, or hormone
 replacement therapy will also be excluded.

 -  Pregnancy will be excluded in women of child bearing potential by measurement of urine
 ß-HCG (it is standard of care to determine if a woman is pregnant prior to elective
 PCI and will be screened as part of their PHI).

 -  For healthy volunteers, pregnancy will be excluded per verbal report.

 -  Data will be collected regarding patient demographics including height and weight,
 abdominal circumference, blood pressure, comorbid medical conditions, triglycerides,
 HDL, fasting glucose and medication use (including prescription of antithrombotic
 agents, ACE inhibitors, angiotensin receptor blockers, beta blockers, calcium channel
 antagonists and HMG-CoA inhibitors).