# Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles

Cora L Alvarez, Arnaud Chêne, Jean-Philippe Semblat, Benoît Gamain, Claudine Lapouméroulie, Claudio M Fader, Claude Hattab, Jean Sévigny, María Florencia Leal Denis, Natalia Lauri, et al.

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## ► To cite this version:

Cora L Alvarez, Arnaud Chêne, Jean-Philippe Semblat, Benoît Gamain, Claudine Lapouméroulie, et al. Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles. Biochimica et Biophysica Acta:Biomembranes, 2022, 1864, 10.1016/j.bbamem.2022.183980. hal-05331833

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Journal Pre-proof

Homeostasis of extracellular ATP in uninfected RBCs from a
Plasmodium falciparum culture and derived microparticles

Cora L. Alvarez, Arnaud Chêne, Jean-Philippe Semblat, Benoît Gamain, Claudine Lapouméroulie, Claudio M. Fader, Claude Hattab, Jean Sévigny, María Florencia Leal Denis, Natalia Lauri, Mariano A. Ostuni, Pablo J. Schwarzbaum

<figure><img src="image_3.png" /></figure>

PII: S0005-2736(22)00118-3

DOI: https://doi.org/10.1016/j.bbamem.2022.183980

Reference: BBAMEM 183980

To appear in: *BBA - Biomembranes*

Received date: 6 April 2022

Revised date: 11 May 2022

Accepted date: 26 May 2022

Please cite this article as: C.L. Alvarez, A. Chêne, J.-P. Semblat, et al., Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles, *BBA - Biomembranes* (2021), https://doi.org/10.1016/j.bbamem.2022.183980

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
**Title:** Homeostasis of extracellular ATP in uninfected RBCs from a *Plasmodium falciparum* culture and derived microparticles

**Authors:** Cora L. Alvarez¹,², Arnaud Chêne³, Jean-Philippe Semblat³, Benoît Gamain³, Claudine Lapouméroulie³, Claudio M. Fader⁴, Claude Hattab³, Jean Sévigny⁵, María Florencia Leal Denis¹,⁶, Natalia Lauri¹,⁷, Mariano A. Ostuni³ and Pablo J. Schwarzbaum¹,⁷*

1. Instituto de Química y Físico-Química Biológicas “Prof. Alejandro C. Paladini”, Universidad de Buenos Aires (UBA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Farmacia y Bioquímica, Junín 956, C1113AAD Buenos Aires, Argentina.

2. Universidad de Buenos Aires (UBA), Facultad de Ciencias Exactas y Naturales, Departamento de Biodiversidad y Biología Experimental, intendente Güiraldes 2160, Ciudad Universitaria, C1428EGA, Buenos Aires, Argentina.

3. Université Paris Cité and Université des Antilles. INSLRM, BIGR, F-75015 Paris, France.

4. Laboratorio de Fisiología y Fisiopatología del Globo Rojo. Instituto de Histología y Embriología (IHEM), Universidad Nacional de Cuyo, CONICET, Mendoza, Argentina. Facultad de Odontología, Universidad Nacional de Cuyo, Mendoza, Argentina.

5. Centre de Recherche du CHU de Québec-Université Laval, Québec, QC, Canada. Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.

6. Universidad de Buenos Aires (UBA) Facultad de Farmacia y Bioquímica, Departamento de Ciencias Químicas, Cátedra de Química Analítica, Junín 956, C1113AAD, Buenos Aires, Argentina.

7. Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Cátedra de Química Biológica, Junín 956, C1113AAD, Buenos Aires, Argentina.

**Keywords:**

RED BLOOD CELLS / MALARIA / EXTRACELLULAR ATP / ECTONUCLEOTIDASES
MICROPARTICLES

**Highlights:**

* ✓ Uninfected RBCs displayed enhanced ATP release when challenged by different stimuli.

* ✓ Uninfected RBCs showed enhanced ectoATPase activity.

* ✓ Microparticles encapsulate ATP in their lumen and were able to hydrolyze extracellular ATP.

<footnote>* Corresponding author</footnote>
ABSTRACT

*Plasmodium falciparum*, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs).

ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space.

Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases.

RBCs were cultured with *P. falciparum* for 24-48 hours prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs.

Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP.

Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria.

The comparison of eATP homeostasis in uninfected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of *P. falciparum* infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.
# 1. INTRODUCTION

*Plasmodium falciparum* is an important etiological agent of malaria; the clinical signs of the disease occur during the invasion and development of *P. falciparum* parasites inside red blood cells (RBCs) (https://www.cdc.gov/malaria/about/biology/index.html). Among the possible mechanisms contributing to the disease severity are those affecting the flow in the microcirculation due to the capacity of infected RBCs (i-RBCs) to adhere to the vascular endothelium and reduce the vascular lumen. Particularly rosetting, a form of cytoadherence of i-RBCs to non-infected RBCs (ui-RBCs), is associated with microvascular obstruction causing severe malaria and cerebral malaria [1].

RBCs may display mechanisms controlling the vascular caliber that depend on both, the release of intracellular ATP and extracellular ATP hydrolysis. Following acidosis, hypoxia, mechanical and/or osmotic stress, RBCs release ATP by regulated transmembrane transport and/or hemolysis, thus resulting in the accumulation of low micromolar concentrations of extracellular ATP (eATP) [2,3]. Extracellular eATP can act on specific P2 purinergic receptors on adjacent endothelial cells [4], triggering the release of endothelial vasodilators that promote the relaxation of smooth muscle cells surrounding the capillaries of the microvasculature, thus causing vasodilation in the brain, coronary arteries and skeletal muscle [2,5].

On the other hand, eATP can be hydrolyzed by ectonucleotidases present on the surface of RBCs and on different leukocytes and vascular cells, thus terminating the action of eATP on P2 receptors.

The time-dependent profile of eATP accumulation (i.e., eATP kinetics) thus depends on a delicate balance between ATP release in the microenvironment and eATP degradation by ectonucleotidases. Infection by protistan parasites may alter eATP kinetics and provoke systemic responses leading to either beneficial or detrimental consequences for the host (reviewed in [6]). Interestingly in this context, exposure of *P. falciparum* infected RBCs to various stimuli caused a strong activation of ATP release and eATP hydrolysis during all stages of the asexual cycle [7]. Thus eATP kinetics of i-RBCs is profoundly upset and research aiming at deciphering the effect of *P. falciparum* infection on purinergic signaling is gaining an increasing attention [8–11].

While the above results highlight the importance of i-RBCs for purinergic signaling, with potential contributions to vasodilation, a complete picture of RBC-eATP dynamics during *P. falciparum* infection requires taking other RBC-dependent processes into account, that is, the contribution to eATP regulation from ui-RBCs and from microparticles released by vesiculation of the plasma membrane.

Due to the low parasitemia usually found in malaria patients (generally 0.1-1%, rarely exceeding 3-4% in humans [12]), ui-RBCs constitute approx. 96% of the whole RBC population. To the extent that ui-RBCs react similarly to control RBCs, they may also contribute to RBCs-eATP dynamics. However, several changes of ui-RBCs were documented, which might anticipate an alteration of either ATP release, eATP degradation, or both. That is, ui-RBCs were shown to display down-regulated glycolytic flux, altered fatty acid composition of the cell membranes, decreased electrophoretic mobility, altered osmotic fragility, and increased exposure to self-antibodies [12–16].

Another consequence of *P. falciparum* infection is the production of plasma membrane vesicles known as microparticles (MPs) [17–19]. *P. falciparum* infection has been shown to induce a strong production of MPs from i-RBCs, a feature also observed when RBCs are exposed to bacterial toxins [20] and other stressors in pathological contexts [21,22].
Although MPs from leukocytes, platelets, RBCs, and endothelial cells are involved in thrombosis, hemostasis, and inflammation [23,24], the potential role of RBCs derived MPs on eATP regulation has not been studied so far. In principle, MPs may contain ATP originating from the RBCs cytosolic pool, and eATP hydrolysis activity due to membrane bound ectonucleotidases. Thus, depending on their potential capacity to release intracellular ATP and/or hydrolyze eATP, these vesicles may contribute to intravascular eATP regulation in malaria.

Thus, complementing our previous study using i-RBCs, in this study, we analyzed how ui-RBCs and MPs may contribute to eATP dynamics induced by infection of RBCs with *P. falciparum*. Ui-RBCs were exposed to several stimuli to induce ATP release. Luciferin-Luciferase luminometry and colorimetric and radioactive methods were used to quantitatively assess eATP kinetics and the rates of ATP release and eATP hydrolysis in ui-RBCs and MPs. In addition, osmotic fragility tests were employed to assess deformability and cell volume changes of ui-RBCs, and their potential effect on ATP release.

## 2. MATERIALS AND METHODS

### 2.1. Chemicals

All reagents in this study were of analytical grade. Firefly Luciferase (EC 1.13.12.7), sorbitol, forskolin, isoproterenol, papaverine, morphine, Bovine serum albumin (BSA), paraformaldehyde, Tween-20, malachite green, adenosine 5'-triphosphate (ATP), 4-(2-hydroxyethyl)-1-piperazineetahnesulfonic acid (HEPES), para-Nitrophenylphosphate (pNPP), Triton X-100, phenylethylsulfonyl fluoride (PMSF), carbenoxolone and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (St Louis, MO, USA). D-luciferin, Albumax II, RPMI 1640 were obtained from Invitrogen/Molecular Probes Inc (Eugene, OR, USA). [γ 32P]ATP (10 Ci/mmol) was purchased from Perkin Elmer Life Sciences (Santa Clara, California, USA). HlyA was kindly gifted from Dra Vanesa Herlax (INIBIOLP-National University of La Plata, Argentina).

### 2.2. Collection and preparation of human RBCs

Human A+ blood was obtained by venipuncture from healthy volunteers. Blood was kept at 4°C for 24 hours. The day before blood collection, plasma, platelets, and leukocytes were removed by centrifugation (900 x g at 20 °C for 3 min). The supernatant and buffy coat were removed and discarded. The RBCs were resuspended and washed three times in supplemented RPMI medium (RPMI 1640 medium (Invitrogen/Molecular Probes Inc. Eugene, OR, USA), 22 mM glucose, 25 mM HEPES, 0.65 mM hypoxanthine and 50 µg/mL gentamicin). RBCs were resuspended at 45% hematocrit in supplemented RPMI medium and stored for 1-10 days at 4°C.

### 2.3. Maintenance, synchronization and collection of uninfected RBCs

*Plasmodium falciparum* strain NF54 was maintained in A+ human RBCs at 3-5% hematocrit in supplemented RPMI 1640 medium with 1% albumax II Invitrogen/Molecular Probes Inc. (Eugene, OR, USA) in a 90%N₂/5%O₂/5%CO₂ atmosphere pH 7.4 at 37°C. Cultures were sorbitol-synchronized at ring-stage as described previously [7]. Parasitemia was monitored by Giemsa-stained blood smears and maintained a maximum 5-7%. No sexual forms were observed during *in vitro* culture. Ui-RBCs were purified from an i-RBC culture using two methods: magnetic columns and Transwell support, as described below.
### 2.3.1. Magnetic columns

Suspensions containing synchronized parasite cultures of trophozoite stage-RBCs at 4-7% parasitemia were passed through a magnetic column (MACS LD column, Miltenyi Biotec Bergisch Gladbach, North Rhine-Westphalia, Germany). This procedure takes advantage of the electromagnetic properties of hemozoin that retains i-RBCs, and allows their separation from ui-RBCs [25]. A schematic drawing of the procedure is shown in Figure 1A. Briefly, LD columns were mounted on a high-gradient magnetic cell separator VarioMACS (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) and washed with 5 mL PBS medium before being used. A suspension of trophozoite stage RBCs (parasitemia at 4-7%) was centrifuged 900 x g for 3 min and the pellet was resuspended in 2 mL of PBS medium supplemented with 2% bovine serum albumin (BSA) and 2 mM EDTA. The suspension ($2 \times 10^8$ i-RBCs mL$^{-1}$) was loaded on and passed through the LD column. The eluate (not retained cells) was centrifuged at 900 x g for 3 min and the pellet was resuspended in 1 mL of PBS medium supplemented with 0.5% BSA. The parasitemia of the final suspension was 0.85 ± 0.12%. The suspension obtained was denoted as ui-RBCs<sup>MC</sup>. Control experiments were performed under similar conditions but in the absence of the parasite and denoted as c-RBCs<sup>Mesh</sup> (Figure 1A).

### 2.3.2. Transwell support

Permeable supports of 12 mm diameter containing a mesh 0.4 µm pore size were used to culture RBCs on a 24-well plate (Corning, Inc, Glendale, Arizona). A schematic drawing of the procedure is shown in Figure 1D. Suspensions containing synchronized parasite cultures of ring stage-RBCs at 1% hematocrit (parasitemia 1%) were placed on the upper chamber. RBCs at 1% hematocrit were placed on the lower chamber. Incubation was done in supplemented RPMI 1640 medium with Albumax II for 24, 36, and 48 hours, without media exchange. Plates were maintained in a 90%N<sub>2</sub>/5%O<sub>2</sub>/5%CO<sub>2</sub> atmosphere, pH 7.4 at 37°C. Using this configuration, i-RBCs in the upper chamber cannot establish physical contact with RBCs in the lower chamber. Experiments were run using RBCs from the lower compartment, denoted as ui-RBCs<sup>Mesh</sup>. Control experiments were performed under similar conditions, with both chambers containing RBCs and denoted as c-RBCs<sup>Mesh</sup> (Figure 1D).

## 2.4. ATP release and detection of Pannexin-1

### 2.4.1. Non lytic ATP release

ATP was measured using firefly Luciferase, which catalyzes the oxidation of luciferin in the presence of ATP and produce light [26,27]. On-line luminometry measurements of eATP were carried out with c-RBCs<sup>MC</sup>, ui-RBCs<sup>MC</sup>, c-RBCs<sup>Mesh</sup> and ui-RBCs<sup>Mesh</sup>. After purification cells were washed with RBC medium. The medium contains (in mM) 137 NaCl, 2.7 KCl, 4.72 Na<sub>2</sub>HPO<sub>4</sub>, 1.50 KH<sub>2</sub>PO<sub>4</sub>, 1.32 CaCl<sub>2</sub>, 1.91 MgSO<sub>4</sub>, 5 glucose, 0.5% BSA, pH 7.4, 300 mOsm/L. The cells were centrifuged at 900 x g for 3 min and the pellet was resuspended in RBC medium at 3% hematocrit. Ten µL of washed cells ($3 \times 10^6$ cells) were mixed with 40 µL of luminometry mix (RBC medium containing 0.01 µM luciferase, 0.2 mM luciferin, and 0.1 mg/ml of Coenzyme A). The total volume was placed in the assay chamber of a custom-built luminometer to determine the light intensity, as previously described [7,28–32]. Since Luciferase activity at 37 °C is only 10% of that observed at 20 °C [33], to maintain full Luciferase activity, ATP measurements were performed in a cool chamber thermostatized at 20 °C. Cells were incubated for 5 min and exposed to an adrenergic cocktail (3V). This cocktail contains 10 µM isoproterenol (beta-adrenergic agonist), 30 µM forskolin (adenylyl cyclase activator), and 100 µM papaverine (inhibitor of
phosphodiesterases). 3V induce an acute increase in intracellular cAMP coupled to activation of ATP, as previously described [7,29]. At the end of the incubation, the time course of light emission was transformed into extracellular ATP concentration [eATP] versus time using a built-in calibration curve (Supplementary Figure 1). For that, increasing concentrations of ATP from 19 to 450 nM were sequentially added to the assay medium from a stock solution of pure ATP dissolved in RBC medium. Results were expressed as nM/10⁶ cells. Alternatively, increases in [eATP] were evaluated as the ratio between [eATP] at 1 min post-stimulus and the basal [eATP], and are indicated as QATP. Since Pannexin-1 has been postulated to facilitate passive transport of ATP across the plasma membrane of RBCs [29,30,32], 100 µM of the Pannexin-1 blocker carbenoxolone (CBX) was used in kinetics experiments of ATP release. In experiments using MST (10 µM), RBCs were attached to coverslips coated with 0.001% poly-D-lysine as previously described [30,32].

The contribution of ATP to the extracellular medium by hemolysis is proportional to the amount of hemoglobin released. The release of hemoglobin (Hb) into the extracellular medium can be quantified by monitoring light absorption at 405 nm. With this procedure, we previously observed that the amount of Hb was proportional to the amount of lysed RBCs, where for every 10⁶ RBCs, 148 pmol of ATP are released [30]. Thus, at the end of the kinetics experiments, the whole content of the chamber was centrifuged at 900 x g for 2 min, and the absorbance of the supernatants was measured (Supplementary Figure 2). The percentage of hemolyzed RBCs (% Hemolysis) with respect to the total number of RBCs was measured.

### 2.4.2. Detection of Pannexin-1: main conduct of ATP release in RBCs

Detection of Pannexin-1 by SDS-PAGE and Western Blotting was performed in ui-RBCsMC and c-RBCsMC. Samples of cells were used to prepare membranes (ghosts). Ghosts were obtained by hypotonic lysis induced by 1 h exposure to ice cold 5P8 buffer (5 mM NaH₂PO₄; 0.35 mM EDTA, pH 8, containing 1 mM phenyl methane sulfonyl fluoride, PMSF) and centrifuged at 36,000 g at 4 °C for 30 min as previously described [34]. Several washings with the same buffer were performed until a clear supernatant was obtained. Protein quantification was performed by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Ghost's proteins (30 µg/lane) contained in 5P8 buffer were solubilized in 1% Triton X-100 and ran in NuPAGE® 4–12% Bis-Tris gels, as indicated, under non-reducing conditions. Then, proteins were transferred to a nitrocellulose membrane using Trans-Blot Turbo Transfer System (BioRad, Hercules, CA, USA). Immunoblots were performed using rabbit polyclonal antibody anti-pannexin 1 (Alomone Labs, Jerusalem, Israel 1:1000 dilution) ON at 4°C. Western Blotting secondary labeling was performed with anti-rabbit antibody conjugated with horseradish peroxidase (Abliance, Compiègne, France). The densities of Pannexin-1 immunogenic band was normalized to β-actin, and relativized to c-RBCsMC. A molecular weight marker was also run (Supplementary Figure 3).

## 2.5. Hemolysis.

Four treatments (2.5.1-2.5.4) were employed to assess hemolysis of RBCs under different conditions. In all cases, hemoglobin content was determined by measuring the absorbance at 405 nm using a BiotrackII (Amersham Biosciences, Buckinghamshire United Kingdom) spectrophotometer, with the corresponding extinction coefficient [35]. Using this procedure, we previously observed that the amount of hemoglobin was proportional to the amount of lysed RBCs [30]. Results were expressed as % hemolysis.

### 2.5.1. Osmotic Fragility Test
This method was used to measure the effect of hypotonic swelling on hemolysis of c- and ui-RBCs<sup>MC</sup>. Cells were exposed to increasingly hypotonic saline solutions (range: 0.9–0 % NaCl). An aliquot of 10 µL from a 10 % hematocrit suspension of c- and ui-RBCs<sup>MC</sup> was incubated in hypotonic solutions for 30 min at 37°C. Results were expressed as a percentage, considering that RBCs exposure to 0% NaCl solutions yields 100% hemolysis. A sigmoidal equation of the form:

$$Y= \frac{\text{Bottom} + \frac{\text{Top-Bottom}}{1 + 10^{(\text{LogEC50}-\text{X})*\text{Slope factor}}}}{1 + 10^{(\text{LogEC50}-\text{X})*\text{Slope factor}}}$$

was fitted to the experimental data by non-linear regression using the Graph Pad Prism Software. The best fit values of two parameters were obtained: 1- EC<sub>50</sub>, the log concentration of NaCl at which 50% of the cells are hemolyzed and 2- the slope factor, accounting for the steepness of the curve.

### 2.5.2. Sorbitol uptake

This method was used to assess hemolysis of c- and ui-RBCs<sup>MC</sup> by exposure to an iso-osmotic medium containing sorbitol [34]. The kinetics curve of hemolysis was initiated by the addition of 0.4 mL of a cell suspension (50% hematocrit) to 3.6 mL of sorbitol iso-osmotic solutions (300 mM sorbitol, 10 mM HEPES, 5 mM glucose, pH 7.4). At different times the reaction was stopped with ice-cold “stopping solution” (400 mM sucrose), and centrifuged at 10000 × g for 30 s. Suspensions of ui-RBCs (trophozoite erythrocytes at parasitemia 4.05 ± 0.45 %) were also tested. Results are expressed as percentage of hemolyzed RBCs (% Hemolysis) with respect to the total number of RBCs.

### 2.5.3. Alpha-hemolysin (HlyA)

This method was used to measure hemolysis of c- and ui-RBCs<sup>MC</sup> caused by exposure to the toxin Alpha-hemolysin (HlyA) dissolved in iso-osmotic RBC medium. HlyA of uropathogenic strains of Escherichia coli irreversibly binds to RBCs, inducing osmotic hemolysis [36]. An aliquot of 200 µL from a 5 % hematocrit suspension of c- and ui-RBCs<sup>MC</sup> was exposed to HlyA 40 ng/µL. Incubations were performed incubating cells to toxin for 0 (basal) or 10 min at 37°C. Results are expressed as percentage of hemolyzed RBCs (% Hemolysis) with respect to the total number of RBCs.

### 2.5.4. Hypo-osmotic media

This method was used to measure hemolysis of c- and ui-RBCs<sup>MC</sup> challenged by hypo-osmotic media. Assay media had a similar composition to the iso-osmotic RBC medium, except that the NaCl concentration was lower to yield final osmolarities of 171 and 206 mOsm/L. Suspensions of c- and ui-RBCs<sup>MC</sup> (10 % hematocrit) were incubated for 10 min at 20 °C at osmolarities 300 (iso-osmotic), 206 and 171 mOsm/L conditions. Samples were centrifuged at 900 × g for 2 min, and the absorbance of the supernatants was measured. Results were expressed as a percentage of hemolyzed RBCs (% Hemolysis) with respect to the total number of RBCs.

## 2.6. ATP hydrolysis

RBCs do not take up exogenous ATP. Therefore, following addition of ATP to a cell suspension, the rate of ATP hydrolysis can be derived by quantifying the decrease of [eATP] (luminometry), or the accumulation of phosphate (released from ATP) in the assay medium (radioactive and colorimetric methods).

### 2.6.1. Hydrolysis of eATP by on-line luminometry
Coverslips with 10 µL of 3% suspensions (c-RBCs<sup>MC</sup>, ui-RBCs<sup>MC</sup>, c-RBCs<sup>Mesh</sup> and ui-RBCs<sup>Mesh</sup>) in RBCs medium containing 0.5% BSA were mounted in the measuring chamber of the luminometer and exposed to the Luciferase reaction mixture. Light emission was measured for 10 min, after which ATP was added to the chamber at final concentrations of 450, 900 and 1800 nM (in separate experiments), and measurements were carried out for another 30 min. Light output was transformed into [eATP] concentration versus time (see section 2.4.1.). The slope of [eATP] decrease, a measure of the rate of eATP hydrolysis by ectoATPase activity (vi), was estimated from [eATP] kinetics. ATPase activity was expressed as nM/(10<sup>6</sup> cells min). Experimental data of ATPase activity vs [ATP] were fitted to a linear function, from which the slope of the curve was obtained and denoted as K<sub>ATP</sub>.

### 2.6.2. Hydrolysis of eATP using [γ-<sup>32</sup>P]ATP

The rate of eATP hydrolysis was also determined by following the accumulation of [<sup>32</sup>P]Pi release from exogenous [γ-<sup>32</sup>P]ATP added to a RBCs suspension of known hematocrit, as described before [7,28,30,37]. Briefly, the reaction was started by the addition of [γ-<sup>32</sup>P]ATP (0.027 Ci/mmol) to cell suspensions incubated at 20°C. At different times, 0.2 mL aliquots of the suspension were withdrawn and centrifuged at 900 x g for 30 s, and 0.1 mL of the supernatants were poured onto 0.75 mL of a top solution containing 4.05 mM (NH<sub>4</sub>)<sub>6</sub>Mo<sub>7</sub>O<sub>24</sub> and 0.83 mM HClO<sub>4</sub>. The ammonium hydrobdate solution formed a complex with the released phosphate, which was then extracted with 0.6 mL of isobutyl alcohol. Phases were separated by centrifugation at 1000 g for 5 min, and 0.2 mL-aliquots of the organic phase containing [<sup>32</sup>P]Pi were transferred to vials containing 2 mL of 0.5 M NaOH, and the radioactivity was measured by the Gernakov effect.

Any hydrolysis of [γ-<sup>32</sup>P]ATP into ADP-<sup>32</sup>PPi in a cell suspension was defined as ectoATPase activity, the time course of which yields a measure of the rate at which one or more types of ectonucleotidases hydrolyze eATP. To calculate ectoATPase activity, time-dependent levels of Pi were fitted to the following exponential equation:

$$v = v_0 + A \cdot (1 - \exp^{-k \cdot t})$$

where v and v<sub>0</sub> are the values of [<sup>32</sup>Pi] at each time (t) and at t=0, respectively; A represents the maximal value for the increase in v with time and k is a rate coefficient. The parameters of best fit resulting from the regression were used to calculate the initial rate of ectoATPase activity (vi) as k x A (i.e. the first derivative of Equation 1 evaluated at t=0). The moles of [<sup>32</sup>Pi] produced from [γ-<sup>32</sup>P]ATP were measured from the ATP specific activity.

### 2.6.3. ATP hydrolysis using Malachite green assay

EctoATPase activity at non-limiting high concentration of ATP estimates the apparent maximal rates of eATP hydrolysis. Maximal ectoATPase activity was determined with a malachite green assay [38]. Briefly, cell suspensions (c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup>, 10-20% hematocrit) washed with reaction medium lacking Pi (containing (in mM) 10 HEPES, 1 KCl, 140 NaCl, 1 CaCl<sub>2</sub>, 4 MgSO<sub>4</sub>, pH 7.4 at 20°C) were incubated with 1000 µM of ATP at 20 °C under continuous agitation. At different times, 100 µL of reaction medium were placed in a tube containing 100 µL of cold 25% charcoal in 0.1 M HCl. The tubes were centrifuged at 1500 x g for 10 min. Aliquots (50 µL) of the supernatants containing the released Pi were mixed with 50 µL of the malachite green reagent (containing 3.05 mM malachite green, 4.72 N H<sub>2</sub>SO<sub>4</sub>, 12 mM Mo<sub>7</sub>O<sub>24</sub>(NH<sub>4</sub>)<sub>6</sub>, and 0.16% Tween 20). Solutions were incubated at 20 °C for 30 min, and absorbance was determined at 620 nm. Absorbance values were converted to phosphate concentrations by means of a calibration
curve (20-100 µM of Pi). To calculate maximal ectoATPase activity, time-dependent levels of Pi were fitted to the exponential equation previously mentioned (section 2.6.2.).

## 2.7. ATP content of the culture medium in Transwell support

During the cycle progress of *Plasmodium falciparum* occurring in transwell supports, some metabolites, including ATP, can be released into the culture medium. The experimental setup described in section 2.3.2., does not allow physical contact between i- and c-RBCs during culture (Supplementary Figure 4), but allows media exchange. The concentration of [eATP] was quantified at the end of the incubation period. After 48 hours culture, 45 µL aliquots of media were mixed with 5 µL of luciferin-Luciferase 10x mixture in a final volume of 50 µL. Light conversion to [ATP] and calibration were similar to those employed in section 2.4.1. For calibration, increasing concentrations of ATP from 16 to 920 nM were sequentially added to the assay medium from a stock solution of pure ATP dissolved in supplemented RPMI 1640 with Albumax II.

## 2.8. Microparticles

Cultures of 3% hematocrit containing 3-5% i-RBCs were used to produce and release MPs. Cultures of i-RBCs at ring stage were maintained for 24 hours in 15 mL of RPMI supplemented with Albumax II previously filtered through a 0.22-µm sterilized filter (Millipore Darmstadt, Germany). MPs from infected cultures, denoted as i-MP, were isolated from the culture medium. Control cultures were performed under similar conditions, (3% hematocrit, 24 hours in 15 mL of filtered RPMI) in the absence of the parasite. MPs from control cultures, denoted as c-MP, were isolated from the culture medium.

### 2.8.1. Purification

i-MPs and c-MPs were isolated from 15 mL of culture medium. For that purpose, the collected medium was centrifuged at 2000 x g at 20°C for 5 min to remove cells and again at 3000 x g at 20°C for 10 min to remove the remaining debris, followed by filtration through a 0.45-µm sterilized filter (Millipore Darmstadt, Germany). MPs were then pelleted by centrifugation for 2 h at 20000 x g at 4 °C. The pellet was washed twice and then suspended in ~150 µL RBC medium.

### 2.8.2. Electron microscopy

Isolated i-MPs were centrifuged at 100000 x g at 4°C for 2 hours to concentrate them. After washing, they were fixed in 2% glutaraldehyde in 0.15 M sodium cacodylate buffer for at least 2 hours. After another washing step MPs were post-fixed with 2% osmium tetroxide reduced with 1.5% potassium ferrocyanide in PBS (pH 7.4), progressively dehydrated in ethanol, and embedded in low-viscosity epoxy resin. 70-nm-thin sections were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate. The samples were observed under a Zeiss 900 electron microscope.

### 2.8.3. Flow cytometry analysis

Flow cytometry analysis was performed to characterize i-MPs and c-MPs. For that purpose, MPs were incubated with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). The dye enters the MP where intracellular esterases cleave the acetate groups to produce fluorescent CFDA. Dye was incubated for 45 min at 4°C under dark conditions. MPs were washed and diluted with PBS medium for flow cytometry analysis. A BD FACScanto II flow cytometer with HTS (Becton-Dickinson) and FACSDiva software
(v6.1.3) were used for data acquisition and analysis. The percentage of fluorescent MPs was determined under the same conditions for all samples. A total of 10000 events were analyzed. Beads (Megamix-Plus SSC BioCytex, France) were used to calibrate the cytometer according to manufacturer recommendations.

## 2.8.4. Production

Production of MPs was quantified by the Bradford assay [39] and expressed as mg protein per mL of cell culture.

## 2.8.5. Intraparticular ATP

The content of ATP inside MPs was quantified after permeabilization of MPs with digitonin (50 µg/mL). Coverslips with 38 µL aliquots of MPs suspensions in RBC medium + 0.5% BSA were mounted in the luminometer and incubated with 2 µl of luciferin-Luciferase 20x mixture as previously described (section 2.4.1.). After permeabilization with digitonin, a calibration curve was performed to transform light emission into [eATP]. Values were expressed as nM/mg protein of both, i-MPs and c-MPs, from which the intraparticular ATP content could be derived and expressed by the ratio between [eATP<sub>MP</sub>] at 1 min post-digitonin and basal [eATP<sub>MP</sub>] i.e., Q<sub>iATP MP</sub>.

## 2.8.6. Hydrolysis of eATP by on-line luminometry

Coverslips with 10-38 µL aliquots of MPs suspensions in RBC medium + 0.5% BSA were mounted in the luminometer and exposed to a luciferase reaction mixture (section 2.4.1.). Light emission was measured for 1-2 min. ATP was then added to the chamber at final concentrations of 50, 100, 450, 900 and 1800 nM (in separate experiments), and measurements were carried out for another 10 min. For each ATP concentration, the slope of [eATP] decay (vi) was estimated from [eATP] kinetics. EctoATPase activity was expressed as nM/(mg protein min). Experimental data of ATPase activity vs [ATP] were fitted to a linear function, from which the slope of the curve was taken and denoted as K<sub>ATP</sub>.

## 2.9. Data analysis

In kinetic graphs, data represent means without standard error to simplify the figure. Statistical significance was determined using Mann Whitney Test and Kruskal-Wallis Test followed by a Dun's Multiple Comparison Test with the GraphPad Prism software (Graphpad Software, CA, USA). Differences were considered significant when p < 0.05. The numbers of determinations (n) from independent preparations (N) are indicated in each legend.

## 2.10. Ethics Statement

All procedures conformed to the Declaration of Helsinki. The collection of human blood samples for this study was conducted according to the protocols approved by the Clinical Research Ethics Committee of the Faculty of Pharmacy and Biochemistry of the University of Buenos Aires (UBA) (RESCD-2020-281-E-UBA-DCT_FFYB).

All healthy donors provided written informed consent for the collection of samples and subsequent use. The use of this material follows long-standing protocols and has not been associated with any adverse or other unforeseen events.
# 3. RESULTS

Results of this study describe the contribution of ui-RBCs and MPs to eATP regulation in primary cultures of *P. falciparum* infected RBCs. Regulation of eATP depends on the dynamic balance between the rates of ATP release and eATP hydrolysis. This balance can be altered by several stimuli and/or by osmotic transmembrane gradients inducing changes in cell shape or cell volume.

## 3.1- Regulated ATP release of ui-RBCs

The following experiments were aimed at showing whether ui-RBCs, as compared to c-RBCs, exhibited changes in the capacity to release ATP, which should result in an altered kinetics of eATP accumulation.

A wide variety of physiological and pharmacological stimuli induce ATP release from RBCs [40,41], and the kinetic pattern of eATP accumulation is stimulus specific [42]. This is why two stimuli were chosen that activate ATP release by different signaling routes. In RBCs, the adrenergic cocktail 3V activate the Gs route, which induces increases in cAMP [29], while the amphipatic peptide mastoparan (MST) acts as a Gi-βγ protein activator producing increases in cAMP and cell volume [30,32].

### 3.1.1 ui-RBCs purified by magnetic columns

The first series of experiments were run using ui-RBCs purified from an i-RBC culture by magnetic columns and denoted as ui-RBCs<sup>MC</sup>, and results compared with those of similar experiments using RBCs from an RBC culture lacking the parasite (c-RBCs<sup>MC</sup>). Using this protocol, primary culture allowed ui-RBCs to establish physical contact and/or exchange solutes or particles with i-RBCs, before purification (Figure 1A).

#### eATP kinetics of ui-RBCs<sup>MC</sup> and c-RBCs<sup>MC</sup>

In the absence of stimuli, [eATP] of ui-RBCs<sup>MC</sup> and c-RBCs<sup>MC</sup> remained steady, with basal levels ranging from 8.10 ± 0.13 to 6.44 ± 2.75 nM /10<sup>6</sup> cells, respectively (Figure 1B). Following stimulation with 3V (Figure 1B up) or MST (Figure 1B down), both cell types showed a fast increase in [eATP] to a maximal value, which remained constant thereafter. To evaluate the magnitude of ATP release, increases of [eATP] were evaluated as Q<sub>ATP</sub>, i.e., the ratio between [eATP] at 1 min post-stimulus and the basal [eATP]. In the presence of MST and 3V, values of Q<sub>ATP</sub> were 1.4-1.9 fold higher for ui-RBCs<sup>MC</sup> than for c-RBCs<sup>MC</sup>. Pannexin-1 facilitates passive transport of ATP across the plasma membrane in RBCs [40][29]; this protein was identified by western blot analysis in membranes from both c- and ui-RBCs<sup>MC</sup>, showing similar band intensities for both cell types (Figure 1C right). 3V dependent ATP release was 30-40% inhibited by carbenoxolone (CBX), a well-known inhibitor of Pannexin-1 (Figure 1C left).

Cell lysis before and after the treatments was similar for both cell types (Supplementary Figure 2A) so that higher ATP release of ui-RBCs<sup>MC</sup> was not due to higher hemolysis.

In general, experiments using 3V and MST showed that eATP kinetics was compatible with strong, but transient activation of ATP release, which was partially dependent on Pannexin-1. To generalize results, similar experiments were performed using cultures containing another *P. falciparum* strain, that is, the 3D7 strain. Similar eATP kinetics and Q<sub>ATP</sub> values were observed with ui-RBCs<sup>MC</sup> (Supplementary Figure 5).

#### ATP content and energy cost of ATP release
In RBCs, the only source of eATP is intracellular ATP (iATP), which is produced continuously by glycolysis. Since iATP concentration constitutes the main driving force for ATP exit in RBCs [29,32], we verified whether the observed higher ATP release of ui-RBCs<sup>MC</sup>, compared to c-RBCs<sup>MC</sup>, could be due to changes in iATP content. Cells were incubated under unstimulated conditions, and then briefly exposed to 50 µg/mL digitonin, to allow ATP release by cell rupture. Digitonin exposure caused a fast 160-fold [eATP] increase. iATP concentrations were roughly similar for c-RBCs<sup>MC</sup> (1.77 ± 0.35 mM) and ui-RBCs<sup>MC</sup> (1.89 ± 0.25 mM). Thus, in principle, the observed differences in the rate of ATP release for both cell types were not due to changes in the transmembrane chemical gradient of ATP (given by the difference between iATP and eATP), and must therefore be due to an enhanced permeability of ATP in ui-RBCs<sup>MC</sup>.

Since iATP is a key molecule controlling the energy metabolism of cells, a too high ATP release may compromise intracellular energetics of RBCs. In this context, by comparing iATP with eATP concentrations along eATP kinetics, we were able to estimate the energy cost of ATP release. Results showed that maximal values of eATP after 3V exposure (Figure 1B up) represented only 0.4% (c-RBCs<sup>MC</sup>) and 1.1% (ui-RBCs<sup>MC</sup>) of iATP concentration. Thus, the energy cost for ui-RBCs<sup>MC</sup> was relatively low, but nevertheless 2.75-fold higher than for c-RBCs<sup>MC</sup>.

In experiments above, ui-RBCs<sup>MC</sup> could have established physical contact with i-RBCs during primary culture, potentially exchanging or affecting lipids and/or proteins at contact sites, which may in turn alter ATP permeability of those cells. In this respect, we wondered whether the observed activation of ATP release of ui-RBCs<sup>MC</sup> can be also observed using a different protocol to isolate cells, one in which ui-RBCs are produced without having contact with i-RBCs, as described below.

### 3.1.2 ui-RBCs produced in semi-permeable supports

RBCs were cultured in wells containing two chambers separated by a permeable mesh support. Suspensions containing high-stage-RBCs (first stage of the intraerythrocytic cycle of the parasite) were placed on the upper chamber, while c-RBCs were placed on the lower chamber (Figure 1D). Considering that the whole intraerythrocytic asexual cycle of *P. falciparum* occurs in approximately 48 hours, cultures were followed for 24, 36, and 48 hours, without media exchange. Using this configuration, i-RBCs in the upper chamber cannot establish physical contact with RBCs in the lower chamber, though soluble factors and/or vesicles can diffuse through the 0.4 µm pores of the mesh. Experiments were run using RBCs from the lower compartment, denoted as ui-RBCs<sup>Mesh</sup>. Control experiments were performed under similar conditions, where both the upper and lower compartments contained control RBCs; i.e., a RBCs culture in the absence of the parasite. Cells from the lower chamber were taken to perform control assays and denoted as c-RBCs<sup>Mesh</sup>.

#### eATP kinetics using c-RBCs<sup>Mesh</sup> and ui-RBCs<sup>Mesh</sup>

In the absence of stimuli, [eATP] of c-RBCs<sup>Mesh</sup> and ui-RBCs<sup>Mesh</sup> remained steady at 7.8 ± 1.8 and 8.6 ± 2.0 nM/10<sup>6</sup> cells, respectively (Figure 1E). Following 3V stimulation, both cell types show fast increases in [eATP] to a maximum at all culture times. In c-RBCs<sup>Mesh</sup>, increases in [eATP], estimated as Q<sub>ATP</sub>, did not change with time of culture. In ui-RBCs<sup>Mesh</sup>, a relatively low Q<sub>ATP</sub> at 24 hours was followed by incremental changes in Q<sub>ATP</sub> with time of culture, so that 3V dependent ATP release was 3-fold higher for ui-RBCs<sup>Mesh</sup> after 48 hours (Figure 1C left). Thus, ui-RBCs<sup>Mesh</sup> showed an 3V-activation of ATP release that increase with the time at which these cells remain in contact with parasitized media. In ui-RBCs<sup>Mesh</sup> ATP release was inhibited by 30% with the pannexin1 inhibitor CBX (Figure 1C left).
Overall results using semipermeable support showed that ui-RBCs<sup>Mesh</sup> display an enhanced ATP permeability that (because of the separating mesh) does not require the physical contact between ui-RBCs and i-RBCs.
A

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>

B

c-RBCs<sup>MC</sup>
ui-RBCs<sup>MC</sup>

<figure><img src="image_6.png" /></figure>

E

c-RBCs<sup>Mesh</sup>

ui-RBCs<sup>Mesh</sup>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

C

<figure><img src="image_10.png" /></figure>

c-RBCs<sup>MC</sup> ui-RBCs<sup>MC</sup>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>
**Figure 1: Extracellular ATP (eATP) kinetics of uninfected RBCs exposed to 3V and Mastoparan**

A) Scheme illustrating the procedure used to obtain control and uninfected RBCs by magnetic columns.

Suspensions of infected cultures were passed through a magnetic column. Infected cells were retained due to the electromagnetic properties of hemozoin. The non-retained fraction constitute the population of uninfected RBCs (ui-RBCs<sup>MC</sup>). A similar protocol was used for RBCs from a culture lacking the parasite (c-RBCs<sup>MC</sup>).

B) eATP kinetics under exposure to 3V and MST.

The time course of [eATP] from c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup> was quantified by on-line luminometry at room temperature. The kinetics curves were suspended for approx. 15 seconds in order to add the stimulus (arrow). At that point, cells were exposed to: 3V (N=4, n=8) and Mastoparan (N=3, n=6). Levels of eATP are mean values expressed as nM ATP/10<sup>6</sup> cells.

3V= 10 µM isoproterenol + 30 µM forskolin +100 µM papaverine.

MST= 10 µM

C) Left: Increases in [eATP] from data of B and D (48 hours) were evaluated as Q<sub>ATP</sub>, i.e., the ratio between [eATP] at 1 min post-stimulus and the basal [eATP] in the absence or presence (+) of 100 µM carbenoxolone (CBX, Pannexin-1 inhibitor). (**) p=0.0017, ***p=0.0002 c- vs ui-RBCs. # p=0.0399, ## p<0.0012 ui-RBCs in the presence vs absence of CBX). MC: cells from magnetic column protocol; Mesh: cells from transwell support protocol.

Right: Immune detection of Pannexin-1. Western Blotting was performed with samples of c- and ui-RBCs<sup>MC</sup>. Immunoblots were performed using rabbit polyclonal antibody anti-pannexin 1 (1:1000 dilution). Samples were revealed with anti-rabbit antibody conjugated with horseradish peroxidase.

D) Scheme illustrating the procedure to obtain control and uninfected RBCs by culturing cells in transwell supports.

Suspensions containing synchronized parasite cultures at ring stage-RBCs were placed on the upper chamber (right). RBCs at 1% hematocrit were placed on the lower chamber. Incubation was done for 24 (up), 36 (middle), and 48 (down) hours, without media exchange. Using this configuration, infected RBCs in the upper chamber cannot establish physical contact with RBCs in the lower chamber. Experiments were run using RBCs from the lower compartment, denoted as ui-RBCs<sup>Mesh</sup>. Control experiments were performed under similar conditions, with both chambers containing RBCs (in the absence of parasites) and denoted as c-RBCs<sup>Mesh</sup> (left).

E) eATP kinetics under 3V exposure

The time course of [eATP] from c-RBCs<sup>Mesh</sup> and ui-RBCs<sup>Mesh</sup> was quantified by on-line luminometry after 24, 36 and 48 hours of culture in transwell supports. At the time indicated by the arrow, cells were exposed to 3V (N=3-9, n=3-9). Levels of eATP are mean values expressed as nM ATP/10<sup>6</sup> cells.

## 3.2- Lytic ATP release of ui-RBCs

In addition to enhanced ATP efflux by regulated ATP release (as observed above), a potential increased fragility of ui-RBCs may also cause ATP release by cell rupture. Evaluating lytic ATP release is important because several types of infection lead to increased fragility of RBCs [43], and hemolytic eATP was postulated to have a pathophysiological role in local purinergic signaling [44]. Thus, if ui-RBCs were more
fragile than c-RBCs, then imposing osmotic treatments should lead to higher lytic ATP release, and consequently a higher eATP signaling exerted on viable ui-RBCs. Accordingly we first assess hemolysis of ui-RBCs<sup>MC</sup> exposed to several treatments causing osmotic stress.

### 3.2.1. Osmotic fragility test.

This test is used to measure the degree of resistance of RBCs to lysis as a result of a decrease in NaCl concentration of saline solutions. Relative hemolysis increased inversely with NaCl concentration. A sigmoidal equation was fitted to the experimental data. The best fit values of two parameters were obtained: 1- the concentration of NaCl at which 50% of the cells were hemolyzed (EC<sub>50</sub>), 2- a slope factor, accounting for the steepness of the curve. Figure 2A shows results for c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup>. The curve for ui-RBCs<sup>MC</sup> was right-shifted compared to that of c-RBCs<sup>MC</sup>, with the former exhibiting significantly higher hemolysis at 0.5 and 0.6 % NaCl. These changes resulted in the slope factor being -11.58 for c-RBCs<sup>MC</sup> and -7.39 for ui-RBCs<sup>MC</sup>. On the other hand, EC<sub>50</sub> values appear higher for ui-RBCs<sup>MC</sup>, though differences with c-RBCs<sup>MC</sup> were not statistically different (Table 1).

### 3.2.2 Exposure to sorbitol.

During the intracellular stage of the malarial parasite new permeation pathways are induced in the host RBCs membrane [45–47], which causes osmotic hemolysis when infected cells are exposed to sorbitol solutions [48]. When thus wondered whether, in addition to the hypo-osmotic fragility observed above, ui-RBCs display an increased membrane permeability, evidenced by enhanced sorbitol uptake coupled to hemolysis. Accordingly, the kinetic curve of sorbitol-induced hemolysis was performed with c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup>. Kinetic curves followed a non-linear function, with the slope of the curve (k) being 7 times higher in ui-RBCs<sup>MC</sup> than in c-RBCs<sup>MC</sup> (2.58 and 0.35 % hemolysis/min, respectively) (Figure 2B). For a comparison purpose, similar experiments using a suspension of i-RBCs (trophozoite FBS suspensions at 4.05 ± 0.45 % parasitemia) were performed, resulting in a k value 3 times higher than in c-RBCs (11.67 vs 0.35 % hemolysis/min) (Figure 2B).

### 3.2.3 Exposure to alpha-hemolysin

Alpha-hemolysin (HlyA) of pathogenic strains of *Escherichia coli* irreversibly binds to human RBCs, producing irreversible swelling, activation of ATP release and metabolic changes ultimately leading to hemolysis [31,36]. Thus, we verified whether the observed increased fragility of ui-RBCs<sup>MC</sup> vs c-RBCs<sup>MC</sup> could result in an equally differential response to HlyA. Exposure to 40 ng/µl HlyA produce a trend towards higher swelling and higher hemolysis in ui-RBCs<sup>MC</sup>, as opposed to c-RBCs<sup>MC</sup>, though these differences were not significant (Figure 2C). Nevertheless, iso-osmotic swelling induced by HlyA seems to follow the same trend observed during fragility tests, i.e., a higher hemolysis of ui-RBCs<sup>MC</sup> vs c-RBCs<sup>MC</sup>.

The next experiments were carried out to check if the observed increased osmotic fragility of ui-RBCs<sup>MC</sup> correlated with increased lytic ATP release in these cells.

### 3.2.4 Effect of hypo-osmotic stress on ATP release

Under strong hypo-osmotic swelling, ATP release of RBCs is mainly caused by cell rupture [30, 40, 49]. We prepared two hypo-osmotic media (171 and 206 mOsm/L) whose osmolarities coincide with those causing significant differences in the osmotic fragility test between ui-RBCs<sup>MC</sup> and c-RBCs<sup>MC</sup> (i.e., data of Figure 2A at 0.5 and 0.6 % NaCl). Under
these conditions, hemolysis and [eATP] (corresponding to lytic ATP release) were
determined. Following the hypo-osmotic challenge, both parameters (hemolysis and
eATP) revealed higher values for ui-RBCs<sup>MC</sup> than for c-RBCs<sup>MC</sup> (Figure 2D and
Supplementary Figure 6).

<figure><img src="image_13.png" /><figcaption>Figure 2: Hemolysis of infected RBCs</figcaption></figure>

A) Osmotic Fragility Test

Hemolysis (%) of c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup> as a function of NaCl concentration. A sigmoidal fit was applied to data to obtain the best fit values of EC<sub>50</sub> and the slope factor (Table 1). Results are means ± SEM (N=8-9). (*p=0.049; **p=0.007).

B) Effect of Sorbitol

RBCs suspensions of c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup> (5% hematocrit) were exposed to iso-osmotic solutions containing sorbitol (300 mM sorbitol, 10 mM HEPES, 5 mM glucose, pH 7.4) at 37 °C. Kinetic of hemolysis was quantified at several time-points by absorption at 405 nm wavelength. Results are expressed as percentage of hemolyzed RBCs (% Hemolysis) with respect to the total number of RBCs. A suspension of infected-RBCs (trophozoite stage, 4.05 ± 0.45 % parasitemia) was also tested (red symbol). Results are mean values ± SEM (N=4). Dotted lines are a fit of a non-linear function to data, with slope of the curve (k): 0.35 ± 0.16 (c-RBCs<sup>MC</sup>), 2.58 ± 1.26 (ui-RBCs<sup>MC</sup>) and 11.67 ± 3.66 (t-RBCs) %hemolysis/min.

C) Effect of HlyA

RBCs suspensions of c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup> (5% hematocrit) were exposed to HlyA 40 ng/µL at 37 °C for 10 min. Results are expressed as percentage of hemolyzed RBCs (% Hemolysis) with
respect to the total number of RBCs. Results are mean values ± SEM (N=4-5). (* p=0.0134; ** p=0.0060; ns: non-significant differences).

D) Effect of hypo-osmotic stress

RBCs suspensions of c-RBCs<sup>MC</sup> and ui-RBCs<sup>MC</sup> (10% hematocrit) were exposed to media of osmolarities 171, 206 and 300 mOsm/L at 20 °C. At 10, min samples were taken to quantify hemolysis. Results are expressed as percentage of hemolyzed RBCs (% Hemolysis) with respect to the total number of RBCs. Results are mean values ± SEM (N=4-7). (* p=0.0414; * p<0.0236; *** p=0.0004; **** p<0.0001; # p=0.0256; ## p=0.0013; ### p=0.0002 ui-RBCs<sup>MC</sup> c- vs ui-RBCs<sup>MC</sup>).

<table><caption>Table 1: EC<sub>50</sub> and Slope factor</caption><thead><tr><th></th><th colspan="2">c-RBCs<sup>MC</sup></th><th colspan="2">ui-RBCs<sup>MC</sup></th></tr></thead><tbody><tr><td>EC<sub>50</sub> (LOG)</td><td>2,202</td><td>± 0,062 (8)</td><td>2,271</td><td>± 0,151 (9)</td></tr><tr><td>Slope factor</td><td>-11,580</td><td>± 1,306 (8)</td><td>-7,387</td><td>± 1,090 (9)</td></tr></tbody></table>

Sigmoidal fit parameters from data of fragility curves (Figure 2A). Results are means ± SEM (N=8-9)

Results above show how eATP regulation of ui-RBCs is altered by activation of both regulated and lytic ATP release. Next, we evaluated whether changes in eATP hydrolysis of ui-RBCs could also influence eATP regulation. This is important since a potential increase in eATP hydrolysis may attenuate increases in eATP kinetics and the effects of eATP on purinergic signaling.

### 3.3- Extracellular ATP hydrolysis by ectoATPase activity

Once intracellular ATP is released from RBCs, the accumulated eATP can be hydrolyzed by one or more ectonucleotidases acting at the cell surface. The rate of eATP hydrolysis facilitated by these ectoenzymes can be best characterized by exposing cells to exogenous ATP. Accordingly, using ui-RBCs and c-RBCs exposed to exogenous ATP, we determined the time course of eATP consumption at very low, but physiologically relevant ATP concentrations (450, 500, and 1800 nM). Determinations were made by on-line luminometry (to detect the rate of [eATP] decrease) and by following the release of <sup>32</sup>P release from [γ-<sup>32</sup>PiATP].

The initial rate values of the kinetics curves (data not shown) were used to calculate ectoATPase activity for each [eATP], so as to build a substrate curve (i.e., ectoATPase activity vs [eATP]; Figure 3). Results are shown for RBCs produced by magnetic columns (MC, Figure 3A) and by permeable supports (Mesh, Figure 3B). EctoATPase activity could be described by a linear function with [ATP], with the slope of the curve (K<sub>ATP</sub>) representing the sensitivity of eATP hydrolysis to [ATP].

As can be seen in Figures 3A and B, K<sub>ATP</sub> amounted to 2.0 x10<sup>-3</sup> (10<sup>6</sup> min)<sup>-1</sup> (c-RBCs<sup>MC</sup>), 7.1 x10<sup>-3</sup> (10<sup>6</sup> min)<sup>-1</sup> (ui-RBCs<sup>MC</sup>), 2.7 x10<sup>-3</sup> (10<sup>6</sup> min)<sup>-1</sup> (c-RBCs<sup>Mesh</sup>) and 3.7 x10<sup>-3</sup> (10<sup>6</sup> min)<sup>-1</sup> (ui-RBCs<sup>Mesh</sup>). Thus, the rate of eATP hydrolysis by ectonucleotidases was up-regulated about two fold in ui-RBCs, as compared to c-RBCs. A similar trend could be observed at much higher, non-physiological concentrations of eATP (Figure 3C), suggesting that the observed higher ectoATPase activity of ui-RBCs is observed along an ample range of eATP concentrations.

#### 3.3.1 ATP content of the culture medium
Results above show that ectoATPase activity was higher in ui-RBCs than in c-RBCs, when assayed at similar exogenous ATP concentrations. Since ectoATPase activity increases proportionally with [ATP] (Figure 3A-B), we wondered whether changes in [ATP] of culture media may induce additional changes on ectoATPase activity along its substrate curve. To test this hypothesis, we used the permeable support protocol to take a sample of medium after 48 hours incubation from control media or parasitized media. Results, expressed by million cells, showed that ATP content of media was 11 nM in CM and 113 nM in PM. This difference in ATP content was not unexpected considering the usual higher mortality in culture media containing i-RBCs (Figure 3D). Thus, the higher eATP content in *P. falciparum* cultures should induce a higher ectoATPase activity by ui-RBCs (as compared to c-RBCs).

### 3.3.2 Potential identification of ectonucleotidases promoting ectoATPase activity

Among NTPDases, subtypes 1 and 2 were reported to facilitate eATP hydrolysis in many cell types [50], while 5'NT promotes hydrolysis of extracellular AMP. To test for the presence of NTPDases, Western-blot analysis was carried out. Samples prepared from c- and ui-RBCs<sup>MC</sup> lysates were immune tested with specific antibodies against human NTPDase1, -2, and 5'NT (=CD73). Caco2- cells lysates were used as positive controls. NTPDase1 and -2 were identified in Caco2- cells lysates as ~77 kDa and ~ 61 kDa bands, respectively; while these enzymes could not be detected in either c- or ui-RBCs<sup>MC</sup> (Supplementary Figure 7A). An immune reactive band for CD73 was identified at ~ 60 kDa with similar intensities in c- or ui-RBCs<sup>MC</sup> (Supplementary Figure 7B-C). Since we could not identify the presence of either NTPDase1 or -2, we verified whether an ectophosphatase was active in RBCs which could account for the observed eATP hydrolysis. For that purpose we followed the time course of p-nitrophenol production from pNPP, a substrate for phosphatases but not for NTPDases or CD73. Initial rate values of the kinetics curves were used to calculate ectophosphatase activity. Results showed that p-nitrophenol production was similar in ui-RBCs<sup>MC</sup> and c-RBCs<sup>MC</sup> (Supplementary Figure 8), so that ectophosphatase activity is indeed present in these cells, but cannot account for differential eATP hydrolysis between c- and ui-RBCs<sup>MC</sup>.
<figure><img src="image_14.png" /><figcaption>Figure 3: EctoATPase activity of uninfected RBCs</figcaption></figure>

A) EctoATPase activity of uninfected RBCs purified by magnetic column. eATP kinetics curves were performed using uninfected RBCs purified by magnetic columns (ui-RBCs<sup>MC</sup>) and exposed to exogenous [eATP] (450, 900, and 1800 nM) (data not shown). The time course of eATP concentration ([eATP]) was quantified by on-line luminometry. Similar experiments were performed with control RBCs (c-RBCs<sup>MC</sup>). The initial rate of [eATP] decay (ectoATPase activity) was measured and used to build a substrate curve (ectoATPase activity vs [eATP]). Results are means ± SEM (N=2-6, n=4-12).

The dotted line is a fit of a linear function to experimental data, with slope of the curve K<sub>ATP</sub>: 2.05 ± 0.05 x10<sup>-3</sup>(10<sup>6</sup>cells min)<sup>-1</sup> (R<sup>2</sup>: 0.90) for c-RBCs<sup>MC</sup> and 7.10 ± 2.34 x10<sup>-3</sup>(10<sup>6</sup>cells min)<sup>-1</sup> (R<sup>2</sup>: 0.97) for ui-RBCs<sup>MC</sup>. The asterisk represents the result of an independent experiment where ATPase activity was determined by following the time course of [³²P]Pi released from [γ-³²P]ATP at 900 nM (N=2, n=2). \* for c-RBCs<sup>MC</sup> and \* for ui-RBCs<sup>MC</sup>.

B) EctoATPase activity of uninfected RBCs obtained from transwell support culture.

Similar to A but eATP kinetics curves were performed using uninfected RBCs from transwell supports (ui-RBCs<sup>Mesh</sup>) and exposed to exogenous [eATP] (450, 900, and 1800 nM). Results are means ± SEM (N=3-5, n=6-10). The dotted line is a fit of a linear function to experimental data, with slope of the curve K<sub>ATP</sub>: 2.70 ± 0.23 x10<sup>-3</sup>(10<sup>6</sup>cells min)<sup>-1</sup> (R<sup>2</sup>: 0.98) for c-RBCs<sup>Mesh</sup> and K<sub>ATP</sub>: 3.73 ± 0.03 x10<sup>-3</sup>(10<sup>6</sup>cells min)<sup>-1</sup> (R<sup>2</sup>: 0.99) for ui-RBCs<sup>Mesh</sup>. The asterisk represents the extrapolation of activities values taking into account the increments in [eATP] occurring during 48 hours of cell culture (see D).

C) Apparent maximal ectoATPase activity.
Suspensions of ui-RBCs<sup>MC</sup> and c-RBCs<sup>MC</sup> were exposed to exogenous ATP (1000 µM) in assay media without Pi. Pi production was quantified by the malachite green assay (Supplementary Figure 9). Values of ectoATPase activity were derived from the initial rate of Pi production.

Right y-axis. EctoATPase activities at 1000 µM ATP taken from Supplementary Figure 9. Left y-axis. EctoATPase activities at 1800 nM ATP taken from Figure 3A. Results are means ± SEM. (*p=0.0446).

D) ATP of the cell culture medium.

Off-line luminometry was used to assess [eATP] of culture media in the absence or presence of i-RBCs after 48 hours incubation in transwell supports. Results are means ± SEM (N=3). (*p=0.0415). CM: Control Media; PM: Parasitized Media.

**3.4. Microparticles: identification, production and eATP regulation**

Considering that RBCs are known to shed MPs in the absence and presence of several stimuli, and that production of these vesicles is enhanced in pathological contexts, we wondered whether MPs from i-RBCs membranes may also contribute to eATP regulation of RBCs during infection.

Accordingly, we purified MPs from an i-RBCs culture (i-MP) or from a control culture (c-MP). Electron micrographs of MPs, obtained from the differential centrifugation of the infected culture supernatant, showed heterogeneous rounded electron-dense vesicles and other single and double membrane vesicles. The size of the vesicles was <500 microns (Figure 4A). Flow cytometry of labeled MPs yielded a diameter in the range of 200-500 nm, consistent with data from electron micrographs (Supplementary Figure 10). From the total number of events, > 40% were positive for CFSE dye, suggesting the presence of esterase activity within MPs (Supplementary Figure 10). Side scatter plots of labelled MP showed that i-MPs were more heterogeneous and displayed greater intensities than c-MPs (Figure 4B). Production of MPs was 2-fold higher for i-RBCs than that for c-RBCs (Figure 4C).

To evaluate the potential contribution of MPs to ATP regulation, we next tested ATP content and eATP hydrolysis of MPs.

**3.4.1 ATP content**

Testing ATP release from MPs with ATP inducers such as 3V or MST is unfeasible since these vesicles are unlikely to preserve signaling mechanisms of intact RBCs. Nevertheless, as MPs are formed by evaginations of the RBC cell membrane, they might encapsulate cytosolic ATP, which could *in vivo* be released upon rupture and therefore participate in eATP regulation.

Accordingly, on-line luminometry experiments were performed using MPs suspended in RBC medium containing the luciferin-luciferase mixture. This enabled continuous detection of ATP accumulating in the extravesicular media (denoted as eATP<sub>MP</sub>). Results were normalized per mg protein, to allow comparison of eATP kinetics of i-MP vs c-MP (Figure 4D) irrespective of the fact that i-MP are produced in higher quantities (Figure 4C). Experiments were run under unstimulated control conditions for 1.5 min (basal trace) after which 50 µg/mL digitonin was added to allow full release of ATP by MPs rupture (Figure 4D). For both i-MP and c-MP, basal eATP<sub>MP</sub> traces were stable. This means that MPs exhibited a good seal and ATP leakage was negligible. Digitonin exposure caused a fast increase of eATP<sub>MP</sub> for both, c- and i-MP.
The inset of Figure 4D shows increases of eATP<sub>MP</sub> denoted as Q<sub>iATP MP</sub>, i.e., the ratio between eATP at 1 min post-digitonin and the basal eATP. Values of Q<sub>ATP</sub> for c-MP and i-MP were not significantly different.

### 3.4.2 Hydrolysis of eATP by microparticles

Infected-MP and c-MP were exposed to various ATP concentrations (50-1800 nM), and ectoATPase activity was measured and plotted as a linear function of [eATP] (Figure 4E), with slope of the curve (K<sub>ATP</sub>) amounting to 0.33 ± 0.02 min<sup>-1</sup> mg<sup>-1</sup> in c-MP and 1.08 ± 0.06 min<sup>-1</sup> mg<sup>-1</sup> in i-MP (Figure 4E). I.e., eATP hydrolysis by ectonucleotidases was upregulated 3-fold in i-MPs, as compared to that of c-MPs.

Overall results of this part show that MPs are able to enclose ATP in sealed vesicles, and are capable of hydrolyzing eATP, with i-MPs exhibiting higher ectoATPase activity than c-MPs.

<figure><img src="image_15.png" /><figcaption>Figure 4: Structure, ATP content and ATP hydrolysis of microparticles</figcaption></figure>

Microparticles (MPs) were obtained from a suspension of RBCs infected by *P. falciparum* (i-MP) or from a control suspension of RBCs (c-MP).

#### A) Electron micrographs

Electron micrographs of MPs showed heterogeneous rounded electron-dense vesicles and other single and double membrane vesicles. The size of the vesicles was less than 500 microns.

#### B) Flow cytometry analysis

Side scatter plot of labelled MPs.
C) Production of MPs

MPs were purified by differential centrifugation of cell culture medium. Production of MPs was quantified by the Bradford assay and expressed as mg protein per mL of cell culture. RBCs were cultured for 24 hours before purification. Results are means ± SEM (N=3-6) (*p=0.0436).

D) Intraparticular ATP

The value of intraparticular ATP was measured after permeabilization of MPs with digitonin (arrow, 50 µg/mL), and detection of ATP accumulation in the assay medium was done by on-line luminometry. Values are expressed as nM/mg protein of both, i-MPs and c-MPs, from which the intraparticular ATP content could be derived ($Q_{iATP MP}$, inset D). $Q_{iATP MP}$, i.e., the ratio between [eATP<sub>MP</sub>] at 1 min post-digitonin and basal [eATP<sub>MP</sub>], is expressed as means ± SEM (N=3-6).

E) ATP hydrolysis of MPs

A suspension of MPs was exposed to exogenous ATP (50-1800 nM). The time course of eATP concentration ([eATP]) was quantified by on-line luminometry. The initial rate of [eATP] decay after ATP addition was measured from kinetics curves (data not shown). Values are expressed as nM ATP/(min mg MPs). Results are means ± SEM (N=2-3, n=4-6). The dotted line is a fit of a linear function to experimental data, with slope of the curve $K_{ATP}: 0.33 \pm 0.0$ (min mg)<sup>-1</sup> (R<sup>2</sup>:0.97) for c-MP and $K_{ATP}: 1.08 \pm 0.06$ (min mg)<sup>-1</sup> (R<sup>2</sup>:0.98) for i-MP. The error bars represent the result of an independent experiment where ATPase activity was determined by following the time course of [<sup>32</sup>P]Pi released from [γ-<sup>32</sup>P]ATP at 900 nM (N=2, n=2). * for c-MP and * for i-MP.

DISCUSSION

The main goal of this study is to provide a quantitative analysis of the dynamic balance between ATP efflux and eATP hydrolysis of ui-RBCs and MPs, yielding particular kinetics of eATP accumulation. The significance of the main findings is discussed below.

4.1 Extracellular ATP kinetics of ui-RBCs

Ui-RBCs from *P. falciparum* in vitro cultures were stimulated with the adrenergic cocktail 3V and the amphipatic peptide nystatin. These and other treatments to increase cAMP systematically lead to intracellular signaling events triggering activation of ATP release in RBCs from humans and several lab animals [5,29,30,32,51,41,52]. Both for ui-RBCs and c-RBCs, eATP kinetics were similar in that 3V and MST triggered strong and acute but transient ATP release. Ui-RBCs showed much higher ATP release than that of c-RBCs, a feature that appeared after > 24 hours of *P. falciparum* infected cell culture.

Changes operating on ui-RBCs do not require their contact with i-RBCs, since eATP kinetics was very similar when these cells were isolated using magnetic columns, which allow contact between ui-RBCs and i-RBCs, or using a separating mesh, where no contact between these cell types was allowed.

Since both 3V and MST produced no lysis, the observed eATP kinetics can be assigned to regulated mechanisms of ATP release. In this respect, Pannexin-1 proved to be an important ATP conduit of RBCs challenged by 3V and/or MST [7,29,30,32], and several other stimuli [40,52]. In agreement with these findings, 3V-dependent ATP release of ui-RBCs was partially inhibited by the Pannexin-1 inhibitor CBX, causing significant flux reductions.

Despite a higher ATP efflux of ui-RBCs as compared to c-RBCs, Pannexin-1 level at the surface of both cell types appeared similar, thus suggesting that the observed higher ATP release of ui-RBCs is not related to a change in plasma membrane concentration of Pannexin-1. Nevertheless, ATP efflux might be also activated indirectly. Indeed, cell membranes of ui-RBCs were shown to have modified fatty acid composition, oxidation state, and bio-mechanical and nano-structural alterations [13,14,53,54]. Oxidative stress
due to either infection by *Plasmodium* spp. or other stressors are known to cause
activation of permeability to ATP and other solutes in i-RBCs [47,55]. Enhanced
membrane permeability is important in as much as it facilitates parasite growth by
supplying incoming nutrients and removing outgoing metabolic waste products [46,56].

The concentration of intracellular ATP was approximately 3 orders higher than the measured concentration of eATP for both ui-RBCs<sup>MC</sup> and c-RBCs<sup>MC</sup>. This chemical gradient provides a strong thermodynamic force to drive significant transmembrane ATP efflux. Although elevated ATP efflux in ui-RBCs could have resulted from even subtle increases in [iATP], no changes in this parameter were observed, strongly suggesting that the higher ATP efflux of ui-RBCs is due to enhanced ATP permeability.

In addition to regulated ATP efflux, a higher fragility of ui-RBCs should explained the observed higher hemolysis of ui-RBCs. Accordingly, osmotic fragility experiments showed a higher osmotic fragility of ui-RBCs<sup>MC</sup>, as compared to c-RBCs<sup>MC</sup>, which correlated with increased permeability to sorbitol, and increased release of ATP when cells were challenged by hypo-osmotic media. This higher fragility implies that substantial lytic ATP can be released upon mechanical stress, suggesting a similar response when *in vivo* ui-RBCs pass through the small branches of the microcirculation.

## 4.2 Extracellular eATP hydrolysis of ui-RBCs

Hydrolysis of eATP by ectoATPase activity of RBCs is an important mechanism to control eATP accumulation, terminating the action of eATP on P receptors of RBCs and other cells of the vascular space [2,3]. EctoATPase activity of ui-RBCs and c-RBCs was directly proportional to [eATP] in the low micromolar range, which is within the range of the concentrations of the nucleotide in the plasma of healthy individuals [33]. This means that any increase in plasma [eATP] should lead to higher eATP hydrolysis by ectonucleotidases of RBCs.

Interestingly in this context, ectoATPase activity was more efficient in ui-RBCs than in c-RBCs. Such an activation of eATP hydrolysis was also observed in i-RBCs [7]. The higher ectoATPase activity observed in ui-RBCs cannot be due to *de novo* expression of ectonucleotidases, since RBCs lack a nucleus and the cellular machinery required for protein synthesis.

Western blot analysis did not show reactive bands for either NTPDase 1 and 2 (Supplementary Figure 7A). We cannot rule out that low amounts of NTPDase are nevertheless present at the RBC surface but are not detected due to the sensitivity limits of our assay. Low concentration of NTPDase with a relatively high $K_{ATP}$ may promote significant eATP hydrolysis. In ui-RBCs, the high kinetic value of ectoATPase is indicative of changes in the membrane environment. In this respect, modifications of the fatty acid composition were reported for ui-RBCs [13], though the correlation of these changes with the activity of membrane-bound enzymes was not studied. Membrane lipid modifications, including a decreased content of polyunsaturated fatty acids, arachidonic, and docosahexaenoic acid, have been described in non-parasitized RBCs co-cultured with parasitized-RBCs [13][14].

## 4.3 ATP content and extraparticular ATP of microparticles (eATP<sub>MP</sub>)

Recent studies have reported enhanced production of MPs both in the circulation of malaria patients and in i-RBCs cultures. MPs are important not only for cell-cell communication [17–19,57,58] but also contribute to vascular dysfunction during malaria infection [59]. Coinciding with previous reports, we show that the production of MPs was also significantly enhanced during infection. In the absence of treatment, basal eATP level of MPs was steady. Exposure to detergent led to an immediate increase of ATP in the
assay medium, thus corroborating the capacity of MPs to retain ATP originally produced RBCs.

As reported before for RBCs exposed to various stimuli causing MP production [17,18,57], the membranes of the resulting particles will not necessarily display an identical composition to that of the originating RBCs. Nevertheless, to evaluate the contribution of MPs to eATP regulation we checked whether MPs membranes exhibited ectoATPase activity. MPs, both from c-RBCs and i-RBCs, displayed a linear dependence of ectoATPase activity with exogenous [ATP]. EctoATPase activity was higher for i-MPs vs c-MPs in two ways: 1- in a per mg basis, since i-MPs showed a higher kinetic efficiency (i.e., K<sub>ATP</sub); 2- in an absolute basis, due to the higher MPs production of infected cultures, as compared to MPs from c-RBCs. Thus MPs not only contribute to eATP degradation of RBCs, but this process is enhanced during infection.

## 5. CONCLUSIONS

Overall results show that ui-RBCs displayed activation of regulated as well as lytic mechanisms of ATP release. These stimuli represent typical stressors found in malaria, where levels of cathecholamines can be high, and mechanical stress and ischemia due to circulatory impairments can occur. The contribution of ui-RBCs to enhanced intravascular ATP can be relevant since these cells constitute ~ 96% of all cell types, occupy ~ 40% of total blood volume in the circulation and contain mM intracellular ATP, whereas only low µM eATP is required for purinergic signaling.

In vivo, eATP signaling will be counteracted by other processes, since intravascular eATP hydrolysis can be facilitated not only by ectonucleotidases of ui-RBCs, i-RBCs, and MPs, but also by ectonucleotidases of other blood cells and the vascular endothelium, and exonucleotidases of the plasma [60]. Nevertheless, provided that paracrine distances from ATP exit from ui-RBCs, i-RBCs and MPs are short, an elevated [eATP] would activate P2 receptors of the vascular endothelium to enhance vasodilation, thereby partially offsetting vessel occlusion [2,5]. On the other hand, eATP hydrolysis coupled to ectoAMPase activity would produce ADO and ADP, two important purinergic signals modulating several purinergic signaling processes [61]. While the consequences to malaria are unknown, high eADP can partially inhibit ATP release, while extracellular adenosine, from eATP dephosphorylation, can ameliorate cell volume increase in RBCs [28]. Elevated eATP of ui-RBCs, and concomitant purinergic signaling, may also be important to compensate for inhibitory signals expected to occur in malaria. E.g., the massive increase in the glycolytic rate of i-RBCs leads to hyperlactatemia [12], and extracellular lactate is known to inhibit ATP release of RBCs *in vitro* [62].

This capacity of ui-RBCs to release ATP is not without costs, since these cells required 2.75-fold more intracellular ATP than c-RBCs to promote ATP exit. Although small in magnitude, this higher energy requirement is relevant for various reasons. I.e., i-RBCs only live ~ 48 hours before bursting, so that it is adaptive for the parasite to fully reset RBCs biochemistry, and use as much metabolic energy as needed in order to complete the parasite life cycle, kill RBCs and re-infect. On the other hand, ui-RBCs are expected to live much longer, even if the lifespan of these cells is decreased when compared to c-RBCs in humans [16,63,64] and also in rodents [65,66]. Thus, for ui-RBCs, full conservation of intracellular ATP content is remarkable, particularly when these cells exhibit decreased glycolytic flux [67] (the only source of iATP in RBCs) and are not stimulated just once (as in this study) to release ATP, but also repeatedly as they travel across the circulatory
system. MPs regulation of ATP is relevant due to the elevated levels of circulating MPs found in malaria patients, a condition associated with the progress towards disease severity [68–70]. In this context, our results show that MPs can act in purinergic signaling by carrying intraparticular ATP across the circulation, where potential rupture may cause ATP release at specific sites in the microvasculature, as postulated before [20].

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7. ACKNOWLEDGMENTS

This work was supported by CONICET (Grant PIP 112 20150100459); Universidad de Buenos Aires (Grant 200201701001 52BA, PDE 45 2020); Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (PICT 2019-0905, ECOS Sud A15S01; PICT 2018-0630); Laboratory of Excellence GR-Ex (Grant ANR-11-LABX-0051). MAO was funded by grants from the French National Research Agency (ANR-11-LABX-005). JS received support from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2016-05867). CLA, CMF,
MFLD and PJS are career researchers at Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). NL is a doctoral student. We are grateful to Dra Vanesa Herlax for providing alpha-hemolysin. We are very grateful to Dr. SIlvia Repetto (Instituto de Investigaciones en Microbiología y Parasitología Médica-IMPAM-CONICET) for providing her laboratory facilities.

## 8. AUTHORS CONTRIBUTIONS

CLA, ACH, BG, MAO, PJS designed the research study. CLA, JPS, CH, MFLD, NL performed the research. CL, CMF, JS contributed essential reagents and tools. CLA, MAO, PJS analyzed the data and CLA, MAO, PJS wrote the paper. All authors participated to the redaction of the manuscript and agree with its contents.

## 9. ABBREVIATIONS

RBCs: red blood cells; eATP: extracellular adenosine triphosphate; ui-RBCs<sup>MC</sup>: uninfected RBCs purified by magnetic columns from *Plasmodium falciparum* culture; c-RBCs<sup>MC</sup>: control RBCs pass through magnetic columns according to a mining protocol performed to obtain ui-RBCs<sup>MC</sup>; 3V, a cAMP activating cocktail containing 10 µM isoproterenol, 30 µM forskolin, and 100 µM papaverine; CBX, carbenoxolone; MCT: mastoparan; DIG: digitonin; ui-RBCs<sup>Mesh</sup>: RBCs taken from the lower compartment of *P. falciparum* cultures done on permeable supports of 12 mm diameter with a membrane of 0.4 µm pore size mesh; c-RBCs<sup>Mesh</sup>: RBCs taken from the lower compartment of control cultures done on permeable supports of 12 mm diameter with a membrane of 0.4 µm pore size mesh; HlyA, α-hemolysin; CM: Control Media; PM: Parasitized Media; MPs: microparticles; ATP<sub>IMP</sub>: intraparticular ATP.
**Declaration of interests**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[ ] The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
<figure><img src="image_16.png" /></figure>

Graphical abstract
Plasmodium falciparum culture

<figure><img src="image_17.png" /></figure>

MICROPARTICLES

<figure><img src="image_18.png" /></figure>

UNINFECTED RBCs

<figure><img src="image_19.png" /></figure>

Graphics Abstract
<figure><img src="image_20.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /><figcaption>Figure 1</figcaption></figure>
A

C

<figure><img src="image_28.png" /></figure>

B

<figure><img src="image_29.png" /></figure>

D

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /><figcaption>Figure 2</figcaption></figure>
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<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /><figcaption>Figure 3</figcaption></figure>
A

<figure><img src="image_36.png" /></figure>

B

<figure><img src="image_37.png" /></figure>

C

<figure><img src="image_38.png" /></figure>

D

<figure><img src="image_39.png" /></figure>

E

<figure><img src="image_40.png" /><figcaption>Figure 4</figcaption></figure>