T. Kendall Harden, Department of Pharmacology, School of Medicine, University of North Carolina, CB# 7365, Chapel Hill, NC 27599-7365, USA. Tel.: +1 919 966 4816; fax: +1 919 966 5640; e-mail: firstname.lastname@example.org
Summary. Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, α2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .
Adenosine diphosphate (ADP) was first reported to be a potent activator of platelet aggregation in 1961  and is now known to be the cognate agonist for the P2Y1, P2Y12, and P2Y13 receptors [2–5]. Both P2Y1 and P2Y12 receptors are present on platelets and simultaneous activation of both receptors is necessary for ADP-induced platelet aggregation [6–14]. Activation of P2Y1 receptors promotes Gαq-mediated activation of phospholipase C, which in turn hydrolyzes membrane PtdIns(4,5)P2 to yield diacylglycerol and inositol(1,4,5) trisphosphate; these two second messengers activate protein kinase C (PKC) and promote release of Ca2+ from intracellular stores, respectively. P2Y1 receptor activation in the presence of a P2Y12 receptor antagonist leads to cytoskeletal rearrangement, shape change, and transient aggregation [8,15]. Activation of P2Y12 receptors initiates Gαi-promoted decreases in intracellular cyclic AMP formation, activation of phosphatidyl inositol-3 kinase via liberated Gβγ, and downstream activation of GPIIb/IIIa involved in sustained platelet aggregation.
Because of lack of selective P2Y receptor agonists, examination of the discrete actions of the P2Y1 receptor in platelet biology has required concomitant use of receptor agonists and P2Y12 receptor antagonists. We recently synthesized (N)-methanocarba-2MeSADP (MRS2365), as a potent P2Y1 receptor agonist . Studies with recombinant P2Y1, P2Y12, and P2Y13 receptors illustrated that MRS2365 specifically activates the P2Y1 receptor without either agonist or antagonist activity at the P2Y12 or P2Y13 receptors . Selective activation of the P2Y1 receptor also was suggested from functional studies of human platelets as MRS2365 potently induced shape change without causing the sustained aggregation that is a hallmark of simultaneous activation of the P2Y1 and P2Y12 receptors by ADP or by previously studied ADP analogues. Thus, MRS2365 allows discrete examination of the actions of the P2Y1 receptor. We have further examined the actions of MRS2365 in human platelets, and have applied this P2Y1 receptor-selective agonist to further delineate the interplay of P2Y1 and P2Y12 receptor activation in the sustained aggregation response to ADP.
Integrilin (2 mg mL−1 eptifibatide; Millennium Pharmaceuticals, Cambridge, MA, USA ) and heparin (1000 U mL−1; Baxter Healthcare, Deerfield, IL, USA ) were obtained from the University of North Carolina Hospital. 2MeSADP, ADP, prostacyclin, fibrinogen, apyrase, bovine serum albumin, and other buffer reagents were purchased from Sigma Chemical Company, St Louis, MO, USA.
Synthesis of (N)-methanocarba-2MeSADP
(N)-methanocarba-2MeSADP (MRS2365) was synthesized as previously described .
Synthesis of 2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate
2-Iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2500) was synthesized as previously described .
Preparation and assay of washed human platelets
Suspensions of washed human platelets were prepared essentially as described previously . The procedure was a modification of the method of Cazenave et al. , which was originally derived from Mustard et al. . Briefly, one hundred and fifty milliliters of blood was drawn from healthy volunteers into 60 mL syringes containing one-sixth final blood volume of 65 mm citric acid, 85 mm sodium citrate, 110 mm dextrose (ACD), pH 4.5. Immediately after collection, the blood was carefully transferred to 50 mL sterile conical tubes (∼30 mL in each) and placed in a water bath at 37 °C for 30 min, mixed by inversion, and centrifuged at 275 × g for 16 min. The supernatant (platelet-rich plasma) was collected and further centrifuged (13 min at 2200 × g), and the platelets were gently resuspended in 40 mL of modified Tyrode's buffer containing 137 mm NaCl, 2.7 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 3 mm NaH2PO4, 5 mm glucose, 10 mm HEPES (pH 7.4), and 0.36% bovine serum albumin. Ten units per milliliter heparin and 5 μm prostacyclin (PGI2) also were included in the first resuspension (to prevent transient platelet activation during the preparation), and the platelets were incubated at 37 °C for 10 min followed by another addition of 5 μm PGI2 prior to centrifugation (8 min at 1900 × g). This washing step was repeated once using modified Tyrode's buffer without heparin, and the platelets were suspended in the same buffer (without PGI2) containing 0.05 U mL−1 apyrase to a final cell density of 5 × 108 cells mL−1. The platelet suspensions were allowed to rest for 90–120 min at 37 °C prior to experiments .
Platelet aggregation was quantified using the optical mode of a two-channel Chrono-Log aggregometer (Model 560-VS; Chrono-Log Corporation, Havertown, PA, USA) at 37 °C using stirred (1000 r.p.m. ) washed platelet suspensions (425 μL; ∼2.2 × 108 cells) as described previously . Relative platelet aggregation response (maximum extent) was determined by comparison of light transmission in platelet resuspension buffer and is expressed as percentage response. To magnify the signal during measurements of platelet shape change, the reference cuvette was replaced with a platelet suspension diluted to 50% (2.5 × 108 cells mL−1) while the preparation to be studied was maintained at a concentration of 5 × 108 cells mL−1. For all measurements, light scatter through resting platelets was recorded for 1 min prior to addition of drugs as indicated (final volume, 500 μL). Shape change/aggregation was monitored for 6–8 min.
Each experiment was repeated at least three times. The data are presented as representative aggregometer tracings from a typical experiment, or in some cases as mean ± SEM of transformed (i.e. percentage control) data pooled from multiple experiments. Dose–response curves were generated using graphpad prism software (Graphpad Software Inc., San Diego, SC, USA).
Scanning electron microscopy
Scanning electron microscopy was performed at the University of North Carolina Microscopy Services Laboratory. Washed platelets, incubated with drugs as indicated, were fixed by addition of 50 μL of platelet suspension to 500 μL 4% glutaraldehyde in 0.1 m sodium cacodylate [cacodylic acid sodium salt – Na(CH3)2AsO23H2O) pH 7.2] followed by rotation for 30 min at room temperature. One hundred microliters of the fixed platelet suspension was spread onto poly-l-lysine-coated glass coverslips and incubated for 30 min. Following adsorption, the coverslips were rinsed once in 0.1 m sodium cacodylate buffer and dehydrated through a graded series of five ethanol washes (30%, 50%, 75%, 100%, and 100%). The coverslips were transferred in 100% ethanol to a critical point dryer (Balzers CPD-020; Bal-Tec AG, Balzers, Liechtenstein ) and dried using carbon dioxide as the transition solvent. The coverslips were mounted on aluminum specimen supports with carbon adhesive tabs and coated with a 15 nm thickness of gold–palladium metal (60:40 alloy) using a Hummer X sputter coater (Anatech Ltd, Alexandria, VA, USA). Samples were examined with a Cambridge Stereoscan 200 scanning electron microscope (LEO Electron Microscopy Inc., Thornwood, NY, USA) operating at an acceleration voltage of 20 kV, 20 mm working distance, and 40° tilt.
Our previous studies with recombinant human P2Y1 and P2Y12 receptors illustrated that MRS2365 is a selective P2Y1 receptor agonist that neither activates nor blocks the P2Y12 receptor . This P2Y receptor selectivity was examined further in functional studies with natively expressed P2Y receptors on washed human platelets. The extent of platelet aggregation was measured over a 6 min period in response to 10 μm ADP as indicated by an increase (downward deflection) in light transmission ( Fig. 1A). In contrast, MRS2365 promoted a shape change (decrease in light transmission, upward deflection; Fig. 1B) previously shown to occur with activation of the Gq-coupled P2Y1 receptor [22,23] without inducing the sustained platelet aggregation previously shown to require simultaneous activation of the Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor as occurring with ADP. A Gi/z-coupled α2A-adrenergic receptor also exists on platelets, and whereas addition of 1 μm epinephrine alone had little effect on platelet aggregation, marked and sustained platelet aggregation occurred upon activation of the P2Y1 receptor by MRS2365 in platelets preincubated with 1 μm epinephrine (Fig. 1C).
Because of the rapid aggregation of platelets in response to ADP, it was not possible to directly compare the P2Y1 receptor-dependent shape change elicited by ADP vs. that observed with MRS2365. However, as platelet aggregation requires the binding of fibrinogen to activated GPIIb/IIIa, we used eptifibatide, an inhibitor of the binding of fibrinogen to GPIIb/IIIa to block aggregation and to compare the shape change induced by ADP with that promoted by MRS2365 in the absence of platelet–platelet interactions. As illustrated in Fig. 2A,B, the kinetics of occurrence, magnitude, and longevity of the shape change induced by MRS2365 was indistinguishable from that induced by ADP after pretreatment of platelets with eptifibatide. The dynamics of platelet shape change in response to MRS2365 was also examined visually using scanning electron microscopy. MRS2365-induced shape change occurred rapidly with the maximum extent attained within 10 s (Fig. 2C).
MRS2500 was recently synthesized as the highest affinity P2Y1 receptor antagonist reported to date . Moreover, Hechler et al.  recently reported that MRS2500 potently and selectively inhibited P2Y1 receptor activity in studies of platelets measured ex vivo and in several models of experimental thrombosis in vivo. Whereas treatment of platelets with 10 μm MRS2500 alone had no effect, preaddition of this high affinity P2Y1 receptor antagonist blocked the effects of MRS2365 on shape change ( Fig. 3A,B). The reversibility of the shape change induced by MRS2365 also was examined using MRS2500. Whereas the shape change induced by MRS2365 was sustained for at least 8 min in the presence of drug, addition of MRS2500 2 min after addition of MRS2365 resulted in reversal of the P2Y1 receptor-promoted shape change to resting levels of light transmission over a 5–10 min period with a t1/2 of 2.66 min (Fig. 3C).
We previously determined the relative potencies of agonists for purified human P2Y1  and P2Y12  receptors using steady-state GTPase assays in model phospholipid vesicles reconstituted with the purified receptors and their cognate heterotrimeric G proteins. Our results indicate that 2MeSADP is approximately 10-fold selective for the P2Y12 receptor over the P2Y1 receptor, and therefore, low concentrations of 2MeSADP activate platelet P2Y12 receptors with little action at P2Y1 receptors. This selectivity of low concentrations of 2MeSADP for activation of the P2Y12 receptor permitted further confirmation of the P2Y1 receptor specificity of MRS2365. Addition of 3 nm 2MeSADP ( Fig. 4A) to washed platelets produced no measurable effect, and addition of 1 μm MRS2365 induced an observable shape change without platelet aggregation (Fig. 4B). In contrast, simultaneous addition of 3 nm 2MeSADP with 1 μm MRS2365 resulted in marked platelet aggregation (Fig. 4C). These results also are consistent with selective activation of the P2Y1 receptor by MRS2365.
The availability of a P2Y1 receptor-selective agonist allowed us to examine directly the influence of independent activation of the P2Y1 receptor on the subsequent response of platelets to ADP. The degree of platelet aggregation was proportional to ADP concentration ( Fig. 5A; EC50 = 4.1 ± 0.6 μm, n = 7). Selective activation of the P2Y1 receptor by preincubation (2 min) with either 1 μm or 3 μm MRS2365 (Fig. 5B,C) resulted in a reduced platelet aggregation response to all submaximal concentrations of ADP tested. As a result, the potency of ADP decreased over 20-fold (EC50 = 98 μm) following a 2 min preincubation with 3 μm MRS2365 (Fig. 5D). Results from scanning electron microscopy of platelets support these aggregometer data. When compared with resting platelets ( Fig. 6A), incubation of platelets with a maximally effective concentration of MRS2365 (3 μm) resulted in a dramatic shape change including pseudopod projections, but no platelet aggregation was observed (Fig. 6B). In contrast, treatment of platelets with 3 μm ADP caused significant aggregation (Fig. 6C). Although MRS2365-induced pseudopodia formation was still observed, platelets pretreated with MRS2365 for 2 min did not aggregate upon challenge with ADP (Fig. 6D). These results are consistent with the idea that preincubation with MRS2365 leads to functional desensitization of the P2Y1 receptor-promoted pathway that synergizes with a P2Y12 receptor-promoted signaling pathway to confer the sustained aggregation response to ADP.
The hypothesis that loss of the aggregation response to ADP after preincubation with MRS2365 occurs as a consequence of diminution of a P2Y1 receptor-promoted Gq-signaling response was tested by examining the capacity of another Gq-coupled receptor to overcome the MRS2365-induced loss of response to ADP. Thus, serotonin, which activates the Gq-coupled 5-HT2A receptor on platelets, caused a shape change but no sustained platelet aggregation ( Fig. 7A). As illustrated above, preincubation of platelets with MRS2365 resulted in a marked decrease in the capacity of ADP to induce aggregation (Fig. 7B,C). However, co-addition of serotonin with ADP to MRS2365-preincubated platelets produced an aggregation response similar to that observed with ADP in platelets in the absence of preincubation with MRS2365 (Fig. 7D). Thus, activation of another Gq-activating receptor largely overcomes the loss of response to ADP observed as a consequence of preactivation of the P2Y1 receptor.
The concentration dependence of MRS2365 for inducing desensitization of the P2Y1 receptor was examined by preincubating washed platelets with various concentrations of MRS2365 for 2 min prior to challenge with 10 μm ADP. Preincubation of platelets with increasing concentrations of MRS2365 resulted in a concentration of MRS2365-dependent decrease in ADP-promoted platelet aggregation ( Fig. 8A). When plotted as a percentage of the control response (ADP only, no preincubation with MRS2365), the calculated potency of MRS2365 for inducing loss of ADP-stimulated aggregation as a consequence of a 2 min preincubation with MRS2365 was 34 nm (Fig. 8B). These data support the notion that occupancy of the human platelet P2Y1 receptor by MRS2365 concurrently induces shape change and desensitization of a P2Y1 receptor-promoted signaling pathway that is necessary in the sustained aggregation response of platelets to ADP.
The time course of induction of desensitization of the P2Y1 receptor by MRS2365 was examined by preincubating washed platelets with a maximally effective concentration (3 μm) of MRS2365 for various times prior to challenge with 10 μm ADP. Significant loss of ADP-induced aggregation response was observed within 15 s of incubation with MRS2365, and little to no response to ADP was observed after 1 min of MRS2365 preincubation ( Fig. 9A). The t1/2 for occurrence of loss of capacity of ADP to promote platelet aggregation following 3 μm MRS2365 was approximately 18 s (Fig. 9B). To verify that the observed MRS2365-induced loss of the aggregation response was independent of the P2Y12 receptor, a similar time course experiment was carried out using epinephrine to activate the Gi/z-coupled α2A-adrenergic receptor. The extent of loss of epinephrine-induced aggregation was similar to that observed with ADP in MRS2365-pretreated platelets (Fig. 9C). Moreover, the time course of loss of aggregation response to epinephrine (t1/2 = 13 s; Fig. 9D) was in close agreement with that observed with ADP (Fig. 9B). Thus, desensitization of the P2Y1 receptor with a receptor-specific agonist has inhibitory consequences on two different receptors that promote aggregation through a Gi-mediated signaling response.
The results presented here further illustrate the P2Y1 receptor selectivity of MRS2365 and introduce a potentially highly useful pharmacological agent for the study of the role of this receptor in platelet function. Capacity to independently activate the P2Y1 receptor in the absence of a P2Y12 receptor antagonist has allowed us to study ADP-promoted aggregation in real time in platelets subjected to selective preactivation of the P2Y1 receptor. Previous studies with selective receptor antagonists [6–9,12,14] and platelets from mice with targeted gene disruption [10,11,13] have established that both the P2Y1 and P2Y12 receptors are necessary for the sustained aggregation response of human and mouse platelets to ADP. The results presented here illustrate that the aggregation response to ADP is rapidly lost as a consequence of selective preactivation of the P2Y1 receptor. That this effect of MRS2365 is a consequence of receptor-specific desensitization of the P2Y1 receptor/Gq-promoted signaling response is supported by the observation that it is circumvented by independent activation of the Gq-coupled 5-HT2A receptor. That the loss of aggregation response is independent of the P2Y12 receptor is supported by observation of a similar MRS2365-induced loss of aggregation response to epinephrine, which activates the Gi/z-coupled α2A-adrenergic receptor.
The development of MRS2365 as a selective agonist of the P2Y1 receptor complements the earlier discovery of adenosine 3′-,5′-bisphosphate as a selective competitive antagonist of the P2Y1 receptor . Adenosine 3′-,5′-bisphosphate and several higher affinity analogues (e.g. MRS2179) were instrumental in pharmacologically resolving the responses of platelets to ADP into the P2Y1 receptor/Gq- and P2Y12 receptor/Gi-mediated signaling events [8,29–31]. Replacement of the ribose group with a cyclopentane ring fused in the Northern conformation with a cyclopropane ring [i.e. an (N)-methanocarba moiety] led to the development of high affinity non-nucleotide antagonists of the P2Y1 receptor [32,33]. Moreover, this (N)-methanocarba substitution also was well tolerated in adenine and uridine nucleotides and in certain other agonist analogues including (N)-methanocarba-2MeSADP (MRS2365) [16,34]. Three ADP-selective P2Y receptors exist (i.e. the P2Y1, P2Y12, and P2Y13 receptors), and the specificity of MRS2365 as an agonist for the P2Y1 receptor was illustrated with recombinant P2Y receptors .
That MRS2365 alone does not promote sustained aggregation but rather induces the shape change known to occur as a consequence of Gq-dependent signaling provides strong indication that the Gq-coupled P2Y1 receptor is activated by this agonist without simultaneous activation of the Gi-linked P2Y12 receptor. We recently illustrated the high selectivity of MRS2500 as an antagonist for the human P2Y1 receptor [24,35], and Hechler et al.  confirmed this selectivity in studies of platelets ex vivo and in vivo. The observation that MRS2500 completely blocks the effects of MRS2365 further establishes the P2Y1 receptor-selective agonist action of MRS2365. Other functional studies also are consistent with this conclusion. For example, whereas neither MRS2365 nor epinephrine alone caused sustained aggregation, marked and sustained aggregation was observed with addition of MRS2365 following epinephrine, which activates a Gi/z-linked α2A-adrenergic receptor. Similarly, selective activation of the P2Y12 receptor with 3 nm 2MeSADP caused little aggregation and no shape change, and a maximally effective concentration of MRS2365 only produced shape change. However, concurrent addition of 3 nm 2MeSADP and MRS2365 induced marked and sustained aggregation, again supporting the idea that the agonist action of MRS2365 is P2Y1 receptor specific.
Platelets rapidly lose their capacity to aggregate in response to ADP [36–38]. The recent studies of Baurand et al. [21,39] illustrated that preincubation of platelets with ADP or ADPβ S results in rapid loss of P2Y1 receptor-promoted elevation of intracellular Ca2+ and loss of the required function of the P2Y1 receptor in ADP-induced aggregation. In contrast, the capacity of the ADP-activated P2Y12 receptor to inhibit adenylyl cyclase or promote aggregation (in the presence of serotonin which activates the Gq-linked 5-HT2A receptor) was largely retained in ADP- or ADPβS-preincubated platelets. Thus, these investigators proposed that the P2Y1 receptor of platelets rapidly desensitizes in the presence of ADP whereas the P2Y12 receptor does not. The results of the current study in which the responses of platelets have been studied after the selective activation of the P2Y1 receptor confirm the conclusions of Baurand et al. [21,39]. Thus, we show under conditions in which only the P2Y1 receptor is activated that responsiveness of this receptor is rapidly lost, that desensitization is apparently receptor-specific as it can be circumvented by activation of another Gq-linked receptor, and that loss of the aggregation response occurs heterologously, as neither ADP by activation of the Gi-linked P2Y12 receptor nor epinephrine by activation of the Gi/z-linked α2A-adrenergic receptor promoted aggregation after preincubation with MRS2365. Given the synergy observed between the P2Y1 and P2Y12 receptors in the aggregation response to ADP, it is important to point out that the remarkably rapid desensitization of the P2Y1 receptor observed here occurs in the absence of coactivation of both the P2Y1 and P2Y12 receptors as was the case in previous studies with the non-selective agonists, ADP or ADPβS. That is, remarkably rapid desensitization of the P2Y1 receptor occurs irrespective of whether the P2Y1 and P2Y12 receptors are activated simultaneously or the P2Y1 receptor is activated independently.
The molecular basis of P2Y receptor desensitization in platelets is unknown. However, Baurand et al.  illustrated with receptor-specific antibodies that incubation of platelets with ADP results in rapid internalization of the P2Y1 receptor to an intracellular compartment, whereas little change in the distribution of the P2Y12 receptor occurs. In contrast to the conclusions of Baurand et al., Hardy et al.  recently reported that preincubation of platelets with ADP results in rapid desensitization of both the P2Y1 and P2Y12 receptors. Moreover, these investigators provide evidence that the P2Y1 receptor is subject to regulation by protein kinase C isozymes whereas P2Y12 receptor desensitization involves a G protein receptor kinase(s). Interestingly, Fam et al.  have identified Thr339 in the carboxy terminus of the human P2Y1 receptor as a phosphorylation site necessary for protein kinase C-mediated attenuation of P2Y1 receptor promoted signaling. Again, capacity to independently activate the P2Y1 receptor with MRS2365 in the absence of activation or inhibition of the P2Y12 receptor should aide in developing the molecular understanding of the regulation of P2Y1 receptor-dependent signaling.
One of the well-recognized effects of P2Y1 receptor activation during ADP-induced aggregation is the promotion of the shape change of platelets, and although the change of shape is undoubtedly involved in the process of aggregation, it is neither sufficient nor the only process triggered by activation of the P2Y1 receptor. Our results illustrate that the platelet shape change induced by P2Y1 receptor activation is sustained for up to 10 min in the presence of agonist and reverses over a 10 min time period upon P2Y1 receptor blockade. In contrast, the P2Y1 receptor signaling response that synergizes with the P2Y12 receptor-promoted signaling to produce sustained aggregation fully desensitizes within 1 min of receptor activation. Targeted gene disruption in mice [10,11] suggests that P2Y1 receptor signaling entirely emanates from activation of Gq, but loss of function could occur at this or multiple downstream steps not restricted to initial mobilization of Ca2+, activation of protein kinase C, or phospholipase C-catalyzed changes secondary to alteration in membrane phosphoinositide levels. That activation of a Gq-linked receptor for 5-HT circumvents MRS2365-induced loss of ADP-induced aggregation is consistent with, but does not prove, that MRS2365-induced desensitization occurs at the level of the P2Y1 receptor per se. A complex and still unclear set of signaling pathways underlie the formation of pseudopodia, and the molecular machinery necessary to reverse the platelet shape change once it occurs remains largely undefined.
In summary, the studies outlined above have demonstrated that MRS2365 activates the P2Y1 receptor with high potency and selectivity as observed by hallmark features (i.e. shape change/pseudopod formation), leads to rapid P2Y1 receptor desensitization with no observable action at the P2Y12 receptor, and attenuates ADP-induced aggregation upon subsequent agonist challenge. MRS2365 is a novel pharmacological tool that should prove highly useful in discrete analysis of the bifurcating ADP-induced signaling pathways in platelets.
This work was supported by National Institutes of Health grants GM38213, HL54889, and HL57391. DMB recognized support from the Pharmaceutical Research and Manufacturers of America Foundation.