Extracellular Ca2+ modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation

ADP is considered a weak platelet agonist due to the limited aggregation responses it induces in vitro at physiological concentrations of extracellular Ca2+ [(Ca2+)o]. Lowering [Ca2+]o paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A2 production. This study examined the role of ectonucleotidases in the [Ca2+]o-dependence of platelet activation. Reducing [Ca2+]o from millimolar to micromolar levels converted ADP (10 μmol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A2 production with aspirin had no effect on this [Ca2+]o-dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca2+]o resulting in a robust and sustained aggregation in both conditions. Measurements of extracellular ADP revealed reduced degradation in both plasma and apyrase-containing saline at micromolar compared to millimolar [Ca2+]o. As reported previously, thromboxane A2 generation was enhanced at low [Ca2+]o, however this was independent of ectonucleotidase activity. P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y12 receptors for sustained ADP-evoked aggregation, with a minor role for P2Y1. In conclusion, Ca2+-dependent ectonucleotidase activity is a major factor determining the extent of platelet aggregation to ADP and must be controlled for in studies of P2Y receptor activation.


Summary
ADP is considered a weak platelet agonist due to the limited aggregation responses it induces in vitro at physiological concentrations of extracellular Ca 2+ [(Ca 2+ ) o ]. Lowering [Ca 2+ ] o paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A 2 production. This study examined the role of ectonucleotidases in the [Ca 2+ ] o -dependence of platelet activation. Reducing [Ca 2+ ] o from millimolar to micromolar levels converted ADP (10 lmol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A 2 production with aspirin had no effect on this [Ca 2+ ] o -dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca 2+ ] o resulting in a robust and sustained aggregation in both conditions. Measurements of extracellular ADP revealed reduced degradation in both plasma and apyrasecontaining saline at micromolar compared to millimolar [Ca 2+ ] o . As reported previously, thromboxane A 2 generation was enhanced at low [Ca 2+ ] o , however this was independent of ectonucleotidase activity . P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y 12 receptors for sustained ADP-evoked aggregation, with a minor role for P2Y 1 .
In conclusion, Ca 2+ -dependent ectonucleotidase activity is a major factor determining the extent of platelet aggregation to ADP and must be controlled for in studies of P2Y receptor activation.

research paper
First published online 21 February 2011 phosphorylation (Garcia et al, 2007), leading to loss of secondary aggregation (Mustard et al, 1975;Packham et al, 1989), however exactly how Ca 2+ achieves this effect is not known.
Following stimulation of platelet P2Y receptors with ADP, the duration and amplitude of the response can be regulated by two principal mechanisms, firstly, desensitization of the P2Y receptors preventing further signalling and, secondly, removal of ADP by ectonucleotidases. Ectonucleotidases comprise a large family of extracellular nucleotide degrading enzymes including ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPs), alkaline phosphatases and 5¢ nucleotidase (Zimmermann, 2000). ADP derived from platelets and other blood cells is thought to predominantly be metabolised by E-NTPDase1 (CD39), a membrane-bound enzyme expressed by endothelial cells, lymphocytes and macrophages (Kansas et al, 1991;Marcus et al, 1997), as well as microparticles that originate from these cell types (Atkinson et al, 2006;Banz et al, 2008). CD39 converts ADP to AMP, which is subsequently converted to adenosine, an inhibitor of platelet function, by 5¢ nucleotidase (CD73) expressed on endothelial cells and in plasma (Coade & Pearson, 1989;Zimmermann, 2000;Heptinstall et al, 2005). There is also evidence that soluble E-NPPases in plasma can degrade ADP directly to adenosine (Birk et al, 2002;Cauwenberghs et al, 2006), thus ectonucleotidases can convert prothrombotic mediators into inhibitors of platelet activation.
In this study we have investigated further the mechanism(s) underlying the differential responses to ADP at physiological compared to low (micromolar) extracellular calcium concentrations. We demonstrate that degradation of ADP by Ca 2+ -dependent ectonucleotidases is an important factor in determining the amplitude and duration of platelet aggregation. The results have consequences for understanding the effectiveness of ADP as a platelet agonist, and in the selection of experimental conditions to explore P2Y receptor activation.

Platelet preparation
Blood was obtained from healthy, aspirin-free, volunteers according to a protocol approved by the local ethical committee of the University of Leicester. Blood was drawn from the forearm by venepuncture into a syringe containing acid citrate dextrose anticoagulant (ACD: 85 mmol/l trisodium citrate, 78 mmol/l citric acid, 111 mmol/l glucose) 9:1 v/ v. Platelet-rich plasma (PRP) was obtained by centrifugation at 700 g for 5 min. When re-calcified, 20 mmol/l CaCl 2 [calculated using a Nomogram (Hastings et al, 1934)] was added to citrated PRP to achieve [Ca 2+ ] o of approximately 2 mmol/l immediately prior to each experiment. The extracellular Ca 2+ in nominally Ca 2+ -free saline and in similar citrated plasma: saline mixtures has been estimated to be approximately 20 and 17 lmol/l respectively Rolf et al, 2001).
To prepare washed platelet suspensions, apyrase (0AE32 u/ml) and, where stated, aspirin (100 lmol/l or 1 mmol/l) were added to the PRP and platelets pelleted by centrifugation at 350 g for 20 min. Platelets were then resuspended in a volume of nominally Ca 2+ -free saline (145 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl 2 10 mmol/l HEPES, 10 mmol/l glucose, 1 g/l fibrinogen pH 7AE35) equal to that of the removed plasma, with or without apyrase (0AE32 u/ml) as required by the specific experiment. In experiments performed at physiological calcium concentrations, 2 mmol/l CaCl 2 was added to the platelets immediately prior to use.

Platelet disaggregation
Washed, apyrase-free platelets were stimulated with ADP (10 lmol/l) at 37°C under stirring conditions in the presence of 2 mmol/l Ca 2+ . After 2 min, apyrase (0AE32 u/ml), the P2Y 1 receptor antagonist MRS2179 (10 lmol/l), the P2Y 12 receptor antagonist AR-C69931MX (1 lmol/l) or a saline control was added to the suspension. Disaggregation was assessed 3 min after the addition of the P2Y receptor antagonists or apyrase and calculated as a percentage of the peak ADP-evoked aggregation.

ADP measurement
The concentration of extracellular ADP was assessed by luciferin:luciferase luminescence measurements after conversion to ATP via a method adapted from Heath (2004). Briefly, 2 min after addition of 10 lmol/l ADP to plasma or apyrasecontaining saline, with or without Ca 2+ , 50 ll samples were removed and added to a mixture of 420 ll Tris-K acetate buffer (100 mmol/l Tris-acetate, 2 mmol/l EDTA, 25 mmol/l potassium acetate), 10 ll pyruvate kinase/phosphoenolpyruvate (prepared by mixing equal volumes of 10 mg/ml pyruvate kinase and 200 mmol/l phosphoenolpyruvate) and 20 ll CHRONO-LUME. Luminescence was measured using a Model 400 lumi-aggregometer (Chronolog) and converted to ATP levels based upon a calibration curve for each batch of CHRONO-LUME.

TXB 2 measurements
TXB 2 synthesis was measured as an indication of TXA 2 production due to the highly labile nature of TXA 2 . Washed platelets were stimulated with ADP (10 lmol/l) at 37°C under stirring conditions for 3 min in the presence and absence of apyrase (0AE32 u/ml), in both physiological Ca 2+ and nominally Ca 2+ -free conditions, and reactions terminated by snap freezing. For analysis of TXB 2 , samples were thawed and centrifuged at 3000 g for 10 min at 4°C. The supernatant was diluted 1:5 using the buffer supplied with the assay kit and TXB 2 determined according to the manufacturer's instructions (Cambridge Bioscience).

Statistics
Records of aggregation are from individual experiments, typical of 3-7 donors. Differences between means ± SEM were assessed using paired Student's t-test and a P value of <0AE05 was considered to be significant. P values are indicated at levels of <0AE05 (*), <0AE01 (**) and <0AE001 (***).

Results
Extracellular Ca 2+ levels regulate ADP-evoked aggregation independently of TXA 2 synthesis ADP (10 lmol/l) evoked a sustained aggregation of platelets in plasma anti-coagulated with citrate that reduced the extracellular Ca 2+ concentration [(Ca 2+ ) o ] to the micromolar range (Fig 1A, E; average peak aggregation of 53AE9 ± 3AE4%). When the medium was recalcified to approximately 2 mmol/l free Ca 2+ , the aggregation was converted to a transient response that returned to baseline levels of transmission ()2AE8 ± 2%) within approximately 2 min (Fig 1A, E). Previous studies of this [Ca 2+ ] o -dependent aggregation response showed that production of TXA 2 was enhanced at micromolar compared to millimolar [Ca 2+ ] o levels and concluded that secondary stimulation of TXA 2 receptors is responsible for the reversible nature of the ADP-evoked aggregation (Mustard et al, 1975;Packham et al, 1989;Garcia et al, 2007). However, we observed a similar effect of [Ca 2+ ] o on aggregation when TXA 2 synthesis was blocked by aspirin ( Fig 1B, E; average values at 2 min of 50AE7 ± 3% and 0AE9 ± 0AE3% in low and physiological Ca 2+ levels respectively). In platelets resuspended in a physiological saline with apyrase, 10 lmol/l ADP evoked a transient aggregation response in the presence of 2 mmol/l [Ca 2+ ] o , which was also converted to a sustained response by omission of CaCl 2 from the saline (Fig 1C, E; transmission levels 2 min after ADP of 0AE6 ± 2% and 53AE2 ± 5AE4%, respectively), in agreement with reports by other groups (Mustard et al, 1975;Packham et al, 1989). As observed for platelets in the presence of plasma, aspirin did not block the sustained aggregation in salines with micromolar [Ca 2+ ] o (Fig 1D, E; average values at 2 min of 55AE9 ± 1AE3% and 7AE1 ± 4AE2% in low and physiological Ca 2+ levels respectively). To ensure that TXA 2 generation was completely inhibited, experiments were repeated in the presence of 1 mmol/l aspirin, which also had no significant effect on the ability of reduced [Ca 2+ ] o to enhance platelet aggregation (P > 0AE05, data not shown). Together, these data suggest that factor(s) other than altered TXA 2 production must contribute to the ability of reduced [Ca 2+ ] o to enhance ADP-evoked aggregation.

Prevention of ADP degradation abolishes the reversal of aggregation by calcium
Given that our washed platelet preparation contained apyrase (E-NTPDase1 isolated from potato to prevent P2Y receptor desensitization) and PRP has been reported to contain endogenous ectonucleotidases, we considered whether degradation of ADP contributed to the transient nature of the ADPevoked aggregation at millimolar Ca 2+ levels. Aggregation evoked by the hydrolysis-resistant analogue ADPbS was not significantly different in the presence or absence of extracellular Ca 2+ (Fig 2A, D; aggregation at 2 min of 45AE8 ± 3AE7% and 42AE9 ± 5AE6% in normal and low [Ca 2+ ] o , respectively; P > 0AE05). Moreover, when platelets were resuspended in the absence of apyrase, and experiments performed rapidly to limit the effects of desensitization, ADP-evoked aggregation was also sustained in the presence of 2 mmol/l extracellular Ca 2+ (Fig 2B, D

ADP degradation is accelerated by millimolar calcium concentrations
To directly assess the extent of ADP degradation in millimolar versus micromolar [Ca 2+ ] o concentrations, ADP (10 lmol/l) was added to apyrase-treated saline or platelet-free plasma and the ADP concentration after 2 min measured by luminescence following conversion to ATP (see Methods). The concentration of ADP remaining in nominally Ca 2+ -free saline was 1AE61 ± 0AE06 lmol/l, which was significantly reduced to 0AE079 ± 0AE03 lmol/l (P < 0AE001) in the presence of 2 mmol/l Ca 2+ , indicating accelerated nucleotidase activity by physiological  (Fig 3A). Similarly, ADP incubated with citrated plasma was degraded, from 10 to 2AE1 ± 0AE27 lmol/l, by enzymes endogenous to plasma, whereas under recalcified conditions the ADP remaining was markedly lower, at 0AE78 ± 0AE14 lmol/l (P < 0AE001) (Fig 3B). A significant effect of [Ca 2+ ] o on degradation of 10 lmol/l ADP was also detected at earlier time points, as shown by measurements after only 10 s ( Figure S1). This suggests that platelets in the presence of millimolar Ca 2+ are exposed to a reduced level of ADP throughout most of the experiment compared to at micromolar Ca 2+ levels. In contrast, addition of 2 mmol/l MgCl 2 to nominally Ca 2+ -free saline did not significantly affect ADP degradation or lead to a transient aggregation response ( Figure S2). Together with the data in Fig 2, these results support the conclusion that reduced ADP degradation substantially contributes to the paradoxical amplifying effect of reducing Ca 2+ on platelet aggregation. This is also consistent with the reported enhancement of ectonucleotidase activity at millimolar concentrations of calcium compared to its nominal absence or in the presence of a chelator, such as EGTA (Christoforidis et al, 1995;Strobel et al, 1996;Marcus et al, 1997).

Reversal of aggregation is due to removal of ADP, not negative feedback by adenosine
In plasma, 5¢ nucleotidases convert AMP generated by the degradation of ATP and ADP to adenosine (Coade & Pearson, 1989;Heptinstall et al, 2005), thus we also considered whether the transient responses to ADP in plasma involved inhibition via G as -coupled adenosine A2a receptors. In citrated PRP, adenosine (10 lmol/l) inhibited ADP (10 lmol/l)-evoked responses, resulting in aggregation responses similar to those observed with ADP in physiological calcium concentrations ( Figure S3). This inhibition by adenosine was abolished by the addition of adenosine deaminase (1 u/ml). In recalcified PRP, the addition of adenosine deaminase had no effect on ADPevoked aggregation (Fig 4), indicating that negative feedback by the generation of adenosine does not contribute to the reversibility of ADP-mediated responses.

TXA 2 generation is enhanced at low extracellular calcium concentrations independently of altered ectonucleotidase activity
To investigate whether the reduced TXA 2 generation previously reported by others (Harfenist et al, 1987;Packham et al, 1987Packham et al, , 1989 at millimolar [Ca 2+ ] o was a consequence of termination of ADP signalling by ectonucleotidases, the effect of apyrase (0AE32 u/ml) at micromolar and millimolar [Ca 2+ ] o was examined on TXB 2 production from washed platelets 3 min after stimulation with 10 lmol/l ADP (Fig 6). As reported previously (Harfenist et al, 1987;Packham et al, 1987Packham et al, , 1989, TXB 2 production from apyrase-treated platelets was markedly reduced at physiological extracellular Ca 2+ concentrations compared to that observed in the nominal absence of Ca 2+ . However, similar results were observed in saline lacking apyrase. This indicates that although increased [Ca 2+ ] o reduces TXB 2 synthesis, this is not dependent on the effect of Ca 2+ on ectonucleotidases. Relative contribution of ADP degradation versus P2Y receptor desensitization in limiting platelet responses to ADP P2Y 1 and P2Y 12 receptors are both susceptible to receptor desensitization after prolonged agonist stimulation (Hardy et al, 2005;Mundell et al, 2006). To determine the relative importance of receptor desensitization versus ectonucleotidase activity in ADP-evoked aggregation, the pan-PKC inhibitor GF109203X was used to attenuate receptor desensitization and aggregation was measured in citrated PRP before and after Reversal of aggregation by ADP degrading enzymes is largely due to the termination of P2Y 12 receptor signalling. (A) Aggregation of washed platelets (apyrase-free) was stimulated with ADP (10 lmol/l) soon after resuspension in the presence of 2 mmol/l Ca 2+ and after 2 min (arrow) one of the following was added: apyrase (0AE32 u/ml), ARC-69931MX (Cang, 1 lmol/l), MRS2179 (MRS, 10 lmol/l), or a vehicle control. (B) Disaggregation was measured 3 min after the addition of the inhibitors or vehicle and calculated as a percentage of peak ADP-evoked aggregation response. TXA 2 generation is enhanced at low extracellular calcium concentrations independently of apyrase activity. ADP (10 lmol/l)evoked thromboxane B 2 production (as a measure of TXA 2 generation) in washed suspensions of platelets with and without apyrase in nominally Ca 2+ -free conditions or in the presence of 2 mmol/l extracellular Ca 2+ . TXB 2 was measured 3 min after addition of ADP. recalcification ( Fig 7A). An intermediate concentration of ADP (2 lmol/l) was used for these experiments to unmask a clear potentiating effect of PKC inhibition, which increased the sustained aggregation at 3 min from 36AE9 ± 6AE5% to 60AE5 ± 5AE6% in micromolar [Ca 2+ ] o (Fig 7B). In contrast, after recalcification, aggregation in the presence of GF109203X or vehicle control, was not significantly different and returned to baseline levels of )3AE5 ± 1AE6% and 2AE7 ± 1AE2% (P > 0AE05), respectively, 3 min after stimulation ( Fig 7B). Thus, ADP degradation overrides any contribution by P2Y receptor desensitization to the reversal of aggregation in physiological external Ca 2+ concentrations.

Discussion
Reports of differential platelet responses to ADP in physiological versus nominally Ca 2+ -free conditions emerged over 20 years ago (Mustard et al, 1975;Packham et al, 1989). These studies concluded that enhanced TXA 2 production accounts for the paradoxical amplifying effect of lowering Ca 2+ on ADP-evoked aggregation. The present study now shows that altered degradation of ADP can also contribute to this phenomenon. The known Ca 2+ -dependence of ecto-ADPases (Marcus et al, 1997;Zimmermann, 2000) provides the basis for the difference observed in millimolar versus micromolar [Ca 2+ ] o and this conclusion is supported by direct measurements of ADP. The sustained aggregation evoked by ADP is largely due to stimulation of P2Y 12 receptors, consistent with previous reports of the more crucial role of this G i -coupled pathway compared to P2Y 1 in amplifying responses to ADP, collagen and thrombin receptors (Trumel et al, 1999;Dorsam & Kunapuli, 2004;Hechler et al, 2005;Jackson et al, 2005;Cosemans et al, 2006). The present results also highlight the importance of controlling for nucleotide breakdown in studies of P2 receptor signalling when the external Ca 2+ concentration is modified. For example, it is common practice to include soluble apyrase to limit P2 receptor desensitization within in vitro experiments and simply to vary the external [Ca 2+ ] to investigate the relative contribution of Ca 2+ entry versus release pathways in nucleotide-evoked signalling events.
The limited aggregation response at normal [Ca 2+ ] o has contributed to the view that ADP is a 'weak platelet agonist'. However, when metabolism of ADP is limited, the ability of this agonist to stimulate sustained aggregation, as shown in the present study, is more consistent with the substantial reduction in platelet activation observed in P2Y 1 and P2Y 12 receptordeficient mice (Fabre et al, 1999;Leon et al, 1999;Andre et al, 2003) and the major role of ADP in amplifying collagen-and thrombin-evoked responses in vitro. It is possible that dense granule secretion evoked by collagen and thrombin provides a more sustained source of ADP compared to the single bolus application used in standard in vitro experiments. Furthermore, ATP (and thus also ADP) remains sustained for a considerable time near sites of vascular injury (Born & Kratzer, 1984), probably reflecting the continual recruitment and activation of platelets during the haemostatic process. Thus, the in vitro experimental condition that limits ADP degradation, such as micromolar [Ca 2+ ] o , may more closely represent the ability of this agonist to stimulate platelet function in vivo. Alternatively, use of a non-hydrolysable analogue, such as ADPbS, or repeated application of ADP to replace degraded agonist should be considered within in vitro studies designed to investigate mechanisms of ADP-dependent platelet activation.
Whilst ADP degradation significantly contributed to the transient nature of the responses to ADP in millimolar [Ca 2+ ] o , we agree with earlier studies (Packham et al, 1989;Garcia et al, 2007) that TXA 2 generation is lower at millimolar compared to micromolar [Ca 2+ ] o . In our experiments, the marked enhancement of TXB 2 generation by lowering [Ca 2+ ] o was also observed in apyrase-free saline, indicating that the Ca 2+dependent modulation of TXA 2 generation can occur independently of effects on ectonucleotidase activity. It has been reported that reduced TXA 2 synthesis in physiological calcium concentrations is the result of inhibited ERK phosphorylation (Garcia et al, 2007), however the process by which this is achieved is unclear, and whether these effects are downstream of an extracellular event or whether calcium influx is required, remains to be investigated. In the present study, 10 lmol/l ADP stimulated sustained aggregation at millimolar [Ca 2+ ] o in the absence of apyrase, conditions under which there was no detectable TXA 2 generation, suggesting that this response is independent of secondary signalling through TXA 2 receptors. At lower concentrations of ADP, however, the release of secondary agonists is required to achieve full aggregation, therefore the effect of extracellular calcium on TXA 2 production may be more significant, and modulation of ADP-evoked aggregation by [Ca 2+ ] o may result from a combination of both altered ectonucleotidase activity and TXA 2 production. Although we did not observe any difference in aggregation within the normal physiological range of extracellular calcium concentrations (0AE5-2 mmol/l) (three donors, data not shown), results from this study demonstrate the impact of variable ectonucleotidase activity on platelet function, which may have profound implications in certain clinical conditions. It has previously been reported that in blood from patients with elevated leucocyte counts, degradation of ADP is accelerated and aggregation in response to ADP is reduced due to increased NTPDase levels (Pulte et al, 2007;Glenn et al, 2008). Moreover, in a rat model of cholestatic liver disease where plasma ectonucleotidase activity is enhanced, reduced aggregation was exhibited in response to ADP and low dose collagen (which is dependent on ADP secretion) (Witters et al, 2010). Conversely, individuals demonstrating reduced ectonucleotidase expression may have more reactive platelets and be more susceptible to thrombotic events. Such patients may benefit from therapeutic intervention with soluble forms of NTPDase1.
In conclusion, the present study shows that reduced degradation of ADP by ectonucleotidases contributes to the paradoxical amplification of ADP-evoked aggregation at micromolar compared to millimolar extracellular Ca 2+ levels. The sustained inside-out activation of fibrinogen receptors that occurs in response to ADP at low [Ca 2+ ] o is likely to be more representative of the potential contribution of ADP to a developing thrombus in vivo, where a constant supply of this P2Y receptor agonist from activated platelets can override enzymatic clearance in the vicinity of a developing thrombus.