Submit a Revision

*your email :**
page name* : such as "Introduction", "Conclusion"..etc
<div> <p> <span>Effect of the Active Metabolite of Clopidogrel on the Oligomeric Forms of P2Y12.</span> P2Y12 receptors tagged at their NH<sub>2</sub> termini with either hemagglutinin (HA) or the c-myc epitope were transiently expressed in HEK293T cells to investigate their state of multimerization by using immunoblotting analyses. As illustrated in <a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F1">Fig. 1</a><em>A</em> (left lanes), anti-HA, anti-cMyc and anti-P2Y12 antibodies all identified monomeric as well as oligomeric receptor species in whole cell lysates. It should be noted that dimers were only detected with the anti-P2Y12 antibody, and not with the anti-tag antibodies. This may be due to a lesser accessibility of NH<sub>2</sub>-located tags by anti-tag antibodies in dimeric structures, as opposed to the accessibility by the anti P2Y12 antiserum to the COOH-located peptidic sequence used to raise the antibody. Upon treatment of the cells with the active metabolite of clopidogrel (Act-Met), the bands corresponding to the oligomeric state of P2Y12 receptors were no longer detected by all three antibodies (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F1">Fig. 1</a><em>A</em>, right lanes). The use of an anti-P2Y12 antiserum recognizing the COOH terminus of this receptor allowed for the immunodetection of dimers in whole cell lysates of Act-Met-treated cells. Using this antiserum, an increase in the intensity of the bands corresponding to dimeric and monomeric forms of P2Y12 was also observed, potentially reflecting a compensation for the loss of the oligomeric forms of P2Y12. As for the inability of the other two anti-tag antibodies to detect the P2Y12 dimers after Act-Met treatment, conformational changes at the NH<sub>2</sub> terminus of the receptors may eliminate recognition by the anti-HA and anti-c-Myc antibodies. </p> <p> Because Act-Met exhibits a free thiol-reactive function (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16835302#B55">55</a>), we compared the Act-Met-induced changes in oligomerization of P2Y12 with those induced by DTT, a commonly used thiol-reducing reagent (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F1">Fig. 1</a><em>B</em>). Both compounds strongly affected the high-molecular-mass species of P2Y12 in a concentration-dependent manner, Act-Met being at least three orders of magnitude more potent than DTT. However, the two compounds induced somewhat different effects on P2Y12 oligomeric organization. Act-Met appeared to preferentially disrupt oligomers into dimers, whereas treatment with DTT only generated monomers. The activity of Act-Met on P2Y12, when compared to DTT activity, seems to be restricted to a limited class of thiol-sensitive chemical functions. </p> <p> <span>Effect of Act-Met on the Localization of P2Y12 in Lipid Rafts.</span> In the course of the biochemical characterization of P2Y12 receptors, we found that greater amounts of P2Y12 oligomeric forms were obtained when octyl-glucoside was used for cell lysis instead of Triton X-100 (data not shown). This observation led us to suggest that P2Y12 oligomers could be located in particular microdomains within the plasma membrane. These microdomains, e.g., lipid rafts (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16835302#B28">28</a>), are known to be insoluble in Triton X-100 due to their lipid composition, rich in cholesterol and sphingolipids. To ascertain the presence of P2Y12 oligomers in lipid rafts, Triton X-100 cell lysates were fractionated by sucrose gradient centrifugation and gradient fractions were analyzed for the presence of P2Y12 by immunoblotting. Lipid raft-containing fractions were monitored by caveolin immunoblotting, these proteins serving as a convenient marker for caveolae, a subpopulation of lipid rafts (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16835302#B59">59</a>). P2Y12 detection using the anti-P2Y12 antibody (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>A Upper Left</em>) showed that oligomers were mainly located in the microdomain-rich fractions (fractions 4 and 5), whereas dimers and monomers were predominantly found in the microdomain-free fraction 6. This fraction contained the monomeric, dimeric, as well as the oligomeric form of P2Y12. </p> <p> After treatment with Act-Met, oligomers were no longer detected in any of the gradient fractions (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>A Upper Right</em>). This loss/disruption of the P2Y12 oligomeric entities was accompanied by an increase in monomers and dimers in fractions 5 and 6 of the sucrose gradient. The vast majority of monomers and dimers were redistributed outside microdomains as attested by their strong enrichment in the gradient fraction 6 that has a very poor content of caveolin. </p> <p> We next wanted to verify the selectivity of the observed disruption and redistribution of P2Y12 complexes induced by Act-Met, and rule out any nonspecific effect on membrane microdomains that might result in artefactual partitioning of proteins out of lipid rafts. Microdomains prepared from HEK293T cells coexpressing c-Myc-P2Y12 and HA-P2Y13 were analyzed by anti-HA immunoblotting. In this heterologous expression system, P2Y13 existed as monomeric and oligomeric species, the latter in far lower amounts than P2Y12 expressed in those same cells (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>A Lower Left</em>). Both monomeric and oligomeric P2Y13 species were mostly associated with lipid rafts (fractions 4 and 5). In nontreated HEK293T cells, P2Y13 monomers were targeted to cellular lipid microdomains, as opposed to P2Y12 monomers that were mainly localized outside such microdomains. When cells were treated with Act-Met, P2Y13 localization remained unchanged (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>A Lower Right</em>) in contrast to P2Y12, thus demonstrating the selective action of the active metabolite of clopidogrel on P2Y12 oligomers. </p> <p> To test the functionality of the various species, we performed binding of [<sup>33</sup>P]2MeS-ADP to the proteins present in the sucrose gradient fractions 3–7. The P2Y12 protein content was evaluated by measuring the intensity on Western blots of bands migrated under reducing conditions (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>B Upper</em>). In this experiment, significant specific binding was detected in fractions 3–5, identified as caveolin-containing fractions (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>B Lower Left</em>), measured in the same amount of proteins deposited. In fraction 6, no binding was detected despite its high content in P2Y12. This was particularly obvious when the data were expressed as ratios (specific binding/P2Y12 content). In fractions 3–7, these ratios were 142, 37, 57, 0, and 1, respectively. Only minimal binding was detected in fractions 3–5 of the gradient fractions prepared from Act-Met treated cells (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>B Lower Right</em>). Sensitivity to Act-Met of P2Y12 oligomers present in lipid raft-enriched fractions was assessed by immunoblotting (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F2">Fig. 2</a><em>B Upper Right</em>), showing a strong reduction in P2Y12 detection in fractions 3–5. </p> <p> <span>Effect of Clopidogrel on P2Y12 Oligomers in Rat Platelets.</span> Because the conclusions of the <em>in vitro</em> experiments performed on cells expressing recombinant proteins may not be relevant <em>in vivo</em>, we examined the effects of an <em>in vivo</em> treatment with clopidogrel on endogenous P2Y12 in platelets, the physiologically targeted cell (see Fig. 4, which is published as supporting information on the PNAS web site). </p> <p> In this set of experiments, rats were treated orally for 2 h with various doses of clopidogrel, then their blood was taken and platelets were prepared. Platelet proteins were resolved by SDS-/PAGE under nonreducing conditions. P2Y12 receptors were detected by Western blotting using anti-P2Y12 rabbit antiserum (Fig. 4<em>A</em>). The main P2Y12 species present in resting platelets corresponded to oligomers that were diffusely resolved on the acrylamide gel. Monomeric and dimeric species were hardly detected under the experimental conditions used. When animals were treated with a single dose of clopidogrel, and their platelets prepared 2 h later, no expression of P2Y12 oligomers was detected in platelet lysates. This loss was accompanied with a dramatic increase in detection of P2Y12 dimers. This increase depended on the dose of clopidogrel administered to the animals and closely correlated with the antiaggregating activity of clopidogrel (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16835302#B42">42</a>), as verified on the same platelet preparation (Fig. 4<em>B</em>). Platelet microdomains were then studied for their P2Y12 content. Because platelet rafts are devoid of caveolin, CD36, a membrane glycoprotein strongly enriched in platelet rafts (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16835302#B60">60</a>), was used to monitor the raft enrichment in platelet extracts separated on sucrose gradients. In resting platelets, P2Y12 receptors appeared to be predominantly expressed as oligomers located in lipid microdomains (Fig. 4<em>C Left</em>). Isolation of such microdomains led to a much better detection of P2Y12 oligomer bands by immunoblotting than that observed with whole platelet extracts. The observation that oligomers represent the main P2Y12 species in platelets (<em>i</em>) reinforces the hypothesis that the raft-associated P2Y12 oligomers actually represent the functional form of the receptor and (<em>ii</em>) emphasizes the critical role of membrane microdomains in ADP-mediated platelet activation (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16835302#B61">61</a>). </p> <p> After <em>in vivo</em> clopidogrel treatment, platelet P2Y12 oligomers were completely converted into dimeric forms and to a lesser extent into monomeric forms of the receptor; then, dimers and monomers were partitioned outside the platelet lipid rafts. Thus, the situation previously observed in the HEK293 heterologous expression system was found in a more physiologically relevant model. </p> <p> <span>Effect of Act-Met on P2Y12 Mutants.</span> Act-Met contains a free thiol functional group that is necessary for its activity, suggesting that an interaction with cysteine residues on P2Y12. We decided to investigate whether a mutation of one of these cysteines could modify the activity of Act-Met on 2MeS-ADP binding as well as P2Y12 oligomerization. P2Y12 contains 10 cysteines. According to seven-transmembrane (7TM) receptor modeling, only four cysteines are predicted to be exposed at the surface of the cell (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F3">Fig. 3</a><em>A</em>). However, because the cell permeability of Act-Met is not currently known, and to study the possible involvement of cysteine residues in the mechanism of action of clopidogrel, 10 P2Y12 receptor mutants were generated in which a cysteine was replaced by an alanine. Mutated receptors were transiently expressed in Cos7 cells and binding of [<sup>33</sup>P]2MeS-ADP was performed. Specific binding was detected with all mutant P2Y12 receptors, except C97A and C175A (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=table&id=T1">Table 1</a>). On cells expressing the eight other mutants, Act-Met was able to inhibit [<sup>33</sup>P]2MeS-ADP binding with efficacies comparable to that measured on cells expressing the wild-type P2Y12 receptor. Because Ding <em>et al</em>. (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16835302#B62">62</a>) suggested that C17 and C270 were the targets of the active metabolite of clopidogrel, we tested the double mutant C17A/C270A receptor. Affinity for 2MeS-ADP dropped 10-fold, but sensitivity to Act-Met was not affected (<a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=table&id=T1">Table 1</a>). </p> <p> Although neither C97A nor C175A mutated receptors were able to bind 2MeS-ADP, we nevertheless tested their sensitivity to Act-Met by measuring P2Y12 oligomer disruption. <a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1635153&rendertype=figure&id=F3">Fig. 3</a><em>B</em> illustrates the fact that both mutants were normally expressed in HEK293T cells, as shown by Western blot detection with anti-P2Y12 antiserum (lanes a). Both mutant oligomers were reduced by the treatment with DTT (lanes c), showing that, as in wild-type P2Y12, disulfide bonds are involved in these oligomeric complex formations. However, the treatment with Act-Met revealed some differences (lanes b). Oligomers of C175A mutants were strongly disrupted, concurrent with an increase in the monomer bands, indicating that the clopidogrel-sensitive disulfide bond was still present in this mutant. Surprisingly, no dimers were detected in this case. With regards to the C97A mutant, Act-Met failed to reduce the amount of oligomers and to increase those of monomers, showing that the mutant oligomers were totally insensitive to Act-Met. </p> <p> This investigation of P2Y12 receptors individually mutated on cysteine residues led us to pinpoint C97 as the amino acid targeted by Act-Met. </p> </div>
Confirmation code: Enter the code exactly as it appears. All letters are case insensitive, there is no zero.