Identification of the Ca2+ entry pathway involved in deoxygenation-induced phosphatidylserine exposure in red blood cells from patients with sickle cell disease

Phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients is increased compared to levels in normal individuals and may participate in the anaemic and ischaemic complications of SCD. Exposure is increased by deoxygenation and occurs with elevation of intracellular Ca2+ to low micromolar levels. The Ca2+ entry step has not been defined but a role for the deoxygenation-induced pathway, Psickle, is postulated. Partial Psickle inhibitors 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS), 4,4′-dithiocyano-2,2′-stilbene-disulphonic acid (DIDS) and dipyridamole inhibited deoxygenation-induced PS exposure (DIDS IC50, 118 nM). Inhibitors and activators of other pathways (including these stimulated by depolarisation, benzodiazepines, glutamate and stretch) were without effect. Zn2+ and Gd3+ stimulated PS exposure to high levels. In the case of Zn2+, this effect was independent of oxygen (and hence HbS polymerisation and RBC sickling) but required extracellular Ca2+. The effect was completely abolished when Zn2+ (100 μM) was added to RBCs suspended in autologous plasma, implying a requirement of high levels of free Zn2+.


Introduction
Phosphatidylserine (PS) is an aminophospholipid which is normally confined to the inner leaflet of lipid bilayers [40].
In red blood cells (RBCs), this is particularly important because its exposure is prothrombotic, encourages phagocytosis and increases RBC adherence [7,29,59,63,64]. In healthy individuals, usually <1 % RBCs expose PS [40]. Programmed externalisation is thought to occur in damaged or senescent RBCs to aid their removal from the circulation. This process has been termed eryptosis [42,45] and is analogous to the apoptosis of nucleated cells. Elevated PS exposure is also seen in a number of disease states, including sickle cell disease (SCD) [17,66].
In SCD, patients show various complications which fall into two main groups: a chronic anaemia and acute vasoocclusive disorders (such as pain, stroke, acute chest syndrome and osteonecrosis). PS exposure is observed in a high, but variable, percentage of RBCs (2-10 % is often quoted [15,17,40,41,66,67]). PS may therefore contribute to both the anaemic and ischaemic complications of SCD [29]. The mechanism by which it becomes externalised has therefore received considerable attention [1,17,40].
Asymmetrical distribution of PS is usually maintained by high activity of an ATP-dependent aminophospholipid translocase (or flippase) in conjunction with relatively low activity of a Ca 2+ -activated scrambling process (or scramblase activity) [4,25]. The presence of the abnormal haemoglobin, HbS, in patients' RBCs somehow perturbs this equilibrium to favour PS exposure. HbS molecules are able to aggregate on deoxygenation forming rigid polymers which distort RBC shape, alter rheology and stimulate a number of other deleterious sequelae [29]. Deoxygenation also stimulates PS exposure [6,49,66]. Damage to the cytoskeleton may free PS from anchor sites resulting in its higher mobility [22,26,52]. In addition, however, deoxygenation-induced PS exposure is Ca 2+ dependent requiring elevation of the concentration of intracellular Ca 2+ ([Ca 2+ ] i ) to low micromolar levels [66]. The high cation permeability of HbS-containing RBCs may be involved in this process [24,31,46]. In particular, a role has been postulated for the deoxygenation-induced cation conductance (sometimes termed P sickle ) which is activated upon deoxygenation, HbS polymerisation and RBC shape change [32,46,50]. This pathway is permeable to divalent cations including Ca 2+ and can therefore mediate Ca 2+ entry. Elevated [Ca 2+ ] i both inhibits the flippase and also activates the scramblase [25]. A number of other RBC cationic permeabilities have also been described, however, including conductances activated by depolarisation, glutamate, benzodiazepines and stretch, as well as following infection with intraerythrocytic parasites like malaria [3,35] (although the latter is more widely considered to increase activity of anionselective pathways [39]). One or more of these, instead of, or as well as P sickle , may contribute to the Ca 2+ entry involved in deoxygenation-induced PS exposure. Clear identification of the pathway(s) involved would be beneficial as it would direct the search for potential inhibitors amenable to clinical use for amelioration of the complications of SCD.
In this study, we use various inhibitors and agonists of RBC pathways in an attempt to clarify the Ca 2+ entry step which results in deoxygenation-induced PS exposure in RBCs from SCD patients. Results indicate that partial inhibitors of P sickle reduced deoxygenation-induced PS exposure, consistent with mediation of Ca 2+ entry via this pathway, but those inhibitors and activators of several other potential entry pathways were without effect.

RBC preparation
Blood samples were collected from HbSS SCD patients, with consent and ethical permission (11/LO/0065), in the anticoagulant EDTA, except for the experiments in which RBCs were incubated in autologous plasma. In this case, heparin was used as an anticoagulant to prevent chelation of Ca 2+ and/or Zn 2+ (Fig. 6). In Fig. 6, only RBCs from normal (HbAA) individuals were used. RBCs were washed three times into HK HBS for 3 min at 600 g and then subsequently twice into LK HBS, unless otherwise indicated, to give a final haematocrit (Hct) of 5 %. RBC suspensions were then deoxygenated for 20 min in Eschweiler tonometers flushed with warmed humidified N 2 , by which time PS exposure was unchanged relative to that measured in untreated RBCs (as shown previously [66]). They were then diluted into test tubes also pre-equilibrated with N 2 (final Hct 0.5 %) in the absence or presence of potential inhibitors/activators of PS exposure. Incubation was carried out at 37°C for up to 60 min in the presence of 1 mM vanadate to inhibit both the flippase and the plasma membrane calcium pump. Solutions of DIDS (stock 5-50 mM in DMSO) were made up fresh every other day as stability decreased after 3 days resulting in strong stimulation of PS exposure, probably through the action of a breakdown product(s). Controls were exposed to the same final concentration of solvent (DMSO or H 2 O, 0.2 %) but otherwise treated in the same way. For experiments with glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and homocysteine, RBCs were kept oxygenated and incubated in Eppendorf tubes (up to 60 min, 37°C). For experiments to test the effect of plasma on the action of Zn 2+ , RBCs were re-suspended in autologous plasma after washing, equilibrated with air and 5 % CO 2 delivered using a Wösthoff gas mixer (Bochum, Germany) at 37°C to give a final pH 7.4. Normal total plasma Zn 2+ levels are about 10-15 μM [16,28,48]. This was not measured, rather additional Zn 2+ at the concentration indicated was added directly to plasma and thus represented the minimum total concentration present.
Labelling of PS exposure Samples of 5 μl aliquots (10 5 RBCs) were placed in 250 μl of LA-FITC binding buffer. PS labelling was carried out in the dark, because of the high sensitivity of FITC to light, at room temperature for 10 min. RBCs were then pelleted by centrifugation for 10 s at 16,100×g and washed once into LK HBS to remove unbound LA-FITC. Unlike annexin-V, LA-FITC binds to PS in a Ca 2+ -independent manner [14,61]. Control experiments showed that binding was irreversible. Samples were then kept on ice until analysed by flow cytometry (FACS). Inhibitors/activators were tested (at 100 μM) for self-fluorescence using unlabelled RBCs. The percentage of RBCs exposing PS is usually normalised to values measured in control RBCs prior to addition of inhibitors, as the absolute magnitude of exposure varied between samples. These control values are given in the figure legends.

FACS acquisition and analysis
Externalised PS was measured in the FL-1 channel, with an emission wavelength for FITC of 519 nm, of a fluorescenceactivated flow cytometer (FACSCalibur, Becton Dickinson, BD) and analysed with BD CellQuest Pro software using the protocol as previously published [66]. In control experiments, forward scatter (FSC, size) and side scatter (SSC, granularity) gates for RBCs were identified using a PElabelled anti-glycophorin A assay. FSC was set with threshold at 512. Measurements were taken under logarithmic gain with voltages set at FSC, E00; SSC, 235; FL-1, 688; FL-2, 630; and FL-3, 590. Compensation was set as FL-1, 2.0 % of FL-2; FL-2, 19.6 % FL-1; FL-2, 0.0 % FL-3; and FL-3, 8.5 % FL-2 to minimise the effect of over spill of fluorescence to adjacent channels. For each measurement 10,000 events were gated. All gated LA-FITC-labelled cells were additionally cross checked against overlap into FL-2 and FL-3 fluorescent channels and for spill out of the size gate and if these occurred events were excluded from analysis. The percentage of such resolved events was noted and for most experiments was ≤1 %. On FL-1/FL-2 dot plot graph, the cut-off quadrants, for negative fluorescent gate, were set using unlabelled cells as x=20 and y=20. The PS-positive cells were represented as the percentage of all gated RBCs with sufficient externalised PS to appear positive fluorescently in the FL-1 channel [14] and thus fallen into the quadrant (x>20 and y<20).

K + flux measurements
Potassium fluxes were measured using 86 Rb + as a tracer for K + [18,62]. In these experiments, RBCs (Hct about 20 %) were first equilibrated in Eschweiler tonometers (Kiel, Germany) in air or N 2 at 37°C, pH 7.4, in LK saline in which Cl − was substituted with NO 3 − and MOPS for HEPES. Cl − substitution prevented K + transport through the Na + -K + -2Cl − cotransporter and K + -Cl − cotransporter. Aliquots were then diluted tenfold into test tubes containing the same saline and also pre-equilibrated with air or N 2 for flux measurements. Bumetanide (10 μM), ouabain (100 μM) and clotrimazole (5 μM) were all also present to inhibit K + transport through the Na + -K + -2Cl − cotransport, Na + /K + pump and Ca 2+ -activated K + channel. 86 Rb + was then added into a KCl solution to give a final [K + ] of 7.5 mM. Influx was performed over 10 min, after which RBCs were washed four times in ice-cold isotonic MgCl 2 wash solution, lysed using Triton X-100 (0.1 %) and protein was precipitated with trichloroacetic acid (5 %) and 5 min centrifugation at 13,000×g. 86 Rb + (K + ) in the supernatant was then counted by liquid scintillation as Čerenkov radiation (Tri-Carb 2100TR, PerkinElmer, MA, USA). The difference in K + influx at the two gas tensions provides a measure of the deoxygenation-induced cation conductance, P sickle .

RBC morphology
Suspensions

Effects of inhibitors of P sickle
Although there are no specific inhibitors of P sickle , stilbenes [30] and the pyrimidine derivative dipyridamole [33] have been shown to inhibit the deoxygenation-induced cation permeability of RBCs from SCD patients. In the first series of experiments, therefore, the effects of 4-acetamido-4′isothiocyanostilbene-2,2′-disulphonic acid (SITS), 4,4′dithiocyano-2,2′-stilbene-disulphonic acid (DIDS) and dipyridamole were tested on deoxygenation-induced PS exposure ( Fig. 1). Each of the three reagents significantly inhibited the deoxygenation-induced PS exposure at both 30 and 60 min, at all concentrations used (10, 50 and 100 μM). The effect of DIDS was explored in more detail. Significant inhibition was observed by 100 nM (p<0.005), with an IC 50 of 118±10 nM (n=9), and was largely complete by 250 nM. It was possible that these reagents acted indirectly through inhibition of HbS polymerisation and sickling. This was tested using morphological studies to assess the percentage of RBCs which underwent sickling following deoxygenation. After 60 min deoxygenation, in the absence and presence of DIDS (100 μM), sickling was 61±8 and 62±5 % (means ± S.E.M., n=4, not significant (N.S.)), respectively. A possible explanation for the effect of DIDS on PS exposure via inhibition of sickling was therefore discounted. Effect of inhibitors of the non-specific cation channels of RBCs A non-selective cation conductance has been described in RBCs from normal individuals, activated by a number of manoeuvres including osmotic or oxidative shock, or Cl − substitution with gluconate (which will also cause RBC shrinkage) [19,43]. Amiloride derivatives represent effective inhibitors of this pathway [44]. The effect of ethylisopropylamiloride (EIPA) was therefore tested on the deoxygenation-induced PS exposure of RBCs from patients with SCD but no significant effect was observed (Fig. 2).

Effects of inhibitors of unusual RBC cation pathways
Recently, it has been proposed that RBCs express a conductance mediated via the peripheral benzodiazepine receptor (PBR) [9]. This pathway has multiple conductance states and is permeable to a number of ions including anions. It has also been postulated that RBCs respond to glutamate receptor agonists, such as NMDA and homocysteine, with the possibility that these ligand-gated channels may also be present [51]. Various agonists and antagonists of these pathways were investigated (Fig. 3). In experiments on deoxygenationinduced PS exposure, however, neither the PBR agonist diazepam (10-100 μM; Fig. 3a) nor its antagonist PK11195 (10-100 μM; Fig. 3b) had any significant effects. When tested on oxygenated RBCs, the glutamate receptor agonists NMDA and homocysteine (both 100 μM; Fig. 3c) did not affect PS exposure. Similarly, the NMDA receptor antagonist (+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801, 50 μM) had no effect on the deoxygenation-induced PS exposure (data not shown).

Effects of inhibitors of stretch-activated channels
Under certain conditions, the aminoglycoside antibiotics, streptomycin and gentamicin [13], and the heavy metals Zn 2+ and Gd 3+ [10,13,50] reduce the permeability of RBCs from SCD patients, possibly acting as stretch-activated channel blockers [60]. Long-term treatment with Zn 2+ has also been observed to induce PS exposure in normal human RBCs [38]. Their effects on deoxygenation-induced PS exposure were investigated (Figs. 4 and 5). In the presence of streptomycin, PS exposure increased, but not significantly (Fig. 4). Both Zn 2+ (up to 100 μM) and Gd 3+ (up to 50 μM) markedly increased PS exposure (Fig. 5a, b). Higher concentrations of Gd 3+ (100 μM) caused RBC aggregation and its effects could not be investigated using FACS. The action of Zn 2+ (100 μM) was studied in more detail (Table 1). Zn 2+ significantly increased PS exposure in both oxygenated and deoxygenated RBCs. Its effect was therefore different to the deoxygenation-induced PS exposure, requiring HbS polymerisation and sickling. The effect, however, was also dependent on the presence of extracellular Ca 2+ (1.1 mM) and largely abolished when it was omitted (Table 1). Additionally, similar effects were observed for both the RBCs from SCD patients and from healthy individuals (cf Figs. 5a (HbSS)) and Fig. 6 (HbAA). In corresponding K + flux measurements, Zn 2+ increased the deoxygenation-induced K + influx from 0.59±0.14 to 1.02±0.13 mmol(l cells h) −1 (means ± S.E.M., n=3; p<0.002) thus almost doubling the activity of the P sickle -like activity.
As negatively charged plasma proteins are likely to chelate Zn 2+ , in the final series of experiments, the effect of Zn 2+ was determined in RBCs (in this case from normal HbAA individuals suspended in autologous plasma, Fig. 6). In these experiments, PS exposure was measured in the absence and presence of added Zn 2+ (100 μM). In saline, as in Fig. 5a, Zn 2+ resulted in considerable PS exposure. In plasma, however, an increase in the percentage of RBCs showing PS exposure remained modest even after 60 min of incubation. Similar findings were observed with RBCs from SCD patients (data not shown).

Discussion
The present findings further define the Ca 2+ entry step responsible for deoxygenation-induced PS exposure in RBCs from SCD patients. In particular, partial P sickle inhibitors (the Time (min) PS exposing RBCs  stilbenes, SITS and DIDS and the pyrimidine derivative dipyridamole) were similarly active against PS externalisation. By contrast, modulators of other pathways (including inhibitors of the non-specific cation channel and agonists or blockers of glutamate-and benzodiazepine-gated channels) were without effect. Heavy metals Zn 2+ and Gd 3+ , rather than inhibiting PS exposure, caused increased levels of externalisation, an effect which in the case of Zn 2+ was shown to be Ca 2+ dependent but independent of oxygen tension, HbS polymerisation and RBC sickling.
RBCs from SCD patients have been known to show increased solute permeability for over 50 years. Seminal experiments by Tosteson and colleagues showed that deoxygenated RBCs from SCD patients lost K + at greater rates than they gained Na + , resulting in net solute loss and shrinkage [65]. This is particularly important as the lag time to HbS polymerisation following deoxygenation is inversely proportional to a very high power of the concentration of HbS [20]. Modest dehydration will markedly increase the likelihood of sickling as RBCs traverse hypoxic areas of the vasculature. Since then, the nature of the altered permeability has been much studied and several pathways have been identified [23,31,46]. Amongst these are the KCl cotransporter (likely KCC1 and KCC3 isoforms) and the Ca 2+ -activated K + channel (or Gardos channel). In addition to increased permeability to univalent cations, RBCs from SCD patients also show elevated permeability to both Ca 2+ and Mg 2+ [21,55,58]. The pathway(s) responsible for passage of these ions remains less certain, but a pre-eminent role for the deoxygenationinduced cation conductance termed P sickle has been proposed [31,46].
Although the permeability characteristics of P sickle have been extensively investigated, its molecular identity remains enigmatic [31,32]. As a flux pathway, its activation is associated with deoxygenation, HbS polymerisation and RBC shape change [31,53,58]. It is permeable to both univalent and divalent cations including Ca 2+ , shows a distinct pH dependence for both activation and permeation [34] Fig. 3 Effect of modulators of novel RBC cation channels on PS exposure in RBCs from SCD patients. a PS exposure is normalised to exposure in RBCs at time 0, just before addition of diazepam, which was 5.3±1.0 % (indicated by the dotted line), and after 30 and 60 min deoxygenation in the absence (solid black histograms) or presence (lighter histograms) of diazepam at 10, 50 and 100 μM. Data represent means ± S.E.M., n=6. All N.S. compared to deoxygenated RBCs in the absence of diazepam. b PS exposure is normalised to exposure in RBCs at time 0, just before addition of PK11195, which was 3.0±0.5 % (indicated by the dotted line), and after 30 and 60 min deoxygenation in the absence (solid black histograms) or presence (lighter histograms) of PK11195 at 10, 50 and 100 μM. Data represent means ± S.E.M., n=7. All N.S. compared to deoxygenated RBCs in the absence of PK11195. c PS exposure is normalised to exposure in oxygenated RBCs at time 0, just before addition of NMDA or homocysteine, which was 4.0±0.9 % (indicated by the dotted line), and after 30 and 60 min of incubation in the absence (black histograms) or presence (lighter histograms) of NMDA or homocysteine (both 100 μM). Data represent means ± S.E.M., n=6. All N.S. compared to oxygenated RBCs in the absence of NMDA or homocysteine and appears randomly (or "stochastically") activated upon deoxygenation [47]. As for its molecular nature, identification of specific inhibitors has proved elusive. Stilbenes [30] and dipyridamole [33], however, are partial inhibitors, with the latter having been used in clinical trials [12].
Deoxygenation of RBCs from SCD patients is known to result in increased PS exposure [6,49,66]. The effect is Ca 2+ dependent, requiring physiological levels of extracellular Ca 2+ and elevation of intracellular Ca 2+ to low micromolar levels [66]. P sickle is therefore an obvious candidate for mediation of Ca 2+ entry with subsequent PS exposure following from inhibition of the aminophospholipid translocase (or flippase) and stimulation of the RBC scramblase-both now known to occur at low micromolar [Ca 2+ ] [5,66]. Notwithstanding, various other RBC cation pathways have been described [35] and it is possible that one or more of these are also involved.
The present results support a role for the P sickle -like pathway in deoxygenation-induced PS exposure. Thus, stilbene derivatives and dipyridamole were active against deoxygenationinduced PS exposure, with no effect on morphological sickling (as also observed previously [30]) implying that they do not act via reduction in HbS polymerisation and subsequent RBC shape change. Inhibition by DIDS, in particular, occurred at low concentrations (IC 50 118 nM), more similar to those required to inhibit anion flux through the anion exchanger AE1, rather than monovalent cation flux through the P sickle pathway described previously [30]. Altered AE1 behaviour has been postulated to play a role in the conductance of deoxygenated sickle cells [46] and has been shown to mediate a cation permeability [11] or alter the activity of other cation transporters [8], at least when mutated, which may be relevant to the present findings. DIDS may also directly inhibit the scrambling transporter, as well as Ca 2+ entry, but this occurs at much lower affinity with an IC 50 of about 10 μM [37]. Here, 250 nM reduced PS exposure during deoxygenation to oxygenated levels.
By contrast, none of the other channel inhibitors/agonists, including those for the more established non-specific cation Effect of heavy metals, Zn 2+ and Gd 3+ , on deoxygenationinduced PS exposure in RBCs from SCD patients. a PS exposure at time 0, just before addition of Zn 2+ , was 3.8±0.4 % (indicated by the dotted line), and is also shown after 30 and 60 min deoxygenation in the absence (solid black histograms) or presence (lighter histograms) of Zn 2+ at 10, 50 and 100 μM. Data represent means ± S.E.M., n=7. # p<0.001, + p<0.03 compared to deoxygenated RBCs in the absence of Zn 2+ . b PS exposure at time 0, just before addition of Gd 3+ , was 3.5±0.6 % (indicated by the dotted line), and is also shown after 30 and 60 min deoxygenation in the absence (solid black histograms) or presence (lighter histograms) of Gd 3+ at 10, 25 and 50 μM. Data represent means ± S.E.M., n=5. # p<0.002, + p<0.02 compared to deoxygenated RBCs in the absence of Gd 3+ channel [2,3,27] or for the more controversial PBR-and glutamate-gated pathways [9,51], had any effect. Streptomycin, which might act as a stretch-activated channel blocker [13,60], was also without effect.
Zn 2+ and Gd 3+ , which also inhibit the cation conductance of RBCs from SCD patients [10,50], did not inhibit PS exposure. Instead, they caused greatly increased levels of externalisation-an effect previously described for Zn 2+ in RBCs from normal individuals following longer term (24 h) exposure [38]. This was not dependent on oxygen tension and appears to be mediated via a different mechanism than the deoxygenation-induced PS exposure under study. Their stimulation also required extracellular Ca 2+ , as described previously [38]. It would appear that both destabilise the RBC permeability, in a similar way to that described previously for lead and aluminium [45]. A possible action is via titration of the surface negative charges. K + influx measurements, however, also showed a rise in the activity of P sicklelike pathway which may also provide increased entry of Ca 2+ for PS scrambling. Notwithstanding, experiments in plasma suggest that chelation of Zn 2+ would reduce its free concentration below values required for the rapid PS exposure described here. This effect may be of some significance as some SCD patients are Zn 2+ deficient and supplementation is sometimes given [56,57]. Ensuring that free plasma [Zn 2+ ] does not rise excessively is an obvious caveat in these cases.
Finally, although the present work is concerned with Ca 2+ entry and deoxygenation-induced PS exposure in RBCs from SCD patients, it is also important to note that in RBCs from normal individuals other pathways may be significant. In this context, it is important to note that the present results concern RBCs from SCD patients whose permeability is altered by the presence of polymerised HbS. Most previous reports on PS exposure have been carried out on RBCs from normal individuals (e.g. [44]). It is likely that Ca 2+ entry, which triggers subsequent PS exposure, can occur via several different routes. Thus, in addition to the P sickle pathway of sickle cells, normal RBCs show marked Ca 2+ elevation in response to lysophosphatidic acid [54,63,68], possibly via the non-selective cation channel [35,36,44]. An alternative Table 1 Effect of Zn 2+ on phosphatidylserine (PS) exposure in red blood cells (RBCs) from patients with sickle cell disease. All RBCs were incubated in low potassium-containing (LK) saline and treated with Zn 2+ (100 μM) for the duration indicated. Extracellular Ca 2+ was also present at 1.1 mM or omitted from saline (with addition of 2 mM EGTA  Fig. 6 Effect of Zn 2+ and autologous plasma on PS exposure in RBCs from normal (HbAA) individuals. RBCs were incubated under oxygenated conditions in LK saline (solid histograms) or in autologous plasma (hatched histograms), in which case pH was maintained by flushing with warm humidified CO 2 (5 %). Percentage PS exposure is shown at time 0, just before addition of Zn 2+ (100 μM), and after 30 and 60 min.
Histograms represent means ± S.E.M., n=4 (paired samples). # p<0.008 compared to values in plasma pathway for PS exposure involving protein kinase C has also been proposed [54,63], as well as the Ca 2+ -mediated one. The role of these pathways in RBCs from SCD patients remains to be investigated; however, the lack of effect of EIPA on deoxygenation-induced PS exposure argues against the involvement of the non-specific cation channel.
In summary, we show that PS exposure in RBCs from SCD patients is stimulated by deoxygenation, via a pathway likely consistent with mediation via P sickle . Future experiments will be aimed at establishing the molecular identity of this pathway and the establishment of inhibitors with potential for clinical use.