Background-We observed that the prodrug clopidogrel was less effective in inhibiting platelet aggregation with coadministration of atorvastatin during point-of-care platelet function testing. Because atorvastatin is metabolized by cytochrome P450 (CYP) 3A4, we hypothesized that clopidogrel might be activated by CYP3A4. Methods and Results-Platelet aggregation was measured in 44 patients undergoing coronary artery stent implantation treated with clopidogrel or clopidogrel plus pravastatin or atorvastatin, and in 27 volunteers treated with clopidogrel and either erythromycin or troleandomycin, CYP3A4 inhibitors, or rifampin, a CYP3A4 inducer. Atorvastatin, but not pravastatin, attenuated the antiplatelet activity of clopidogrel in a dose-dependent manner. Percent platelet aggregation was 34Ϯ23, 58Ϯ15 (Pϭ0.027), 74Ϯ10 (Pϭ0.002), and 89Ϯ7 (Pϭ0.001) in the presence of clopidogrel and 0, 10, 20, and 40 mg of atorvastatin, respectively. Erythromycin attenuated platelet aggregation inhibition (55Ϯ12 versus 42Ϯ12% platelet aggregation; Pϭ0.002), as did troleandomycin (78Ϯ18 versus 45Ϯ18% platelet aggregation; PϽ0.0003), whereas rifampin enhanced platelet aggregation inhibition (33Ϯ18 versus 56Ϯ20% platelet aggregation, Pϭ0.001). Conclusions-CYP3A4 activates clopidogrel. Atorvastatin, another CYP3A4 substrate, competitively inhibits this activation. Use of a statin not metabolized by CYP3A4 and point-of-care platelet function testing may be warranted in patients treated with clopidogrel. (Circulation. 2003;107:32-37.)Key Words: drugs Ⅲ pharmacology Ⅲ platelets Ⅲ statins C lopidogrel inhibits platelet aggregation. 1 It decreases the incidence of coronary artery stent thrombosis and is approved for reduction of myocardial infarction, stroke, and vascular death in patients with atherosclerotic vascular disease. [2][3][4] Clopidogrel is an inactive thienopyridine prodrug that requires in vivo conversion in the liver to an active metabolite that exerts its antiplatelet effect by forming an inactivating disulfide bond with the platelet P2Yac (P2Y12) adenosine diphosphate (ADP) receptor. [5][6][7][8] The P2Yac ADP receptor is a guanosine triphosphate (GTP)-coupled 7 transmembrane protein that mediates platelet aggregation by inhibiting adenyl cyclase. 8 In rats, it has been suggested that clopidogrel is activated by cytochrome P450 1A2, 6 whereas an analogue of clopidogrel, CS-747, is speculated to be activated by human cytochrome P450 3A4 (CYP3A4). 9 In humans, it is not known how clopidogrel is activated.Atorvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor widely used to treat hypercholesteremia. It is metabolized by CYP3A4, 10 the most abundant cytochrome P450 in human liver. Patients with atherosclerotic disease are frequently treated for hypercholesteremia with both clopidogrel and atorvastatin or another statin.During the course of evaluating the effect of clopidogrel on platelet function using a novel bedside platelet aggregometer, it was noted that the antiplatelet activity of clopidogrel...

NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. NADPH-cytochrome P450 oxidoreductase (CYPOR)4 is a ϳ78-kDa, multidomain, microsomal diflavin protein that shuttles electrons from NADPH 3 FAD 3 FMN to members of the ubiquitous cytochrome P450 superfamily (1, 2). In humans, the cytochromes P450 (cyt P450) are one of the most important families of proteins involved in the biosynthesis and degradation of a vast number of endogenous compounds and the detoxification and biodegradation of most foreign compounds. CYPOR also donates electrons to heme oxygenase (3), cytochrome b 5 (4), and cytochrome c (5).The FAD receives a hydride anion from the obligate two electron donor NADPH and passes the electrons one at a time to FMN. The FMN then donates electrons to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts electrons at two different steps in its complex reaction cycle. Ferric cyt P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the second of the two electrons to form the peroxo (Fe ϩ3 OO) 2Ϫ cyt P450 intermediate (6). In vivo, CYPOR cycles between the one-and three-electron reduced forms (7,8). Although the one-electron reduced form is an air-stable, neutral blue semiquinone (FMN ox/sq , Ϫ110 mV), it is the FMN hydroquinone (FMN sq/hq , Ϫ270 mV), not the semiquinone, that donates an electron to its redox partners (8 -11). CYPOR is the prototype of the mammalian diflavin-containing enzyme family, which includes nitric-oxide synthase (12), methionine synthase reductase (13,14), and a novel reductase expressed in the cytoplasm of certain cancer cells (15). CYPOR is also a target for anticancer therapy, because it reductively activates anticancer prodrugs (16).CYPOR consists of an N-terminal single ␣-helical transmembrane anchor (ϳ6 kDa) responsible for its local...

A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119 -129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b 5 . Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b 5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b 5 . All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b 5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.The cytochromes P450 (P450s) 1 are a ubiquitous superfamily of mixed function oxidases that catalyze the oxidation of a large number of hydrophobic endogenous and xenobiotic substrates. Known substrates number in the thousands, whereas unique P450 sequences are counted in the hundreds at this time (2-4).The versatility of these oxidases and their potential for industrial purposes has generated a great deal of interest in understanding their structure, function, and redox reactions. The reaction catalyzed by P450 is shown in Reaction 1.where RH is the substrate and ROH is the oxidized product.The enzymatic cycle includes substrate binding, first electron transfer, oxygen binding, second electron transfer, substrate oxidation, and finally product dissociation. The redox partners for the microsomal P450s are cytochrome P450 reductase (P450 reductase) which contains both a FAD and FMN cofactor and cytochrome b 5 (cyt b 5 ). The crystal structure of P450 reductase has recently been published, and the two domains of the enzyme have been individually expressed and characterized (5, 6). In contrast, the crystal structure of cyt b 5 has been known for many years but has just recently been refined (7,8). The first and second electrons are donated to P450 by P450 reductase. Because of its redox potential (Х ϩ 25 mV), cyt b 5 can only donate the second electron to P450 (9). In fact, it has been suggested that cyt b 5 may be able to transfer the second electron to selected P450s even faster than P450 reductase, thereby decreasing the amount of superoxide produced (1...