(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Abstract

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, alpha2A-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 .

Fig. 1

Agonist-induced responses of human platelets. Platelets were prepared as described in Methods. Ten micromolar adenosine diphosphate (ADP) was added after a 1 min (A) while 3 μM MRS2365 was added either alone (B) or following 1 μM epinephrine (C). Aggregometer traces are from a single experiment and are representative of at least three experiments using different platelet preparations.

Fig. 2

Comparison of ADP and MRS2365-induced platelet shape changes. Agonist-induced shape change of washed platelets was measured as described in Methods. One hundred twenty-five micromolar eptifibatide (GP_IIb/IIIa antagonist) was added 2 min prior to agonist treatment. Either 10 μM ADP (A) or 3 μM MRS2365 (B) was added and the resulting shape changes recorded. Aggregometer traces are from a single experiment and are representative of at least three experiments. Scanning electron micrographs of platelet shape change at time points following selective activation of the P2Y₁ receptor with 3 μM MRS2365 (C). The maximum extent of shape change was observed at 15 s and was maintained through the measurement period of 2 min. Bar = 2 μm, 10 000×.

Fig. 3

Reversal of MRS2365-promoted platelet shape change with MRS2500. Platelet shape change was measured following the addition of 3 μM MRS2365 alone (A), 10 μM MRS2500 followed by 3 μM MRS2365 (B), or 3 μM MRS2365 followed by 10 μM MRS2500 (C). The t_1/2 for reversal of MRS2365-induced shape change following the addition of MRS2500 was 2.7 min (r² = 0.89).

Fig. 4

Requirement of simultaneous addition of 2MeSADP and MRS2365 to elicit platelet aggregation. After 1 min of recording, compounds were added to the platelet suspensions (arrow) and shape change or aggregation was recorded for 6–8 min. (A) 3 nM 2MeSADP, (B) 1 μM MRS2365, (C) 3 nM 2MeSADP and 1 μM MRS2365 added simultaneously. Aggregometer traces are from a single experiment and are representative of at least three experiments using different washed platelet preparations.

Fig. 5

Decreased ADP-induced aggregation following treatment with MRS2365. Platelet aggregation was measured in response to the indicated concentrations of ADP added (arrow) after a 1 min recording period (A) or an additional 2 min pretreatment with either 1 μM MRS2365 (B) or 3 μM MRS2365 (C). Maximal aggregation values were used to generate concentration–response curves (D). Concentration–response curves under control conditions (panel A) were identical for agonist added at either 1 min or 3 min. Aggregometer traces are from a single experiment and are representative of at least three experiments using different platelet preparations.

Fig. 6

Scanning electron microscopy of human platelets following exposure to (N)-methanocarba-2MeSADP (MRS2365). Electron microscopy was performed as described in Methods. (A) resting platelets, (B) 3 μM MRS2365 alone (2 min), (C) 3 μM ADP for 30 s, (D) 3 μM MRS2365 (2 min) followed by 3 μM ADP for an additional 2 min.

Fig. 7

Restoration of MRS2365-promoted inhibition of ADP-induced platelet aggregation by independent stimulation of a G_q signaling pathway. Suspensions of human washed platelets were treated with vehicle (PBS; A,B) or 3 μM MRS2365 (C,D) two minutes after 1 μM 5-HT (A), 10 μM ADP (B,C), or 5 μM 5-HT + 10 μM ADP (D) were added and platelet aggregation was recorded for a total of 8 min. Aggregometer traces are from a single experiment and are representative of results from at least three experiments.

Fig. 8

Concentration dependence of MRS2365 for induction of loss of the ADP-induced platelet aggregation response. Platelets were preincu-bated with the indicated concentrations of MRS2365 for 2 min followed by challenge with 10 μM ADP (A). Tracing amplitudes for each condition were transformed with respect to control (no MRS2365) to produce a concentration–response curve (B). Aggregometer traces are from a single experiment and are representative of results from at least two experiments.

Fig. 9

Time dependence of MRS2365-promoted reduction in ADP- or epinephrine-induced platelet aggregation. Platelets were pretreated for the indicated times with 3 μM MRS2365 followed by challenge with 10 μM ADP (A) or 1 μM epinephrine (C). The maximum amplitude measured in each tracing was compared with control [0:00; (A), 10 μM ADP; (C), simultaneous addition of 1 μM epinephrine and 3 μM MRS2365] to generate time course curves in which the t_1/2 for loss of response was calculated. Aggregometer traces are from a single experiment and are representative of results from four (B) or two (D) experiments.