Thrombin Regulated Platelet Activation

Thrombin is the major protease in the coagulation cascade whose pleiotropic actions can
ultimately lead to thrombosis and tissue injury. Thrombin is the key effector of the
coagulation cascade and converts fibrinogen to fibrin which is essential for laying the mesh
work for clot formation. Further, thrombin also provides positive feedback by converting
inactive coagulation factors into their active state, thereby generating more thrombin. In
addition, thrombin displays a diverse range of effects in vascular cells that functionally
connects tissue damage to both hemostatic and inflammatory responses. Many of the cellular
effects of thrombin are initiated via activation of a family of Protease-Activated Receptors
(PARs) which are coupled to heterotrimeric G proteins.

Cardiovascular disease remains the leading cause of death in the United States, accounting
for over 39% of deaths and over $350 billion in annual health care costs in this country.
Current pharmacological therapy for treatment of diseases caused by blood clots, such as
heart disease and stroke, often involve the use of drugs that do not reflect current
scientific understanding of these pathologies. Acute coronary syndrome is a thrombotic
event, and platelet activation plays a critical role in the formation of intravascular
thrombus at the site of arterial injury or plaque rupture. Medical management of acute
coronary syndrome is centered on anti-platelet therapies, however, current anti-platelet
drugs do not fully attenuate platelet activation, can have delayed onset and long durations
of action and may result in significant morbidity due to bleeding complications. With
increasing numbers of percutaneous coronary intervention in patients with ACS, bleeding
complications become a major concern in this new era of anti-platelet and coagulation
therapies. The direct thrombin inhibitor bivalirudin was associated with a significant
reduction of in-hospital bleeding complication as compared to heparin plus IIbIIIa
inhibitors in patients undergoing PCI, while composite clinical endpoints (death, MI,
repeated revascularization) were maintained. An explanation for the lack of superiority of
direct thrombin inhibitors when compared to heparin, could be that direct thrombin
inhibitors also block the anticoagulation and anti-inflammatory effects of thrombin thereby
potentially altering the risk/benefit ratio. In addition, all of the agents used in treating
ACS have undesirable side effects such as bleeding. An agent that would selectively block
the inflammatory and thrombotic effects of thrombin by selectively blocking PAR activation,
without altering the protective APC pathway or inhibiting fibrin generation (bleeding) would
potentially have more of a desirable risk/benefit ratio.

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
this 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.

In defining which G protein mediates the effects of different PARs on platelets, we will
target that particular receptor-G protein interface using the C-terminus of the particular G
alpha that mediates the response. We have used overexpression of the native C-terminal
peptides of different G proteins to determine which G protein signaling pathway is involved
in physiological responses in endothelial cells. As this method is not applicable to
platelets, which are not transfectable, we propose to create membrane-permeable versions of
Gα C-terminal peptides that would be acutely deliverable to platelets to tease out signaling
pathways. The roles of G proteins in platelet activation in mice has been studied
extensively through the use of knockout technology. The most striking phenotype was in mice
lacking G alpha q. They demonstrate an absence of platelet aggregation and are protected
against thromboembolism. Recently, the role of G alpha 13 in platelets has been uncovered
through the use of an inducible mouse line lacking G alpha 13. Previously the role of G
alpha 13 was unknown as these mice die in utero from defects in angiogenesis. Surprisingly,
absence of G13 in mice platelets leads to a reduced potency of thrombin, TXA2 and collagen
to induce platelet shape change and aggregation. The specific roles of G proteins in
platelet activation in humans has been less well studied, because of the lack of a tractable
genetic approach and the inability to transfect human platelets. One study has identified a
patient with diminished Gαq activity (50% of Gαq immunoreactivity compared to normal) who
also had impaired agonist-induced platelet aggregation and secretion.

Significant differences exist in the expression patterns of G proteins and PARs between
mouse and human platelets. Perhaps the biggest difference is that thrombin activation of
mouse platelets is mediated through PAR3 and PAR4, while thrombin activation of human
platelets is mediated through PAR1 and PAR4. This was made very clear as thrombin was still
able to activate platelets in PAR1 knockout mice. Murine PAR3 is unable to signal to
downstream effectors and functions to present thrombin to PAR4 in platelets, as PAR4 lacks
the hirudin-like sequence and does not bind thrombin efficiently. By contrast, human PAR3
has been proposed to signal through thrombin-triggered phosphoinositide hydrolysis, however,
its functional role remains to be established. Mice platelets only express Gαq while not
expressing Gα11, which most likely explains the more severe phenotype with Gαq knockout mice
compared to the other knockout mice. However, human platelets may contain G alpha 11, in
addition to G alpha q, although this has been viewed with controversy. Our dominant-negative
G protein peptide strategy should now allow us to directly study PAR-G protein signaling in
human platelets.

Coronary artery disease (CAD) is the leading cause of death in the world. The lifetime risk
of having coronary heart disease after age 40 is 49% for men and 32% for women. The AHA
estimates that more than 12 million people have some form of CAD history. This will increase
with the aging of the baby boom generation. Research spanning nearly three decades has
firmly established the role of platelet activation in the pathophysiology of ACS. In
contrast to the central role of platelet activation in the pathogenesis of unstable angina
and acute myocardial infarction, a major role for platelet activation in the setting of
chronic stable angina has not been widely acknowledged. Coronary angioplasty leads to vessel
wall injury and exposure of subendothelial structures with resultant platelet activation
that may limit the early and late success of the procedure. In contrast with the findings in
patients with stable angina, patients referred for angioplasty exhibit more consistent
increases in platelet activation prior to their intervention. Platelet aggregation and the
expression of activated GPIIbIIIa and P-selectin are increased in patients referred for
coronary artery stenting or angioplasty. In contrast, increased concentrations of
circulating markers of platelet activation in patients following angioplasty have been
reported by some but not all groups. Based on the REPLACE-2 trial of bivalirudin,
interventional cardiologists at Vanderbilt and around the world are increasingly using
bivalirudin as the anticoagulant of choice for PCI. We will examine the effects of blocking
thrombin on PAR signaling in platelets from subjects with normal coronary arteries as well
as patients undergoing PCI. We have carried out preliminary studies of platelet aggregation
before and after bivalirudin infusion in patients from Vanderbilt's Coronary Catheterization
Laboratory. As expected, thrombin mediated aggregation of ex vivo platelets is inhibited by
bivalirudin (data not shown). Therefore, we hypothesize that removing the protective effect
of basal thrombin on the vasculature could change the platelet PAR1 or PAR4 signaling state.
This possibility has not been examined, and it is important that such studies be conducted,
as direct thrombin inhibition becomes a more widely used therapeutic strategy in acute
coronary syndrome.

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).