Jacqueline R. Glenn

Adenine nucleotide metabolism in human blood – important roles for leukocytes and erythrocytes

Summary.  Adenosine diphosphate (ADP) released into blood induces platelet aggregation and contributes to hemostasis and thrombosis. Released ATP can also induce platelet aggregation and there is evidence that blood leukocytes and also erythrocytes play important roles in this. Rapid metabolism of ADP and ATP by endothelial cells is important in protecting platelets from their effects. Here we have performed a systematic investigation of adenine nucleotide metabolism in human blood and the involvement of blood cells. Conversion of ATP to ADP in blood was due almost exclusively to the presence of leukocytes; plasma, platelets and erythrocytes made little or no contribution. Mononuclear leukocytes (MNLs) and polymorphonuclear leukocytes (PMNLs) were equally effective. Conversion of ADP to AMP was also promoted by leukocytes, with no involvement of platelets or erythrocytes. Some ADP was also converted to ATP in blood, apparently via an enzyme present in plasma, but ATP was then rapidly removed by the leukocytes. Conversion of AMP to adenosine occurred via a plasma enzyme with little or no contribution from any cellular element. As expected, in blood the adenosine produced was removed very rapidly by erythrocytes and then converted to inosine and then hypoxanthine. In the absence of erythrocytes plasma supported only a slow conversion of adenosine to inosine and hypoxanthine, which was not influenced by platelets or leukocytes. This study has demonstrated that leukocytes and erythrocytes play a major role in adenine nucleotide metabolism in blood and that these cells, as well as endothelial cells, may be important determinants of the effects of ATP and ADP on platelets.

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 &mgr;mol/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 &mgr;mol/L) may be a consequence of inhibition by ATP of ADP action at ADP receptors.

GPIIb-IIIa antagonists cause rapid disaggregation of platelets pre-treated with cytochalasin D. Evidence that the stability of platelet aggregates depends on normal cytoskeletal assembly.

Platelet activation is accompanied by changes in the composition of the platelet cytoskeleton with rapid incorporation and displacement of certain proteins. Here we have inhibited cytoskeletal assembly by pretreating platelets with cytochalasin D (CyD) and investigated the effect on the stability of the aggregates that form. The experiments were performed in both citrated and hirudinized platelet-rich plasma (PRP) and aggregation was induced by adenosine diphosphate (ADP), collagen, the TXA2-mimetic U46619 and adrenaline. Platelets in the aggregates that formed, underwent rapid disaggregation on addition of EDTA or a GpIIb-IIIa antagonist such as MK-852 and GR144053F, all of which are agents that interfere with the ability of fibrinogen to interact with GpIIb-IIIa. This was the case irrespective of the aggregating agent used and occurred in both citrated and hirudinized PRP. In contrast, the rate of disaggregation brought about by some other agents, iloprost and ARL 66096, appeared to be unaffected by CyD. Information was also obtained on the effects of CyD on the cytoskeletal changes brought about by ADP and the effects on the cytoskeleton of subsequent addition of M K-852. The results show that CyD retards the incorporation of certain proteins (actin, myosin, alpha -actinin, actin binding protein and a 66 K protein) into the cytoskeleton and that subsequent addition of MK-852 results in rapid displacement of some of these with re-incorporation of a 31 K protein. The results suggest that the early changes in the cytoskeleton following platelet activation contribute to the stability of the aggregates that form, and that interference with these early changes results in aggregates that are easily disassembled by agents that interfere with GpIIb-IIIa-fibrinogen complex formation.

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 46.2 × 103 leukocytes/µl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1–10 µM) and ATP (10–100 µM) was markedly reduced, but to ATP (1000 µM) 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.

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: A new finding that may influence the design of anti-thrombotic agents

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y1 receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y1 antagonist) and AR-C69931 (a P2Y12 antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y1 but also with partial antagonism at P2Y12 receptors. Antagonism at P2Y12 receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X1 receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.

PGE2 reverses Gs-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-Gs-mediated inhibition of platelet aggregation by interaction with EP4 receptors

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.

‘VASPFix’ for measurement of VASP phosphorylation in platelets and for monitoring effects of P2Y12 antagonists

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.

Leukocytosis, Vascular Disease, and Adenine Nucleotide Metabolism

To 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.6 It was recognized that leukocytes are active in the metabolism of adenine nucleotides many years …

The composition of the platelet cytoskeleton following activation by ADP: effects of various agents that modulate platelet function.

Platelet activation by adenosine diphosphate (ADP) results in changes in the composition of the large cytoskeletal fragments that can be isolated following solubilization of platelets with Triton X-100 and low speed centrifugation. Here we have used several different agents that modify platelet responses to investigate some of the factors that affect these cytoskeletal changes. All the experiments involved use of hirudinized platelet-rich plasma in which TXA, synthesis and release of dense body constituents does not occur following platelet activation with ADP. ADP alone caused a significant and sustained increase in the cytoskeletal content of actin binding protein (ABP), myosin, α-actinin, a 66K protein and actin, and a significant decrease in a 31K protein. In the presence of MK-852 or GR 144053 (GpIIbDIIa antagonists), in samples merely left unstirred and in Glanzmanns thrombasthesenia, ADP produced no increase in ABP or the 66K protein and no decrease in the 31K protein. The increase in myosin and α-actinin became reversible but there was still incorporation of actin into the cytoskeleton. In the presence of ARL 66096 (a P(2T) purinoceptor antagonist that inhibits aggregation but not shape change) there was no increase in ABP or the 66K protein and no decrease in the 31K protein. ARL 66096 also prevented incorporation of α-actinin and actin. As with MK-852, myosin incorporation became reversible. Iloprost inhibited all the cytoskeletal changes, the effects of MgCI(2) were similar to those of MK-852, and acetylsalicylic acid (ASA) had no effect. In some experiments MK-852, ARL 66096, iloprost or MgCI, were added 0.5 min after the ADP. They all produced disaggregation and this was accompanied by reversal of the changes in the composition of the cytoskeleton that had occurred initially on stimulating the platelets with ADP. The results suggest that: (1) myosin is incorporated into the cytoskeleton transiently during shape change; (2) ADP interaction with the P(2T) receptor leads to incorporation of α-actinin and actin into the cytoskeleton as well as platelet aggregation; (3) further incorporation of α-actinin and myosin and incorporation of ABP and the 66K protein occur consequent to fibrinogen binding and platelet aggregation; (4) displacement of the 31K protein from the cytoskeleton is also a consequence of fibrinogen binding and platelet aggregation; (5) platelet disaggregation is accompanied by reversal of any cytoskeletal changes that have already occurred; (6) continuous occupation of the P(2T) receptor is required for maintenance of the cytoskeletal changes; (7) CAMP inhibits and reverses cytoskeletal assembly; and (8) MgCl(2) acts similarly to a GpIIb/IIIa antagonist under these experimental conditions.

Synthesis of novel (benzimidazolyl)isoquinolinols and evaluation as adenosine A1 receptor tools

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in eukaryotes. The adenosine A1 receptor (A1AR) is a class A GPCR that is of interest as a therapeutic target particularly in the treatment of cardiovascular disease and neuropathic pain. Increased knowledge of the role A1AR plays in mediating these pathophysiological processes will help realise the therapeutic potential of this receptor. There is a lack of enabling tools such as selective fluorescent probes to study A1AR, therefore we designed a series of (benzimidazolyl)isoquinolinols conjugated to a fluorescent dye (31–35, 42–43). An improved procedure for the synthesis of isoquinolinols from tetrahydroisoquinolinols via oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and atmospheric oxygen is reported. This synthetic method offers advantages over previous metal-based methods for the preparation of isoquinolinols and isoquinolines, which are important scaffolds found in many biologically active compounds and natural products. We report the first synthesis of the (benzimidazolyl)isoquinolinol compound class, however the fluorescent conjugates were not successful as A1AR fluorescent ligands.