A Comparative Analysis of the Vascular Effects of Clopidogrel Metabolites

Abstract

Dual antiplatelet therapy using low‐dose aspirin with a purinergic receptor 2Y12 (P2Y12) antagonist is the most common preventative method for arterial thrombosis. Clopidogrel, a P2Y12 antagonist, effectively inhibits ADP‐induced platelet aggregation. However, it also increases adverse bleeding side effects which do not correlate with the antiplatelet effect. Of patients who have taken clopidogrel for at least one year, 31% have cerebral microbleeds and 47% of those patients also develop intracerebral hemorrhage. Through clopidogrel’s complex metabolism only 5% of the active metabolite responsible for the antiplatelet effect is generated. In order to avoid the complex metabolism associated with clopidogrel, DT678 was developed. DT678, a conjugate of the clopidogrel active metabolite, is completely converted into the active metabolite to inhibit platelet aggregation. We have previously reported that DT678 is equally as effective in preventing platelet aggregation however, it has a reduced bleeding risk compared to clopidogrel in a rabbit model. A more complete mechanistic understanding of the cellular pathways affected by clopidogrel and its metabolites are required in order to explain the difference in bleeding risk.The purpose of this study was to determine the effects of clopidogrel on spontaneous myogenic tone generation, vasoconstriction or vasodilation in middle cerebral arteries (MCA). Rabbits were anesthetized and the jugular vein was cannulated for blood collection and intravenous drug administration of various doses of DT678, clopidogrel or vehicle. Blood collections were taken at baseline and two‐hour post IV drug administration. The blood was then used in a platelet aggregometry assay to determine the inhibition of platelet aggregation. Two‐hours post treatment the brain was collected and placed in Ca2+ free physiological salt solution. The MCA was isolated and cannulated on an arteriography chamber. The cannulated artery, in Ca2+ physiological salt solution, was pressurized to 80mmHg with a flow rate of 0.25ml/min through the lumen at 37°C. Spontaneous myogenic tone was allowed to develop before assessing the vascular effects of increasing concentrations (10−9–10−5M) of ADP or specific purinergic receptor agonists (P2Y1: MRS 2365; P2Y2 MRS 2768; P2Y4 MRS 4062; P2Y6: MRS 2693; P2Y11: NF 546; or P2Y14: MRS 2905). If spontaneous myogenic tone was not generated the MCA was preconstricted with 10−6M serotonin. Spontaneous myogenic tone and ADP‐induced vasodilation was inhibited in clopidogrel‐treated, but not DT678‐treated middle cerebral arteries. These data suggest that clopidogrel has non‐platelet mediated effects on purinergic receptors expressed in the MCA and may explain the cerebral bleeding side effects observed.Support or Funding InformationThis work was supported in part by MSU PHM/TOX and CVM Startup Funds, Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation Research Starter Grant and NIH/NHLBI R43HL139380.