Project Summary:
Dual antiplatelet therapy using low-dose aspirin with a purinergic receptor 2Y12 (P2Y12) antagonist is the most
common preventative method for arterial thrombosis. Antiplatelet agents, such as clopidogrel, cause an
increased risk of cerebral microbleeds and intracerebral hemorrhage. Of patients who have taken clopidogrel for
at least one year, 31% have cerebral microbleeds and 47% of those patients also develop intracerebral
hemorrhage. Clopidogrel inhibits ADP-induced platelet aggregation, but this effect does not correlate with the
increase in adverse bleeding. This suggests adverse effects of clopidogrel are not mediated by the active
metabolite and that the parent compound or one of the many inactive metabolites might be responsible. A more
complete understanding of the cellular pathways affected by clopidogrel and its metabolites are required in order
to decrease this risk. Clopidogrel is converted into the active metabolite by cytochrome P450 enzymes
(CYP450's). Only approximately 5% of clopidogrel is activated and loss of function polymorphisms in CYP450's
reduce this further. DT678 is a conjugate of the clopidogrel active metabolite that is converted into the active
form through a non-enzymatic thiol exchange reaction with glutathione. We have previously reported that DT678
has a reduced bleeding risk compared to clopidogrel in a rabbit model. My preliminary data revealed that
spontaneous myogenic tone (MT) and ADP-induced vasodilation are inhibited in clopidogrel-treated, but not
DT678-treated middle cerebral arteries (MCA's). My central hypothesis is that clopidogrel modulates P2Y1,
P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 signaling in the vasculature, and this impairs vascular function
which results in the increased bleeding side effects without causing a correlated decrease in platelet
aggregation. In order to test my central hypothesis, I will pursue the following three specific aims: 1. Identify
the receptor signaling pathways modulated by clopidogrel and DT678. My working hypothesis is that
clopidogrel inhibits P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 as well as P2Y12 in stably transfected cells.
Additionally, I hypothesize that DT-678 only inhibits the P2Y12 receptor without having effects on other purinergic
receptors. 2. Determine the effect of specific purinergic agonists in the middle cerebral artery from
clopidogrel- and DT678-treated rabbits. My working hypothesis is that clopidogrel metabolites interact with
P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 in the MCA to impair MT generation and dilation. 3. Evaluate the
effects of clopidogrel and DT678 in the parenchymal arterioles of P2Y12-/- mice. My working hypothesis
that clopidogrel modulates P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and/or P2Y14 in the vasculature to inhibit myogenic
tone, constriction and dilation in P2Y12-/- mice. The information obtained from this project will allow for
development of new antiplatelet therapies that have improved efficacy with a decreased risk of intracerebral
hemorrhage.