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 P2Y(12) 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 P2Y(12) 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 P2Y(12) Test were also high with the LTA, VerifyNow, Multiplate, and BioCytex VASP P2Y(12) Tests. Similarly, low residual platelet function using the P2Y(12) 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.

PAMFix is a fixative that was developed to stabilise the platelet biomarker P-selectin on activated platelets. Stabilisation of this biomarker introduced the possibility of performing measurements of platelet function remotelywhere there was no access to special equipment or technical expertise. The idea was to use PAMFix to fix the platelets after stimulating platelets in blood, after which the stabilised samples would be transported to a central laboratory for ana`lysis by flow cytometry. This would obviate the need for special equipment or technical expertise at or near the point at which the blood was collected and processed. Here we demonstrate the stability of samples of blood treated with PAMFix and describe several applications which the use of PAMFix has proved to be particularly beneficial.

Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated and dephosphorylated consequent to increases and decreases in cyclic nucleotide levels. Monitoring changes in VASP phosphorylation is an established method for indirect measurement of cyclic nucleotides. Here we describe the use of an innovative cocktail, VASPFix, which allows sensitive and reproducible measurement of phosphorylated VASP (VASP-P) in a simple, single-step procedure using cytometric bead technology. Frozen VASPFix-treated samples are stable for at least six months prior to analysis. We successfully used VASPFix to measure VASP-P in platelets in both platelet-rich plasma and blood in response to compounds that increase (dibutyryl cAMP, adenosine, iloprost, PGE1) and decrease (ADP, PGE1) cAMP, and to determine the effects of certain receptor antagonists on the results obtained. The change in VASP-P brought about by adding ADP to PGE1-stimulated platelets is a combination of the effect of ADP at the P2Y12 receptor and of PGE1 at both IP and EP3 receptors. For iloprost-stimulated platelets EP3 receptors are not involved. A procedure in which iloprost, ADP and VASPFix were used to determine effectiveness of clopidogrel and prasugrel in patients was compared with an established commercial procedure that uses PGE1 and ADP; the latter produced higher platelet reactivity values that were the result of PGE1 interacting with platelet EP3 receptors. We conclude that VASPFix can be used both as a research tool and for clinical investigations and provides better specificity for P2Y12 receptor inhibition. The latter confers a distinct advantage over existing methods used to monitor effects of P2Y12 antagonists on platelet function.

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.

The role of prostanoid receptors in mediating the effects of PGE3 on human platelet function -

Prostaglandin E2 (PGE2) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3′5′-monophosphate (cAMP). PGE2 reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a Gs-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 PGE2. 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 PGE2 were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through Gs-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. PGE2 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 PGE2 is mediated via the EP3 receptor. In contrast, PGE2 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 PGE2 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 PGE2 may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y(12) 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 P2Y(12) 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 TXA(2) 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 (PGI(2), iloprost, PGD(2), adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE(1) and PGE(2) 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, P2Y(12) 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.

Objective— To investigate whether adenosine diphosphate (ADP)–derived adenosine might inhibit platelet aggregation, especially in the presence of a P2Y 12 antagonist, where the effects of ADP at the P2Y 12 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 P2Y 12 antagonists cangrelor, prasugrel active metabolite, and ticagrelor. In the presence of a P2Y 12 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 P2Y 12 antagonist used. Conclusion— ADP inhibits platelet aggregation in the presence of a P2Y 12 antagonist through conversion to adenosine. Inhibition occurs in PRP but not in whole blood except when adenosine uptake is inhibited. None of the P2Y 12 antagonists studied replicated the effects of dipyridamole in the experiments that were performed.

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.

There is evidence that the overall effects of prostaglandin E2 (PGE2) on human platelet function are the consequence of a balance between promotory effects of PGE2 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 E1 (PGE1), 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 A2 mimetic U46619 to compare the effects of PGE1 and PGE2 on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, P receptors latelet function -selectin ASP phosphorylation 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 PGE2 interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE1 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.

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 PGE2. 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).

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.

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.

SummaryThe 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°C and 37°C. Spontaneous aggregation was also measured after addition of saline alone. In citrated blood, spontaneous aggregation was markedly enhanced at 28°C compared with 37°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°C. The P2Y12 antagonist AR-C69931 inhibited all spontaneous aggregation at 28°C and reduced all ADP-induced aggregation responses to small, reversible responses. Aspirin had no effect. Aggregation was also enhanced at 28°C compared with 37°C with low but not high concentrations of TRAP and U46619. PAF-induced aggregation was maximal at all concentrations when measured at 28°C, but reversal of aggregation was seen at 37°C. Baseline levels of platelet CD62P and CD63 were significantly enhanced at 28°C compared with 37°C. Expression was significantly increased at 28°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°C compared with 37°C, particularly in the presence of ADP.

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 26, No. 2Leukocytosis, Vascular Disease, and Adenine Nucleotide Metabolism Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBLeukocytosis, Vascular Disease, and Adenine Nucleotide Metabolism Stan Heptinstall, Jacqueline R. Glenn, Andrew Johnson, Bethan Myers, Ann E. White, and Lian Zhao Stan HeptinstallStan Heptinstall Departments of Cardiovascular Medicine and Haematology, Queens Medical Centre, Nottingham, University of Nottingham and University Hospital NHS Trust, Nottingham, United Kingdom Search for more papers by this author , Jacqueline R. GlennJacqueline R. Glenn Departments of Cardiovascular Medicine and Haematology, Queens Medical Centre, Nottingham, University of Nottingham and University Hospital NHS Trust, Nottingham, United Kingdom Search for more papers by this author , Andrew JohnsonAndrew Johnson Departments of Cardiovascular Medicine and Haematology, Queens Medical Centre, Nottingham, University of Nottingham and University Hospital NHS Trust, Nottingham, United Kingdom Search for more papers by this author , Bethan MyersBethan Myers Departments of Cardiovascular Medicine and Haematology, Queens Medical Centre, Nottingham, University of Nottingham and University Hospital NHS Trust, Nottingham, United Kingdom Search for more papers by this author , Ann E. WhiteAnn E. White Departments of Cardiovascular Medicine and Haematology, Queens Medical Centre, Nottingham, University of Nottingham and University Hospital NHS Trust, Nottingham, United Kingdom Search for more papers by this author , and Lian ZhaoLian Zhao Departments of Cardiovascular Medicine and Haematology, Queens Medical Centre, Nottingham, University of Nottingham and University Hospital NHS Trust, Nottingham, United Kingdom Search for more papers by this author Originally published1 Feb 2006https://doi.org/10.1161/01.ATV.0000197801.28944.41Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:e22–e23To the Editor:We read with interest the paper by Barry S. Coller on leukocytosis and its relationship with vascular disease morbidity and mortality1 and the subsequent correspondence.2,3 We are intrigued to learn that relative leukocytopenia may be associated with increased morbidity and mortality in patients with acute myocardial infarction1,4 and in those undergoing percutaneous coronary intervention.5 We note the discussion on whether leukocyte count is merely a marker of general disturbances in inflammation and general poor health, or whether leukocytes might contribute directly to thrombosis and atherosclerosis.Although several mechanisms through which leukocytes may contribute to thrombosis and atherosclerosis were discussed,1–3 nothing has been said about the role of leukocytes in adenine nucleotide metabolism. Adenosine diphosphate (ADP) is, of course, a major contributor to the thrombotic mechanism as evidenced by the successful use of ADP antagonists which reduce ADP-induced platelet activation and aggregation and thereby act as anti-thrombotic agents.6It was recognized that leukocytes are active in the metabolism of adenine nucleotides many years ago7 but recent papers from our own group have re-emphasized their importance.8–10 In summary, first we found that adenosine triphosphate (ATP) added to blood induces platelet aggregation via a mechanism involving leukocytes and ADP formation.8 We then found that leukocytes (all neutrophils, all monocytes and a subset of lymphocytes) test positive for an antibody to CD39, the NTPDase which converts ATP into ADP and also ADP into adenosine monophosphate (AMP).9 We also saw that leukocytosis results in modified platelet aggregation responses to both ATP and ADP; platelet aggregation in blood induced by ATP is more rapid in leukocytosis while aggregation induced by ADP is followed by rapid disaggregation. A systematic analysis of the role of blood cells and plasma enzymes in adenine nucleotide metabolism by high-performance liquid chromatography (HPLC) was then performed.10 This confirmed that leukocytes are the principle means through which ATP and ADP added to blood are broken down to ADP and AMP respectively. Thus leukocytes provide a means of metabolising adenine nucleotides that is additional to that provided by vascular endothelial cells and thus provide a clearance mechanism that is active within the blood itself as well as at the blood periphery.In view of the current discussion on leukocytosis we show in Figure 1 the results of new experiments in which we added ATP or ADP to normal blood, blood to which autologous leukocytes had been added to increase the count from 4.4 to 26×103/μL and blood from a patient with hyperleukocytosis with a white cell count of 126×103/μL. Nucleotides and products were determined by HPLC. It can be seen that in the presence of normal numbers of leukocytes ATP was converted to ADP which peaked at about 15 minutes and was then subsequently broken down to AMP; ADP was converted directly to AMP. With added leukocytes the rate of ATP metabolism is markedly enhanced (t1/2 from &15 minutes to &4 minutes) with ADP produced more quickly, but present for a shorter duration before conversion to AMP; the rate of ADP conversion to AMP was also markedly enhanced. In hyperleukocytosis both ATP and ADP were metabolized very quickly with rapid conversion to AMP which was subsequently removed. Download figureDownload PowerPointFigure 1. Metabolism of ATP (a, c and e) and ADP (b, d and f) in blood (anticoagulated with hirudin) from normal volunteers (a, b; WCC=4.4×103/μL), blood to which autologous leukocytes had been added (c, d; WCC=26×103/μL) and blood from a patient with hyperleukocytosis (e, f; WCC=126×103/μL). Measurement of ATP, ADP and AMP was by HPLC.10 Results are mean±SEM, n=3 (a–d) or a single determination (e, f). ATP (100 μmol/L) or ADP (100 μmol/L, except for f where 30 μmol/L ADP was used) was added to blood samples which were incubated at 37°C for up to 30 minutes.It seems to us that differences in adenine nucleotide metabolism as determined by leukocyte count may well have relevance to thrombosis and also to hemostasis. On the one hand, leukocytes in blood provide a mechanism for platelet activation via ATP, as well as ADP. Further, because high leukocyte counts provide a means of converting ATP to ADP more rapidly, earlier platelet activation can occur. In this regard, leukocytes can be thought of as being prothrombotic. On the other hand, leukocytes also provide an effective means of removing ADP thus limiting platelet responses to this important nucleotide, possibly an important anti-hemostatic role.We believe the extent to which the increased morbidity and mortality associated with leukocytosis may be a consequence of altered adenine nucleotide metabolism needs to be investigated further. Erythrocytes are a huge source of ATP which can be released physiologically in response to hypoxia11 and via cell damage. Activated platelets also release ATP as well as ADP. There is also the possibility that the increased morbidity and mortality associated with leukocytopenia in acute myocardial infarction and in percutaneous coronary intervention may be a consequence of ineffective ADP removal. In which case there would be further justification for the use of ADP antagonists in these conditions.1 Coller BS. Leukocytosis and ischemic vascular disease morbidity and mortality: is it time to intervene? Arterioscler Thromb Vasc Biol. 2005; 25: 658–670.LinkGoogle Scholar2 Kaski JC, Avanzas P, Arroyo-Espliguero R. Neutrophil count and complex lesions in patients with coronary artery disease. Arterioscler Thromb Vasc Biol. 2005; 25: e112.LinkGoogle Scholar3 Coller BS. Neutrophil count and complex lesions in patients with coronary artery disease. Arterioscler Thromb Vasc Biol. 2005; 25: e112.LinkGoogle Scholar4 Grzybowski M, Welch RD, Parsons L, Ndumele CE, Chen E, Zalenski R, Barron HV. The association between white blood cell count and acute myocardial infarction in-hospital mortality: findings from the NationalRegistry of Myocardial Infarction. Acad Emerg Med. 2004; 11: 1049–1060.CrossrefMedlineGoogle Scholar5 Gurm HS, Bhatt DL, Gupta R, Ellis SG, Topol EJ, Lauer MS. Preprocedural white blood cell count and death after percutaneous coronary intervention. Am Heart J. 2003; 146: 692–698.CrossrefMedlineGoogle Scholar6 Hacke W. From CURE to MATCH: ADP receptor antagonists as the treatment of choice for high-risk atherothrombotic patients. Cerebrovasc Dis,. 2002; 13: 22–26.CrossrefMedlineGoogle Scholar7 Coade SB, Pearson JD. Metabolism of adenine nucleotides in human blood. Circ Res. 1989; 65: 531–537.CrossrefMedlineGoogle Scholar8 Stafford NP, Pink AE, White AE, Glenn JR, Heptinstall S. Mechanisms involved in adenosine triphosphate-induced platelet aggregation in whole blood. Arterioscler Thromb Vasc Biol. 2003; 23: 1928–1933.LinkGoogle Scholar9 Glenn JR, White AE, Johnson A, Fox SC, Behan MWH, Dolan G, Heptinstall S. Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood. Platelets. 2005; 16: 159–170.CrossrefMedlineGoogle Scholar10 Heptinstall S, Johnson A, Glenn JR, White AE. Adenine nucleotide metabolism in human blood -important roles for leukocytes and erythrocytes. J Thromb Haemost. 2005; 3: 2331–2339.CrossrefMedlineGoogle Scholar11 Wang L, Olivecrona G, Götberg M, Olsson ML, Winzell MS, Erlinge D. ADP acting on P2Y13 receptors is a negative feedback pathway for ATP release from human red blood cells. Circ Res. 2005; 96: 189–196.LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails February 2006Vol 26, Issue 2Article InformationMetrics Download: 82 https://doi.org/10.1161/01.ATV.0000197801.28944.41PMID: 16424358 Originally publishedFebruary 1, 2006 PDF download