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

Abstract

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.

ADP is an important platelet agonist during haemostasis and thrombosis, exerting its effects through two G-protein-coupled receptors P2Y₁ and P2Y₁₂ (Gachet, 2008). P2Y₁ is coupled to G_αq, leading to an increase in cytosolic calcium through stimulation of phospholipase C_β (PLC_β) (Jin et al, 1998; Savi et al, 1998; Leon et al. 1999), whereas P2Y₁₂ is coupled to G_αi, leading to activation of phosphatidylinositol 3-Kinase (PI3-K) (Trumel et al, 1999; Jackson et al, 2005) and inhibition of adenylate cyclase. For platelet-platelet adhesion, and thus thrombus formation, activation of the fibrinogen receptor α_IIbβ₃ is required, a process that depends upon concomitant stimulation of G_αq and G_αi signalling pathways (Jin & Kunapuli, 1998). Several platelet agonists bind to receptors coupled to G_αq, including thrombin, thromboxane A₂ (TXA₂) and ADP. However, ADP is the only platelet agonist that stimulates G_αi signalling at physiological concentrations and is therefore an essential co-stimulus to achieve full functional responses for all known platelet agonists (Dorsam & Kunapuli, 2004; Gachet, 2008). Despite the central role of ADP in platelet aggregation and thrombogenesis, it is normally considered to be a weak platelet agonist due to the reversible nature of the aggregation response observed in vitro at physiological levels of external Ca²⁺ (Gachet, 2008). Extremely low levels of extracellular Ca²⁺ abolish fibrinogen binding to α_IIbβ₃ integrin, however at micromolar extracellular calcium concentrations, ADP-evoked aggregation is enhanced compared to physiological Ca²⁺ levels and not readily reversible (Mustard et al, 1975; Packham et al, 1989). Although this paradoxical effect was reported more than two decades ago, the underlying basis whereby extracellular calcium modulates ADP-evoked aggregation remains unclear. It has been proposed that millimolar Ca²⁺ levels inhibit TXA₂ generation via altered ERK phosphorylation (Garcia et al, 2007), leading to loss of secondary aggregation (Mustard et al, 1975; Packham et al, 1989), however exactly how Ca²⁺ 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 Ca²⁺-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.

Materials and methods

Materials

CHRONO-LUME was purchased from Labmedics (Manchester, UK), Pyruvate kinase was obtained from Roche Diagnostics Limited (East Sussex, UK), GF109203X and MRS2179 were purchased from Tocris (Bristol, UK). Thromboxane B₂ (TXB₂) assay kits were purchased from Cambridge Bioscience LTD (Cambridge, UK). Cangrelor (ARC-69931MX) was a kind gift from AstraZeneca (Moindal, Sweden). ADP, apyrase (gradeVII), aspirin, phosphoenolpyruvate and all other chemicals were purchased from Sigma (Poole, UK).

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 CaCl₂ [calculated using a Nomogram (Hastings et al, 1934)] was added to citrated PRP to achieve [Ca²⁺]_o of approximately 2 mmol/l immediately prior to each experiment. The extracellular Ca²⁺ in nominally Ca²⁺-free saline and in similar citrated plasma:saline mixtures has been estimated to be approximately 20 and 17 μmol/l respectively (Packham et al, 1987; Rolf et al, 2001).

To prepare washed platelet suspensions, apyrase (0·32 u/ml) and, where stated, aspirin (100 μmol/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 Ca²⁺-free saline (145 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl₂ 10 mmol/l HEPES, 10 mmol/l glucose, 1 g/l fibrinogen pH 7·35) equal to that of the removed plasma, with or without apyrase (0·32 u/ml) as required by the specific experiment. In experiments performed at physiological calcium concentrations, 2 mmol/l CaCl₂ was added to the platelets immediately prior to use.

Platelet aggregation

PRP or washed platelet suspensions were diluted (1:1) in saline with or without apyrase (0·32 u/ml) and stimulated with ADP at 37°C under stirring conditions. Aggregation was measured using optical aggregometry (Model 400 lumi-aggregometer; Chronolog, Havertown, PA, USA).

Platelet disaggregation

Washed, apyrase-free platelets were stimulated with ADP (10 μmol/l) at 37°C under stirring conditions in the presence of 2 mmol/l Ca²⁺. After 2 min, apyrase (0·32 u/ml), the P2Y₁ receptor antagonist MRS2179 (10 μmol/l), the P2Y₁₂ receptor antagonist AR-C69931MX (1 μmol/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 μmol/l ADP to plasma or apyrase-containing saline, with or without Ca²⁺, 50 μl samples were removed and added to a mixture of 420 μl Tris-K acetate buffer (100 mmol/l Tris-acetate, 2 mmol/l EDTA, 25 mmol/l potassium acetate), 10 μl pyruvate kinase/phosphoenolpyruvate (prepared by mixing equal volumes of 10 mg/ml pyruvate kinase and 200 mmol/l phosphoenolpyruvate) and 20 μl 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.

TXB₂ measurements

TXB₂ synthesis was measured as an indication of TXA₂ production due to the highly labile nature of TXA₂. Washed platelets were stimulated with ADP (10 μmol/l) at 37°C under stirring conditions for 3 min in the presence and absence of apyrase (0·32 u/ml), in both physiological Ca²⁺ and nominally Ca²⁺-free conditions, and reactions terminated by snap freezing. For analysis of TXB₂, 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 TXB₂ 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 <0·05 was considered to be significant. P values are indicated at levels of <0·05 (*), <0·01 (**) and <0·001 (***).

Results

Extracellular Ca²⁺ levels regulate ADP-evoked aggregation independently of TXA₂ synthesis

ADP (10 μmol/l) evoked a sustained aggregation of platelets in plasma anti-coagulated with citrate that reduced the extracellular Ca²⁺ concentration [(Ca²⁺)_o] to the micromolar range (Fig 1A, E; average peak aggregation of 53·9 ± 3·4%). When the medium was recalcified to approximately 2 mmol/l free Ca²⁺, the aggregation was converted to a transient response that returned to baseline levels of transmission (−2·8 ± 2%) within approximately 2 min (Fig 1A, E). Previous studies of this [Ca²⁺]_o-dependent aggregation response showed that production of TXA₂ was enhanced at micromolar compared to millimolar [Ca²⁺]_o levels and concluded that secondary stimulation of TXA₂ 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 [Ca²⁺]_o on aggregation when TXA₂ synthesis was blocked by aspirin (Fig 1B, E; average values at 2 min of 50·7 ± 3% and 0·9 ± 0·3% in low and physiological Ca²⁺ levels respectively). In platelets resuspended in a physiological saline with apyrase, 10 μmol/l ADP evoked a transient aggregation response in the presence of 2 mmol/l [Ca²⁺]_o, which was also converted to a sustained response by omission of CaCl₂ from the saline (Fig 1C, E; transmission levels 2 min after ADP of 0·6 ± 2% and 53·2 ± 5·4%, 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 [Ca²⁺]_o (Fig 1D, E; average values at 2 min of 55·9 ± 1·3% and 7·1 ± 4·2% in low and physiological Ca²⁺ levels respectively). To ensure that TXA₂ 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 [Ca²⁺]_o to enhance platelet aggregation (P > 0·05, data not shown). Together, these data suggest that factor(s) other than altered TXA₂ production must contribute to the ability of reduced [Ca²⁺]_o to enhance ADP-evoked aggregation.

Fig 1.

Platelet responses to ADP are sustained at low extracellular calcium concentrations. Sample (A–D) or average (E) responses to 10 μmol/l ADP without added extracellular Ca²⁺ (low Ca²⁺) or in the presence of approximately 2 mmol/l external Ca²⁺. (A) citrated PRP; (B) citrated PRP treated with aspirin (100 μmol/l); (C) platelets resuspended in saline containing apyrase (0·32 u/ml); (D) platelets resuspended in saline containing apyrase (0·32 u/ml) and aspirin (100 μmol/l); (E) Aggregation measured at 2 min.

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 ADP-evoked aggregation at millimolar Ca²⁺ levels. Aggregation evoked by the hydrolysis-resistant analogue ADPβS was not significantly different in the presence or absence of extracellular Ca²⁺ (Fig 2A, D; aggregation at 2 min of 45·8 ± 3·7% and 42·9 ± 5·6% in normal and low [Ca²⁺]_o, respectively; P > 0·05). 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 Ca²⁺ (Fig 2B, D; 59·3 ± 3·8 and 61·9 ± 2·4% in normal and low Ca²⁺ respectively). Finally, the [Ca²⁺]_o-dependence of ADP-evoked aggregation responses in plasma was abolished when platelets were resuspended in autologous heat-treated plasma (60°C, 30 min) to destroy enzymatic activity (Fig 2C, D; 41·1 ± 1·1 and 41·5 ± 1·9% in millimolar versus micromolar [Ca²⁺]_o respectively). Together, these observations are consistent with a role for Ca²⁺-dependent nucleotidase activity in the [Ca²⁺]_o-dependence to ADP-evoked sustained aggregation responses.

Fig 2.

Prevention of ADP degradation leads to sustained aggregation at physiological extracellular calcium concentrations. Sample (A–C) or average (D) aggregation responses without added extracellular Ca²⁺ (low Ca²⁺) or in the presence of approximately 2 mmol/l external Ca²⁺. (A) Washed platelets (containing 0·32 u/ml apyrase) stimulated with the hydrolysis-resistant analogue ADPβS (10 μmol/l); (B) platelets soon after resuspension in apyrase-free saline stimulated with ADP (10 μmol/l); (C) platelets resuspended in heat-treated citrated plasma stimulated with ADP (10 μmol/l); (D) Average aggregation measured at 2 min.

ADP degradation is accelerated by millimolar calcium concentrations

To directly assess the extent of ADP degradation in millimolar versus micromolar [Ca²⁺]_o concentrations, ADP (10 μmol/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 Ca²⁺-free saline was 1·61 ± 0·06 μmol/l, which was significantly reduced to 0·079 ± 0·03 μmol/l (P < 0·001) in the presence of 2 mmol/l Ca²⁺, indicating accelerated nucleotidase activity by physiological [Ca²⁺]_o (Fig 3A). Similarly, ADP incubated with citrated plasma was degraded, from 10 to 2·1 ± 0·27 μmol/l, by enzymes endogenous to plasma, whereas under recalcified conditions the ADP remaining was markedly lower, at 0·78 ± 0·14 μmol/l (P < 0·001) (Fig 3B). A significant effect of [Ca²⁺]_o on degradation of 10 μmol/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 Ca²⁺ are exposed to a reduced level of ADP throughout most of the experiment compared to at micromolar Ca²⁺ levels. In contrast, addition of 2 mmol/l MgCl₂ to nominally Ca²⁺-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 Ca²⁺ 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).

Fig 3.

ADP degradation by apyrase and endogenous ectonucleotidases present in plasma is reduced at micromolar calcium concentrations. Degradation of a single bolus of 10 μmol/l ADP was assessed by measurements of the concentration of ADP remaining after 2 min in (A) saline containing apyrase (0·32 u/ml) in the nominal absence of Ca²⁺ or in the presence of 2 mmol/l Ca²⁺; and (B) citrated plasma before and after re-calcification to 2 mmol/l.

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_αs-coupled adenosine A2a receptors. In citrated PRP, adenosine (10 μmol/l) inhibited ADP (10 μmol/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 ADP-evoked aggregation (Fig 4), indicating that negative feedback by the generation of adenosine does not contribute to the reversibility of ADP-mediated responses.

Fig 4.

Transient ADP-evoked aggregation responses resulting from Ca²⁺-dependent ectonucleotidase activity do not involve negative feedback by adenosine. Platelet aggregation stimulated by ADP (10 μmol/l) in nominally Ca²⁺-free conditions or in the presence of 2 mmol/l Ca²⁺ with or without adenosine deaminase (AD, 1 u/ml).

Sustained aggregation to ADP requires constant P2Y₁₂ receptor stimulation

We next sought to determine whether reversal of aggregation by Ca²⁺-dependent nucleotidases was due to loss of activation of P2Y₁, P2Y₁₂ or both receptors. Disaggregation of ADP (10 μmol/l)-stimulated platelets was measured in response to apyrase (0·32 u/ml), the P2Y₁₂ antagonist cangrelor (1 μmol/l) or the P2Y₁ antagonist MRS2179 (10 μmol/l) (Fig 5A). Cangrelor reversed aggregation by 74·6 ± 5·9%, comparable to that observed with apyrase 74·9 ± 1·2% (Fig 5B). In contrast, MRS2179 caused a more moderate reversal of aggregation (reduction of 24·0 ± 7·8%; Fig 5B). Thus, reversal of ADP-induced aggregation by ectonucleotidases is largely due to loss of signalling through P2Y₁₂ receptors.

Fig 5.

Reversal of aggregation by ADP degrading enzymes is largely due to the termination of P2Y₁₂ receptor signalling. (A) Aggregation of washed platelets (apyrase-free) was stimulated with ADP (10 μmol/l) soon after resuspension in the presence of 2 mmol/l Ca²⁺ and after 2 min (arrow) one of the following was added: apyrase (0·32 u/ml), ARC-69931MX (Cang, 1 μmol/l), MRS2179 (MRS, 10 μmol/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.

TXA₂ generation is enhanced at low extracellular calcium concentrations independently of altered ectonucleotidase activity

To investigate whether the reduced TXA₂ generation previously reported by others (Harfenist et al, 1987; Packham et al, 1987, 1989) at millimolar [Ca²⁺]_o was a consequence of termination of ADP signalling by ectonucleotidases, the effect of apyrase (0·32 u/ml) at micromolar and millimolar [Ca²⁺]_o was examined on TXB₂ production from washed platelets 3 min after stimulation with 10 μmol/l ADP (Fig 6). As reported previously (Harfenist et al, 1987; Packham et al, 1987, 1989), TXB₂ production from apyrase-treated platelets was markedly reduced at physiological extracellular Ca²⁺ concentrations compared to that observed in the nominal absence of Ca²⁺. However, similar results were observed in saline lacking apyrase. This indicates that although increased [Ca²⁺]_o reduces TXB₂ synthesis, this is not dependent on the effect of Ca²⁺ on ectonucleotidases.

Fig 6.

TXA₂ generation is enhanced at low extracellular calcium concentrations independently of apyrase activity. ADP (10 μmol/l)-evoked thromboxane B₂ production (as a measure of TXA₂ generation) in washed suspensions of platelets with and without apyrase in nominally Ca²⁺-free conditions or in the presence of 2 mmol/l extracellular Ca²⁺. TXB₂ was measured 3 min after addition of ADP.

Relative contribution of ADP degradation versus P2Y receptor desensitization in limiting platelet responses to ADP

P2Y₁ and P2Y₁₂ 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 recalcification (Fig 7A). An intermediate concentration of ADP (2 μmol/l) was used for these experiments to unmask a clear potentiating effect of PKC inhibition, which increased the sustained aggregation at 3 min from 36·9 ± 6·5% to 60·5 ± 5·6% in micromolar [Ca²⁺]_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 −3·5 ± 1·6% and 2·7 ± 1·2% (P > 0·05), 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 Ca²⁺ concentrations.

Fig 7.

ADP degradation is the predominant mechanism regulating ADP-mediated platelet aggregation at physiological extracellular Ca²⁺ concentrations. (A) Platelet aggregation evoked by 2 μmol/l ADP in PRP before and after re-calcification, in the presence and absence of the PKC inhibitor GF 109203X (10 μmol/l). (B) Average aggregation 3 min after addition of ADP.

Discussion

Reports of differential platelet responses to ADP in physiological versus nominally Ca²⁺-free conditions emerged over 20 years ago (Mustard et al, 1975; Packham et al, 1989). These studies concluded that enhanced TXA₂ production accounts for the paradoxical amplifying effect of lowering Ca²⁺ on ADP-evoked aggregation. The present study now shows that altered degradation of ADP can also contribute to this phenomenon. The known Ca²⁺-dependence of ecto-ADPases (Marcus et al, 1997; Zimmermann, 2000) provides the basis for the difference observed in millimolar versus micromolar [Ca²⁺]_o and this conclusion is supported by direct measurements of ADP. The sustained aggregation evoked by ADP is largely due to stimulation of P2Y₁₂ receptors, consistent with previous reports of the more crucial role of this G_i-coupled pathway compared to P2Y₁ 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 Ca²⁺ 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 [Ca²⁺] to investigate the relative contribution of Ca²⁺ entry versus release pathways in nucleotide-evoked signalling events.

The limited aggregation response at normal [Ca²⁺]_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 P2Y₁ and P2Y₁₂ receptor-deficient 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 [Ca²⁺]_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 ADPβS, 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 [Ca²⁺]_o, we agree with earlier studies (Packham et al, 1989; Garcia et al, 2007) that TXA₂ generation is lower at millimolar compared to micromolar [Ca²⁺]_o. In our experiments, the marked enhancement of TXB₂ generation by lowering [Ca²⁺]_o was also observed in apyrase-free saline, indicating that the Ca²⁺-dependent modulation of TXA₂ generation can occur independently of effects on ectonucleotidase activity. It has been reported that reduced TXA₂ 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 μmol/l ADP stimulated sustained aggregation at millimolar [Ca²⁺]_o in the absence of apyrase, conditions under which there was no detectable TXA₂ generation, suggesting that this response is independent of secondary signalling through TXA₂ 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 TXA₂ production may be more significant, and modulation of ADP-evoked aggregation by [Ca²⁺]_o may result from a combination of both altered ectonucleotidase activity and TXA₂ production.

Although we did not observe any difference in aggregation within the normal physiological range of extracellular calcium concentrations (0·5–2 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 Ca²⁺ levels. The sustained inside-out activation of fibrinogen receptors that occurs in response to ADP at low [Ca²⁺]_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.

Conflict of interest

The authors declare they have no conflict of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Fig S1. Reduced ADP degradation in low (micromolar) calcium concentrations is evident after 10 s. (A) Degradation of ADP (10 μmol/l) by apyrase (0.32 u/ml) in nominally Ca²⁺ free conditions or in the presence of 2 mmol/l Ca²⁺ measured 10 s after ADP addition. (B) Degradation of ADP (10 μmol/l) by ectonucleotidases present in plasma was measured before and after re-calcification, 10 s after ADP addition.

Fig S2. Elevated extracellular Mg²⁺ does not accelerate ADP degradation. (A) Washed platelets stimulated with ADP (10 μmol/l) in nominally Ca²⁺-free saline, or following addition of 2 mmol/l Mg²⁺. (b) Degradation of ADP (10 μmol/l) by apyrase (0.32 u/ml) present in the platelet saline buffer measured after 2 min in nominally Ca²⁺-free saline or following addition of 2 mmol/l Mg²⁺.

Fig S3. Adenosine deaminase blocks the inhibition of ADP-mediated aggregation by adenosine. Aggregation of washed platelets stimulated with ADP (10 μmol/l) in the presence of adenosine (10 μmol/l) with or without prior addition of adenosine deaminase (AD) 1 u/ml.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.