Pleiotropic effects of clopidogrel Kuszynski, Dawn S.; Lauver, D. Adam
Purinergic signalling,
09/2022, Letnik:
18, Številka:
3
Journal Article
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Clopidogrel is a widely prescribed prodrug with anti-thrombotic activity through irreversible inhibition of the P2Y
12
receptor on platelets. It is FDA-approved for the clinical management of ...thrombotic diseases like unstable angina, myocardial infarction, stroke, and during percutaneous coronary interventions. Hepatic clopidogrel metabolism generates several distinct metabolites. Only one of these metabolites is responsible for inhibiting the platelet P2Y
12
receptor. Importantly, various non-hemostatic effects of clopidogrel therapy have been described. These non-hemostatic effects are perhaps unsurprising, as P2Y
12
receptor expression has been reported in multiple tissues, including osteoblasts, leukocytes, as well as vascular endothelium and smooth muscle. While the “inactive” metabolites have been commonly thought to be biologically inert, recent findings have uncovered P2Y
12
receptor-independent effects of clopidogrel treatment that may be mediated by understudied metabolites. In this review, we summarize both the P2Y
12
receptor-mediated and non-P2Y
12
receptor-mediated effects of clopidogrel and its metabolites in various tissues.
Clopidogrel is an effective purinergic 2Y12 receptor (P2Y12) antagonist used to prevent arterial thrombosis, but its use is associated with adverse bleeding. Clinical studies have demonstrated that ...clopidogrel users have an increased risk of cerebral microbleeds and intracerebral hemorrhage. Our previous studies suggest that non-platelet mechanisms mediate these adverse bleeding events; we hypothesize that clopidogrel or one of its metabolites interacts with blood vessels directly to cause bleeding. New Zealand white rabbits (1.9–2.7 kg) were treated orally with vehicle or clopidogrel (3 or 10 mg/kg) for three days. On the fourth day, the rabbits were anesthetized for blood collection and then euthanized. The brain was collected, and the middle cerebral arteries were isolated. We used light transmission aggregometry and pressure myography to elucidate the mechanisms of the off-target effects associated with clopidogrel treatment. We confirmed that inhibition of P2Y12 activation by clopidogrel inhibited ADP-induced platelet aggregation but had no impact on P2Y12-independent arachidonic acid- or collagen-induced platelet aggregation. Analysis of middle cerebral arteries from clopidogrel treated rabbits showed that clopidogrel did not affect P2Y4, P2Y6, and P2Y14 receptor-mediated contraction but attenuated the contractile response after P2Y2 receptor activation. Further analysis determined P2Y2-mediated constriction was endothelium-dependent. Vasoconstriction is a primary component of hemostasis, and impaired vasoconstriction can prolong bleeding. These results suggest clopidogrel inhibits the endothelial P2Y2 receptor in the middle cerebral artery, which provides a mechanistic explanation for the adverse cerebral bleeding associated with the drug.
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•Clopidogrel pretreatment does not significantly inhibit mean myogenic tone generation in MCAs, but reduces the percentage of MCAs that generated tone•Clopidogrel significantly inhibits vasoconstriction resulting from P2Y2 receptor activation•P2Y2-mediated vasoconstriction is endothelium-dependent in the rabbit MCA
The novel clopidogrel conjugate, DT‐678, is an effective inhibitor of platelets and thrombosis in preclinical studies. However, a comparison of the bleeding risk with DT‐678 and currently approved ...P2Y12 antagonists has yet to be determined. The objective of this study was to evaluate the bleeding tendency of animals treated with clopidogrel, ticagrelor, and DT‐678. Ninety‐one New Zealand white rabbits were randomized to one of 13 treatment groups (n = 7). Platelet activation was assessed by flow cytometry and light transmission aggregometry before and after the administration of various doses of DT‐678, clopidogrel, and ticagrelor. Tongue template bleeding times were also measured before and after drug treatment. Treatment with P2Y12 receptor antagonists caused a dose‐dependent reduction in markers of platelet activation (P‐selectin and integrin αIIbβ3) and aggregation in response to adenosine diphosphate stimulation. At the same doses required for platelet inhibition, clopidogrel and ticagrelor significantly prolonged bleeding times, while DT‐678 did not. DT‐678 and the FDA‐approved P2Y12 antagonists clopidogrel and ticagrelor are effective inhibitors of platelet activation and aggregation. However, unlike clopidogrel and ticagrelor, DT‐678 did not prolong bleeding times at equally effective antiplatelet doses. The results suggest a more favorable benefit/risk ratio for DT‐678 and potential utility as part of a dual antiplatelet therapy regimen.
Abstract only
Dual antiplatelet therapy using low‐dose aspirin with a purinergic receptor 2Y
12
(P2Y
12
) antagonist is the most common preventative method for arterial thrombosis. Clopidogrel, a P2Y
...12
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 Ca
2+
free physiological salt solution. The MCA was isolated and cannulated on an arteriography chamber. The cannulated artery, in Ca
2+
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
−5
M) of ADP or specific purinergic receptor agonists (P2Y
1
: MRS 2365; P2Y
2
MRS 2768; P2Y
4
MRS 4062; P2Y
6
: MRS 2693; P2Y
11
: NF 546; or P2Y
14
: MRS 2905). If spontaneous myogenic tone was not generated the MCA was preconstricted with 10
−6
M 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 Information
This 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.
The novel clopidogrel conjugate, DT-678, is an effective inhibitor of platelets and thrombosis in preclinical studies. However, a comparison of the bleeding risk with DT-678 and currently approved ...P2Y
antagonists has yet to be determined. The objective of this study was to evaluate the bleeding tendency of animals treated with clopidogrel, ticagrelor, and DT-678. Ninety-one New Zealand white rabbits were randomized to one of 13 treatment groups (n = 7). Platelet activation was assessed by flow cytometry and light transmission aggregometry before and after the administration of various doses of DT-678, clopidogrel, and ticagrelor. Tongue template bleeding times were also measured before and after drug treatment. Treatment with P2Y
receptor antagonists caused a dose-dependent reduction in markers of platelet activation (P-selectin and integrin α
β
) and aggregation in response to adenosine diphosphate stimulation. At the same doses required for platelet inhibition, clopidogrel and ticagrelor significantly prolonged bleeding times, while DT-678 did not. DT-678 and the FDA-approved P2Y
antagonists clopidogrel and ticagrelor are effective inhibitors of platelet activation and aggregation. However, unlike clopidogrel and ticagrelor, DT-678 did not prolong bleeding times at equally effective antiplatelet doses. The results suggest a more favorable benefit/risk ratio for DT-678 and potential utility as part of a dual antiplatelet therapy regimen.
Clopidogrel is an effective purinergic 2Y
receptor (P2Y
) antagonist used to prevent arterial thrombosis, but its use is associated with adverse bleeding. Clinical studies have demonstrated that ...clopidogrel users have an increased risk of cerebral microbleeds and intracerebral hemorrhage. Our previous studies suggest that non-platelet mechanisms mediate these adverse bleeding events; we hypothesize that clopidogrel or one of its metabolites interacts with blood vessels directly to cause bleeding. New Zealand white rabbits (1.9-2.7 kg) were treated orally with vehicle or clopidogrel (3 or 10 mg/kg) for three days. On the fourth day, the rabbits were anesthetized for blood collection and then euthanized. The brain was collected, and the middle cerebral arteries were isolated. We used light transmission aggregometry and pressure myography to elucidate the mechanisms of the off-target effects associated with clopidogrel treatment. We confirmed that inhibition of P2Y
activation by clopidogrel inhibited ADP-induced platelet aggregation but had no impact on P2Y
-independent arachidonic acid- or collagen-induced platelet aggregation. Analysis of middle cerebral arteries from clopidogrel treated rabbits showed that clopidogrel did not affect P2Y
, P2Y
, and P2Y
receptor-mediated contraction but attenuated the contractile response after P2Y
receptor activation. Further analysis determined P2Y
-mediated constriction was endothelium-dependent. Vasoconstriction is a primary component of hemostasis, and impaired vasoconstriction can prolong bleeding. These results suggest clopidogrel inhibits the endothelial P2Y
receptor in the middle cerebral artery, which provides a mechanistic explanation for the adverse cerebral bleeding associated with the drug.
Hypertension is a common risk factor for thrombotic diseases like heart attack and stroke. Chronic high blood pressure causes endothelial dysfunction which accelerates the atherosclerotic process ...resulting in plaque instability. Since hypertensive patients have an increased risk of thrombotic diseases, many are prescribed the antiplatelet drug, clopidogrel. The present study sought to determine clopidogrel’s effect on blood pressure, cerebral blood flow, and vascular remodeling in angiotensin II (Ang II)‐induced hypertensive mice. Previous results from our laboratory demonstrated that clopidogrel attenuated P2Y2‐induced vasoconstriction in cerebral arteries which could result in an increase in downstream cerebral blood flow. Therefore, we hypothesized that clopidogrel increases cerebral blood flow and decreases vascular remodeling in Ang II‐induced hypertensive mice. Ang II infusion pump implantation or sham surgeries were performed in 48 C57BL/6 mice at 16‐18 weeks of age. Ang II was infused continuously at a rate of 800ng/kg/min for 4 weeks. The mice were also administered vehicle or clopidogrel (10mg/kg/day) starting the day after surgery. Data are presented as means ± SEM, n = 8‐12 mice/group. Ang II significantly reduced blood flow, and clopidogrel‐treated mice had blood flow that was not different from either the sham or Ang II mice (sham vehicle: 305.6 ± 16.17; sham clopidogrel: 313.0 ± 15.27; Ang II vehicle: 244.1 ± 12.76; Ang II clopidogrel: 267.6 ± 14.96). Posterior cerebral arteries were harvested from animals at the end of the experiment. The outer and lumen diameters were reduced in the vessels in Ang II treated mice. However, clopidogrel treatment did not modify this vascular remodeling in the Ang II treated mice. The results suggest that clopidogrel demonstrates protective potential in hypertensive mice. Further studies are required to determine the long‐term effects of clopidogrel on hypertension associated vascular remodeling.
Clopidogrel is an effective P2Y12 antagonist used to prevent arterial thrombosis, however it's use is associated with adverse bleeding events. Clinical studies have demonstrated that clopidogrel ...users have an increased risk of both cerebral microbleeds and intracerebral hemorrhage. Recent reports from our laboratory and others suggest that the adverse bleeding events associated with clopidogrel may not be due to the inhibition of platelets alone. To elucidate the non‐platelet effects of clopidogrel treatment, we used pressure myography to measure changes in vascular function. Male New Zealand white rabbits (2.0‐2.7kg) were treated with vehicle or 10mg/kg clopidogrel for 3 days by oral gavage. These doses were chosen because they are clinically relevant and effective at inhibiting platelet aggregation. On the 4th day, the middle cerebral artery (MCA) was isolated and mounted in a pressure myograph. Cumulative concentrations (10‐9‐10‐5M) of purinergic receptor agonists (MRS 2768 – P2Y2, MRS 4062 – P2Y4, MRS 2693 – P2Y6, and MRS 2905 – P2Y14) were added through the MCA lumen and the artery inner diameter was tracked. Analysis of MCAs from vehicle‐treated rabbits demonstrated constriction in response to P2Y2, P2Y4, P2Y6 and P2Y14 activation. Three days of clopidogrel treatment did not affect the contraction induced by P2Y4, P2Y6, and P2Y14 activation. However, clopidogrel treatment did reduce the P2Y2 mediated contraction (vehicle: ‐22.486% +/‐ 2.107 vs. 10mg/kg clopidogrel: ‐14.159% +/‐ 3.785 percent baseline diameter change at 10‐5M MRS 2768; p < 0.01). Removal of the endothelium revealed that endothelial P2Y2 receptors were responsible for the constriction observed upon receptor activation. Vasoconstriction is a key component of the hemostatic process. Attenuated vasoconstriction could result in an increase in bleeding. These results suggest clopidogrel inhibits P2Y2 receptor vasoconstriction in the MCA which provides a mechanistic explanation for the adverse cerebral bleeding associated with the drug.
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