How does measurement of platelet P-selectin compare with other methods of measuring platelet function as a means of determining the effectiveness of antiplatelet therapy?

Measurement of P-selectin on activated platelets as a means of measuring platelet function utilizing the technology described here has the advantage of not requiring immediate access to specialist equipment and expertise. Blood samples are activated, fixed, stored, and transported to a central laboratory for flow cytometric analysis. Here we have compared P-selectin with other more traditional approaches to measuring platelet function in blood and/or platelet-rich plasma (PRP) from patients with acute coronary syndromes on treatment for at least 1 month with either aspirin and clopidogrel or aspirin with prasugrel. The comparators were light transmission aggregometry (LTA), VerifyNow and Multiplate aggregometry (for determining the effects of aspirin) and LTA, VerifyNow and Multiplate together with the BioCytex VASP phosphorylation assay (for the P2Y12 antagonists). The P-selectin Aspirin Test revealed substantial inhibition of platelet function in all but three of 96 patients receiving aspirin with clopidogrel and in none of 51 patients receiving aspirin and prasugrel. The results were very similar to those obtained using LTA. There was only one patient with high residual platelet aggregation and low P-selectin expression. The same patients identified as "non-responders" to aspirin also presented with the highest residual platelet activity as measured using the VerifyNow system, although not quite as well separated from the other values. With the Multiplate test only one of these patients clearly stood out from the others. The results obtained using the P-selectin P2Y12 Test in 102 patients taking aspirin and clopidogrel were similar to the more traditional approaches in that a wide scatter of results was obtained. Generally, high values seen with the P-selectin P2Y12 Test were also high with the LTA, VerifyNow, Multiplate, and BioCytex VASP P2Y12 Tests. Similarly, low residual platelet function using the P2Y12 test was seen irrespective of the testing procedure used. However, there were differences in some patients. Prasugrel was always more effective than clopidogrel in inhibiting platelet function with none of 56 patients (P-selectin and VerifyNow), only 2 of 56 patients (Multiplate) and only 3 of 56 patients (Biocytex VASP) demonstrating high on-treatment residual platelet reactivity (HRPR) defined using previously published cut-off values. The exception was LTA where there were 11 of 56 patients with HRPR. It remains to be seen which experimental approach provides the most useful information regarding outcomes after adjusting therapies in treated patients.

PGE(2) reverses G(s)-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-G(s)-mediated inhibition of platelet aggregation by interaction with EP4 receptors.

Prostaglandin E(2) (PGE(2)) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3'5'-monophosphate (cAMP). PGE(2) reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a G(s)-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE(2). Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE(2) were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through G(s)-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE(2) reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE(2) is mediated via the EP3 receptor. In contrast, PGE(2) added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE(2) is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE(2) may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP.

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y12 and EP3 receptors, act as antagonists at G(i)-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G(i)-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y12 antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA2 mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI2, iloprost, PGD2, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE1 and PGE2 as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y12 antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.

Mode of action of P2Y(12 ) antagonists as inhibitors of platelet function.

P2Y(12) receptor antagonists are antithrombotic agents that inhibit platelet function by blocking the effects of adenosine diphosphate (ADP) at P2Y (12)receptors. However, some P2Y(12) receptor antagonists may affect platelet function through additional mechanisms. It was the objective of this study to investigate the possibility that P2Y(12) antagonists inhibit platelet function through interaction with G-protein-coupled receptors other than P2Y(12) receptors. We compared the effects of cangrelor, ticagrelor and the prasugrel active metabolite on platelet aggregation and on phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We compared their effects with those of selective IP, EP4 and A2A agonists, which act at Gs-coupled receptors. All three P2Y(12) antagonists were strong inhibitors of ADP-induced platelet aggregation but only partial inhibitors of aggregation induced by thrombin receptor activating peptide (TRAP) or the thromboxane A2 mimetic U46619. Further, after removing ADP and its metabolites using apyrase and adenosine deaminase, the P2Y(12) antagonists produced only minor additional inhibition of TRAP or U46619-induced aggregation. Conversely, the Gs-coupled receptor agonists always produced strong inhibition of aggregation irrespective of whether ADP was removed. Other experiments using selective receptor agonists and antagonists provided no evidence of any of the P2Y(12) antagonists acting through PAR1, TP, IP, EP4, A2A or EP3 receptors. All three P2Y (12)antagonists enhanced VASP-phosphorylation to a small and equal extent but the effects were much smaller than those of the IP, EP4 and A2A agonists. The effects of cangrelor, ticagrelor and prasugrel on platelet function are mediated mainly through P2Y(12)receptors and not through another G-protein-coupled receptor.

PGE1 and PGE2 modify platelet function through different prostanoid receptors.

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.

Adenosine derived from ADP can contribute to inhibition of platelet aggregation in the presence of a P2Y12 antagonist.

OBJECTIVE: To investigate whether adenosine diphosphate (ADP)-derived adenosine might inhibit platelet aggregation, especially in the presence of a P2Y12 antagonist, where the effects of ADP at the P2Y12 receptor would be prevented. METHODS AND RESULTS: Platelet aggregation was measured in response to thrombin receptor activator peptide by platelet counting in platelet-rich plasma (PRP) and whole blood in the presence of ADP and the P2Y12 antagonists cangrelor, prasugrel active metabolite, and ticagrelor. In the presence of a P2Y12 antagonist, preincubation of PRP with ADP inhibited aggregation; this effect was abolished by adenosine deaminase. No inhibition of aggregation occurred in whole blood except when dipyridamole was added to inhibit adenosine uptake into erythrocytes. The effects of ADP in PRP and whole blood were replicated using adenosine and were directly related to changes in cAMP (assessed by vasodilator-stimulated phosphoprotein phosphorylation). All results were the same irrespective of the P2Y12 antagonist used. CONCLUSIONS: ADP inhibits platelet aggregation in the presence of a P2Y12 antagonist through conversion to adenosine. Inhibition occurs in PRP but not in whole blood except when adenosine uptake is inhibited. None of the P2Y12 antagonists studied replicated the effects of dipyridamole in the experiments that were performed.

The role of prostanoid receptors in mediating the effects of PGE(2) on human platelet function.

The effects of prostaglandin E(2) (PGE(2)) on platelet function are believed to be the result of opposing mechanisms that lead to both enhancement and inhibition of platelet function. Enhancement of platelet function is known to be via EP3 receptors linked to G(i) and inhibition of adenylyl cyclase. However, the receptors involved in inhibition of platelet function have not been fully defined. Here we have used measurements of platelet aggregation, calcium signaling and P-selectin expression to assess platelet function induced by platelet activating factor (PAF), thrombin receptor activating peptide (TRAP-6) and the thromboxane A(2) mimetic U46619 respectively, to determine the effects of PGE(2) and of selective prostanoid receptor agonists on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation were also determined. We also assessed the ability of selective prostanoid receptor antagonists to modify the effects of PGE(2). The agonists and antagonists used were iloprost (IP agonist), ONO-DI-004 (EP1 agonist), ONO-AE1-259 (EP2 agonist), sulprostone (EP3 agonist), ONO-AE1-329 (EP4 agonist), CAY10441 (IP antagonist), ONO-8713 (EP1 antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist). Using the agonists available to us we demonstrated that EP3, EP4 and IP receptors elicit functional responses in platelets. The EP3 receptor agonist promoted platelet aggregation, calcium signaling and P-selectin expression and this was associated with a reduction in VASP phosphorylation. Conversely agonists acting at IP and EP4 receptors inhibited platelet function and this was associated with an increase in VASP phosphorylation. The effects on platelet function and VASP phosphorylation of the selective prostanoid receptor antagonists used in conjunction with PGE(2) were consistent with PGE(2) interacting with EP3 receptors to enhance platelet function and with EP4 receptors (but not IP receptors) to inhibit platelet function. This is the first demonstration of the involvement of EP4 receptors in platelet responses to PGE(2).

DG-041 inhibits the EP3 prostanoid receptor--a new target for inhibition of platelet function in atherothrombotic disease.

Receptors for prostanoids on platelets include the EP3 receptor for which the natural agonist is the inflammatory mediator prostaglandin E(2) (PGE(2)) produced in atherosclerotic plaques. EP3 is implicated in atherothrombosis and an EP3 antagonist might provide atherosclerotic lesion-specific antithrombotic therapy. DG-041 (2,3-dichlorothiophene-5-sulfonic acid, 3-[1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-indol-7-yl]acryloylamide) is a direct-acting EP3 antagonist currently being evaluated in Phase 2 clinical trials. We have examined the contributions of EP3 to platelet function using the selective EP3 agonist sulprostone and also PGE(2), and determined the effects of DG-041 on these. Studies were in human platelet-rich plasma or whole blood and included aggregometry and flow cytometry. Sulprostone enhanced aggregation induced by primary agonists including collagen, TRAP, platelet activating factor, U46619, serotonin and adenosine diphosphate, and enhanced P-selectin expression and platelet-leukocyte conjugate formation. It inhibited adenylate cyclase (measured by vasodilator-stimulated phosphoprotein phosphorylation) and enhanced Ca(2+) mobilization. It potentiated platelet function even in the presence of aspirin and/or AR-C69931 (a P2Y(12) antagonist). DG-041 antagonized the effects of sulprostone on platelet function. The effect of PGE(2) on platelet aggregation depended on the nature of the agonist and the concentration of PGE(2) used as a consequence of both pro-aggregatory effects via EP3 and anti-aggregatory effects via other receptors. DG-041 potentiated the protective effects of PGE(2) on platelet aggregation by inhibiting the pro-aggregatory effect via EP3 stimulation. DG-041 remained effective in the presence of a P2Y(12) antagonist and aspirin. DG-041 warrants continued investigation as a potential agent for the treatment of atherothrombosis without inducing unwanted bleeding risk.

Detection of P2Y(14) protein in platelets and investigation of the role of P2Y(14) in platelet function in comparison with the EP(3) receptor.

mRNA encoding the recently discovered P2Y(14) receptor has been reported in platelets, but the presence of P2Y(14) receptor protein and its functionality have not been studied. If P2Y(14) is expressed along with P2Y(1) and P2Y(12) receptors it may have a role in haemostasis. It was the objective of this study to investigate the presence of the P2Y(14) receptor in platelets and its role in platelet function. The effects of the agonist UDP-glucose were compared with those of sulprostone, a selective EP(3) receptor agonist. Expression of P2Y(14) receptor was investigated by immunoblotting and confocal microscopy. Platelet aggregation in platelet-rich plasma (PRP) and whole blood was measured using light absorbance and platelet counting. VASP phosphorylation was investigated using flow cytometry. Immunoblotting provided evidence for P2Y(14) receptor protein and microscopy confirmed its presence on platelets. Despite this, UDP-glucose (up to 100 muM) did not induce platelet aggregation in either PRP or whole blood, and did not potentiate aggregation induced by other agonists. P2Y(14) did not substitute for P2Y(12) in experiments using the P2Y(12) antagonist AR-C69931. No effect of UDP-glucose was seen on adenylate cyclase activity as measured by VASP phosphorylation. In contrast, sulprostone acting via the EP(3) receptor promoted platelet aggregation with effects on adenylate cyclase activity. EP(3) also partially substituted for P2Y(12) receptor. We have demonstrated the presence of P2Y(14) receptor protein in platelets, but no contribution of this receptor to several measures of platelet function has been observed. Further studies are necessary to determine whether the P2Y(14) receptor in platelets has any functionality.

Enhanced platelet aggregation and activation under conditions of hypothermia.

The effects on platelet function of temperatures attained during hypothermia used in cardiac surgery are controversial. Here we have performed studies on platelet aggregation in whole blood and platelet-rich plasma after stimulation with a range of concentrations of ADP, TRAP, U46619 and PAF at both 28 degrees C and 37 degrees C. Spontaneous aggregation was also measured after addition of saline alone. In citrated blood, spontaneous aggregation was markedly enhanced at 28 degrees C compared with 37 degrees C. Aggregation induced by ADP was also enhanced. Similar results were obtained in hirudinised blood. There was no spontaneous aggregation in PRP but ADP-induced aggregation was enhanced at 28 degrees C. The P2Y12 antagonist AR-C69931 inhibited all spontaneous aggregation at 28 degrees C and reduced all ADP-induced aggregation responses to small, reversible responses. Aspirin had no effect. Aggregation was also enhanced at 28 degrees C compared with 37 degrees C with low but not high concentrations of TRAP and U46619. PAF-induced aggregation was maximal at all concentrations when measured at 28 degrees C, but reversal of aggregation was seen at 37 degrees C. Baseline levels of platelet CD62P and CD63 were significantly enhanced at 28 degrees C compared with 37 degrees C. Expression was significantly increased at 28 degrees C after stimulation with ADP, PAF and TRAP but not after stimulation with U46619. Overall, our results demonstrate an enhancement of platelet function at 28 degrees C compared with 37 degrees C, particularly in the presence of ADP.

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood.

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2x10(3) leukocytes/microl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1-10 microM) and ATP (10-100 microM) was markedly reduced, but to ATP (1000 microM) was enhanced (all p<0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t1/2=4 min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4 min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.

Mechanisms involved in adenosine triphosphate--induced platelet aggregation in whole blood.

OBJECTIVE: Effects on platelet aggregation of adenosine triphosphate (ATP) released from damaged cells and from platelets undergoing exocytosis have not been clearly established. In this study we report on the effects of ATP on platelet aggregation in whole blood. METHODS AND RESULTS: Aggregation, measured using a platelet-counting technique, occurred in response to ATP and was maximal at 10 to 100 micromol/L. It was abolished by MRS2179, AR-C69931, and creatine phosphate/creatine phosphokinase, implying that conversion to adenosine diphosphate (ADP) is required. ATP did not induce aggregation in platelet-rich plasma, but aggregation did occur when apyrase or hexokinase was added. Aggregation also occurred after addition of leukocytes to platelet-rich plasma (as a source of ecto-ATPase), and this was potentiated on removal of adenosine by adenosine deaminase, indicating that adenosine production modulates the response. Dipyridamole, which inhibits adenosine uptake into erythrocytes, inhibited aggregation induced by ATP in whole blood, and adenosine deaminase reversed this. DN9693 and forskolin synergized with dipyridamole to inhibit ATP-induced aggregation. CONCLUSIONS: ATP induces aggregation in whole blood via conversion of ATP to ADP by ecto-ATPases on leukocytes. This is inhibited by agents that prevent adenosine removal. Reduced aggregation at high concentrations of ATP (>100 micromol/L) may be a consequence of inhibition by ATP of ADP action at ADP receptors.