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. Author manuscript; available in PMC: 2018 Oct 11.
Published in final edited form as: Blood. 2018 Jul 19;132(12):1332–1344. doi: 10.1182/blood-2018-05-849307

Platelets kill circulating parasites of all major Plasmodium species in human malaria

Steven Kho 1, Bridget E Barber 1,2, Edison Johar 3, Benediktus Andries 4, Jeanne R Poespoprodjo 4,5,6, Enny Kenangalem 4,5, Kim A Piera 1, Anna Ehmann 3, Ric N Price 1,7, Timothy William 2,8,9, Tonia Woodberry 1, Simon Foote 3, Gabriela Minigo 1, Tsin W Yeo 1,2, Matthew J Grigg 1,2, Nicholas M Anstey 1,2,#, Brendan J McMorran 3,*,#
PMCID: PMC6161646  EMSID: EMS79922  PMID: 30026183

Abstract

Platelets are understood to assist host innate immune responses against infection, although direct evidence of this function in any human disease, including malaria, is unknown. Here we characterized platelet-erythrocyte interactions by microscopy and flow cytometry in malaria patients naturally infected with Plasmodium falciparum, P. vivax, P. malariae or P. knowlesi. Blood samples from 376 participants were collected from malaria-endemic areas of Papua, Indonesia, and Sabah, Malaysia. Platelets were observed binding directly with and killing intraerythrocytic parasites of each of the Plasmodium species studied, particularly mature stages, and was greatest in P. vivax patients. Platelets preferentially bound to infected more than uninfected erythrocytes in the bloodstream. Analysis of intraerythrocytic parasites indicated the frequent occurrence of platelet-associated parasite killing, characterized by the intraerythrocytic accumulation of platelet factor-4 and terminal deoxynucleotidyl transferase dUTP nick-end labeling of parasite nuclei (PF4+TUNEL+ parasites). These PF4+TUNEL+ parasites were not associated with measures of systemic platelet activation. Importantly, patient platelet counts, infected erythrocyte-platelet complexes and platelet-associated parasite killing correlated inversely with patient parasite loads. These relationships, taken together with the frequency of platelet-associated parasite killing observed amongst the different patients and Plasmodium species, suggest that platelets may control the growth of between 5 and 60% of circulating parasites. Platelet-erythrocyte complexes comprised a major proportion of the total platelet pool in malaria patients and may therefore contribute considerably to malarial thrombocytopenia. Parasite-killing was demonstrated to be platelet factor-4-mediated in P. knowlesi culture. Collectively, our results indicate that platelets directly contribute to innate control of Plasmodium infection in human malaria.

Keywords: platelets, malaria, Plasmodium, killing, falciparum, vivax, malariae, knowlesi, platelet factor-4, thrombocytopenia

Introduction

Platelets are the second most abundant cell of the circulation after red blood cells (RBC) and the principle regulators of hemostasis. Platelets can also integrate host immune responses through production of immunomodulatory molecules and via cell-to-cell interactions with white blood cells (WBC), and may have host-protective roles in infectious disease.1 Platelets are an abundant source of antimicrobial molecules2,3, have broad-spectrum pathogen killing activities49 and are required for host-mediated pathogen control and host survival in some infectious disease models.1013 Clinically, low platelet counts are often associated with a poor prognosis and increased risk of infection.1416 However, to date direct evidence that platelets contribute to host protection in any human infectious disease is lacking.

Malaria kills over 400 000 people each year.17 While most deaths are caused by Plasmodium falciparum, all Plasmodium species can cause severe and fatal infection.1821 Malaria pathogenesis is driven primarily by parasite biomass2224 and modulated by host innate and adaptive immune responses.25,26 Thrombocytopenia is common in all malarias and is a risk factor for mortality in African children with falciparum malaria27, Southeast Asian adults and children with falciparum and vivax malaria28, and adults with knowlesi malaria.21 However, the mechanisms leading to thrombocytopenia are not fully understood and its subsequent effects on parasite biomass, disease control or progression has yet to be quantified for any of the human Plasmodium species.

Indirect evidence from separate sources suggests that platelets may contribute to the host protection in malaria. This includes observations of human platelets directly binding to and killing P. falciparum-infected RBC (iRBC) in culture, and reduced survival in Plasmodium-infected mice depleted of platelets.12,13,2931 A mechanism for the direct killing of Plasmodium involves platelet factor-4 (PF4), an abundant antimicrobial protein secreted by platelets, that, upon entering the cell via the Duffy-antigen – a chemokine receptor expressed by RBC30 – kills P. falciparum parasites by selectively lyzing the parasite digestive vacuole.29 However, a recent study did not reproduce the parasite killing effect of platelets in P. chabaudi-infected mice or P. falciparum-iRBC32, showing the difficulties in using in vitro and in vivo disease models to study these phenomena33, and highlighting the need for additional research in people with malaria.34

No clinical studies have addressed the role of platelets in killing P. falciparum parasites across the spectrum of clinical disease, nor their role in protection and pathogenesis in human malaria from non-falciparum Plasmodium species. Here we characterized cell-to-cell platelet interactions in patients naturally infected with P. falciparum, P. vivax, P. malariae or P. knowlesi, and examined relationships between platelet-associated parasite killing and parasite biomass. We also demonstrate the mechanism by which human platelets can kill P. knowlesi, a second culturable human Plasmodium species.

Methods

Study participants

In Papua, malaria patients attending the Mitra Masyarakat Hospital in Timika were enrolled between 2014-2016. This lowland forest region has perennial transmission of P. falciparum, P. vivax and P. malariae. In Sabah, patients were enrolled between 2012-2016 from 3 district hospitals in Kudat Division as part of concurrent prospective clinical studies.35 Sabah is an area of low malaria transmission, which during this period was co-endemic for P. falciparum, P. vivax, and the zoonotic parasite, P. knowlesi.

Criteria for enrolment in both cohorts included: a blood film positive by microscopy for any Plasmodium species, including mixed infections in Papua, fever or history of fever in the last 48 h, no major concurrent illness or co-morbidity, and no prior antimalarial therapy in the preceding 24 h. Patients were excluded if pregnant or lactating. In Papua patients aged <16 or >60 years or with a hemoglobin concentration ≤7 g/dL were also excluded. Severe malaria was defined according to WHO 2014 research criteria.20 Hospitalization was at the discretion of the treating clinician in Papua and was mandatory in all malaria patients in Sabah. All malaria patients were treated according to local guidelines as described previously.20,3638 Controls were selected from visitors or relatives of malaria patients, with no fever or history of fever in the preceding 14 d and with a blood film negative for malaria parasites.

Details of blood collection and analysis procedures are described in Supplementary Materials.

Platelet binding to iRBC and uRBC

Blood samples were analyzed by flow cytometry to measure platelet-bound iRBC or uninfected RBC (uRBC), and expressed as frequencies and absolute numbers. In Papua, assays were performed on fresh samples and in Sabah on fixed blood (Cytofix™, BD Biosciences). Fluorescent antibody panels comprised anti-CD45 (white cell exclusion marker), anti-CD41 or CD42b (platelet marker), anti-CD235ab (RBC marker), and either Hoechst 33342 or DRAQ-5 (parasite nuclear marker). The gating strategy for measuring platelet-iRBC/uRBC complexes in Papua is illustrated in Fig. 1B and in Sabah in Fig. S1A. Refer to Supplementary Materials for details of the staining materials and procedures.

Fig. 1. Platelet binding in clinical malaria blood samples.

Fig. 1

A) Photos of platelet-bound iRBC from patient Giemsa smears (black arrowhead = platelet). Images were taken at 1000x magnification using a Samsung Note-3 camera attached to an Olympus CX31 microscope. Scale bar = 5 μm. B) Representative flow cytometry gating strategy to measure platelet binding in a P. falciparum patient and healthy control. C) Frequency of platelet-bound uRBC by flow cytometry in malaria patients compared to controls in samples from Ci) Papua (controls n=17, Pf n=23, Pv n=26, Pm n=9, mixed n=7) and Cii) Sabah (controls n=28, Pf n=14, Pv n=85, Pk n=106) (Kruskal-Wallis, *significantly different to all other groups). D) Frequency of platelet-bound iRBC and uRBC by flow cytometry in samples from Di) Papua (n as per Ci) and Dii) Sabah (n as per Cii) (Wilcoxon test). E) Inverse correlation of platelet-bound iRBC with parasitemia in samples from Ei) Papua and Eii) Sabah (Spearman). Scatterplots indicate median ± interquartile range for each group. Parasitemia values are log transformed. Data presented in Table S1. Abbreviations: Pf, P. falciparum; Pv, P. vivax; Pm, P. malariae; Pk, P. knowlesi.

Platelet-associated parasite killing

Immunofluorescent microscopy of Cytofix™-treated blood was used to quantify platelet-associated parasite killing, based on a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay described elsewhere30 that labels degraded or sheared DNA, an indication of apoptosis or necrosis. Platelets and intraerythrocytic localization of PF4 were detected with an anti-human PF4 Ab (Abcam, UK) and intraerythrocytic Plasmodium DNA was identified using DAPI. Only high parasitemia samples (>5000 parasites/μL) were analyzed and the number of parasites counted per sample recorded. In a subset of patients, iRBC were distinguished into rings (DAPI+ without parasite pigment) or mature stages (DAPI+ with parasite pigment) by immunofluorescence, and the proportion of stages confirmed with Giemsa-stained blood smears performed in parallel. Refer to Supplementary Materials for more detail.

Platelet activation

In Papua, platelet activation markers including platelet PAC-1 binding (recognizing the glycoprotein IIb/IIIa complex of activated platelets), P-selectin (CD62P) surface expression, and platelet-monocyte and platelet-neutrophil aggregate formation, were measured using two flow cytometry panels. Plasma PF4 concentrations were measured by ELISA. In Sabah, platelet-WBC aggregates were determined from the RBC-binding panel. The platelet activation gating strategies are illustrated in Fig. S1, B and C. Refer to Supplementary Materials for details of the staining and analysis procedures.

In vitro parasite and platelet experiments

P. knowlesi and P. falciparum were cultured in 2.5% hematocrit O+ human RBC as described elsewhere.39,40 Platelets were purified from freshly collected human blood as described previously.12 Refer to Supplementary Materials for details of the culture conditions and platelet preparation.

Platelet count and quiescence were determined using an ADVIA 2120i hematology analyzer (Siemens, Australia). Platelet activation capacity was tested by treating platelets with 1 U/mL of human thrombin for 10 min, then measuring PAC-1 binding and P-selectin surface expression by flow cytometry. All platelet preparations in this study (8 preparations from six donors) satisfied the criteria for quiescence (mean platelet volume <9 fL and mean platelet component >23 g/dL) and activation capacity (>1000-fold and >250-fold increase in PAC-1 and CD62P MFI, respectively, compared to untreated rested platelets) (Fig. S4).

For the parasite-platelet co-culture experiments, P. falciparum parasites were synchronized for ring stages the day before experiments using 5% (w/v) D-sorbitol.41 Late-stage P. knowlesi and P. falciparum trophozoites were purified on the day of experiment using 70% Percoll® gradient centrifugation (3500 RPM without brake for 10 min) and washed twice with complete culture medium (CCM). Parasitemia was adjusted to 0.5% at 2% hematocrit and platelets added; equal volumes of Tyrode’s buffer were added to untreated controls. In some experiments before adding to the parasites, recombinant human PF4 (Peprotech, US) and platelet lysates were incubated with 0.5 mg/mL of preservative-free rabbit anti-human PF4 Ab (Abcam, UK) or preservative-free rabbit serum (Novus Biologicals, US) for 15 min at 4°C. For transwell experiments, parasites were separated from 50 million platelets/mL, platelet lysate or 0.5 μM PF4 using transwell inserts with 0.2 μm pore size (Anopore™ membrane, Nalge Nunc International, Denmark). The well inserts were pre-soaked with CCM prior to use. P. knowlesi was harvested after 24 h, and P. falciparum after 48 h culture with platelets, lysate or protein. Giemsa-stained thin smears were prepared, and 1000 RBC were counted per slide at 1000x magnification. Percent parasite growth was calculated using the formula below.

% Parasite Growth = [(treatment parasitemia − initial parasitemia) ÷ (untreated parasitemia − initial parasitemia)] x 100

Platelet binding to parasites was determined in 24 h cultures containing 2% asynchronous parasites and 60 million platelets/mL. Harvested co-cultures were fixed in diluted Cytofix™ (Biosciences, Australia) for at least 24 h and stored at 4°C. For flow cytometry quantitation, fixed cells were washed once with 1% (w/v) BSA/PBS, then stained with mouse anti-human CD42b conjugated to phycoerythrin and mouse anti-human CD235ab conjugated to allophycocyanin for 20 min at 4°C, followed by 5 μg/mL Hoechst 33342 for 5 min at 4°C. Fluorescence signals were measured using the LSR Fortessa (BD Biosciences). At least 100 000 events were collected per sample.

Statistics

The Mann-Whitney test or the Kruskal-Wallis test followed by Dunn’s multiple comparisons was used for between-group comparisons of clinical data. The Wilcoxon matched-pairs signed rank test was applied to paired datasets. One-way ANOVA with Sidak’s multiple comparisons was used for in vitro data. Associations between two variables were assessed using Spearman correlation. Bonferroni corrections to P-values were applied where appropriate. Data were log transformed for multivariate comparisons. All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA) or Stata 14 (StataCorp, Texas, US).

Study approval

The study was approved by the Human Research Ethics Committees of Gadjah Mada University, Indonesia (Papua), the Malaysian Ministry of Health (Sabah), Menzies School of Health Research, Australia (Papua and Sabah), the Macquarie University and the Australian National University. Written informed consent was obtained from all participants.

Results

Study participants

Separate cohorts of malaria patients and healthy controls were enrolled in Papua, Indonesia (n=143), and Sabah, Malaysia (n=233). Patients were categorized according to Plasmodium species, and baseline characteristics, including parasitemia, platelet counts and disease severity presented (Tables 1 and 2). The majority of malaria patients at each site had non-severe malaria, 92% in Papua and 97% in Sabah. Relative to controls, platelet counts were significantly lower in malaria from all Plasmodium species at both study sites and correlated inversely with parasitemia in the P. vivax groups (Papua, r=-0.37, p=0.022, n=38; Sabah, r=-0.36, p=0.0008, n=84) and with histidine-rich protein-2 (HRP2) in falciparum malaria in Papua (r=-0.45, p=0.0007, n=54).

Table 1. Baseline characteristics of n=143 malaria patients and healthy controls from Papua.

Controls
n =24
P. falciparum
n=55
P. vivax
n=38
P. malariae
n=14
Mixed infection
n=12 a
Kruskal-Wallis test p-value
Age, yr 28 [26-33.8] 29 [19-37] 24 [19-32.8] 33.5 [26-47] 22 [19.3-33] 0.027
Males, n (%) 10 (42%) 29 (53%) 23 (61%) 4 (29%) 7 (58%) -
Ethnicity, Highland/Lowland/non-Papuan, n 12/11/1 50/2/3 33/3/2 14/0/0 12/0/0 -
n with severe malaria b - 8 1 - - -
Parasitemia (parasites/μL) Not detected by microscopy 15,600 [3,660-105,000] c 6,160 [2,120-11,400] 1,230 [815-2,540] 7,160 [4,590-106,000] <0.0001
Hemoglobin (g/dL) 12.3 [11.5-14.6] 12.4 [10.2-13.4] 11.9 [10.5-13.1] 9.6** [8.6-10.6] 11.7 [8.8-13.3] 0.0007
WBC count (x1000/μL) 7.4 [6.2-8.2] 5.1** [3.7-6.6] 5.5 [4.8-7.5] 4.2** [3.2-5.1] 5.8 [4.4-7.2] <0.0001
Monocyte count (x1000/μL) 0.45 [0.42-0.55] 0.53 [0.30-0.77] 0.54 [0.40-0.67] 0.54 [0.37-0.68] 0.51 [0.20-0.66] 0.780
Neutrophil count (x1000/μL) 3.5 [2.5-4.4] 2.8 [2.0-4.8] 3.8 [2.8-4.3] 1.6* [1.5-2.4] 3.9 [2.6-4.8] 0.0006
Platelet count (x1000/μL) 202 [171-228] 66** [39-99] d 78** [53-113] 80** [56-101] 60** [44-120] <0.0001

Footnotes:

All values are median [interquartile range] unless otherwise indicated.

Kruskal-Wallis with Dunn’s multiple comparisons test, significantly different to controls (**p<0.0005, *p<0.005).

a

Mixed infection of P. falciparum and P. vivax.

b

Severe malaria criteria encountered: cerebral malaria (Glasgow Coma Score ≤10 for >30 min), jaundice (visible jaundice OR creatinine >1.5 mg/dL OR bilirubin >3 mg/dL AND >100,000 parasites/μL), acute renal failure (creatinine >3 mg/dL +/- urine output <400 mL/day OR urea > 20 mM), hypoglycemia (plasma glucose <40 mg/dL), hyperparasitemia (asexual parasitemia >10%), hypotension (blood pressure <80 mmHg AND cool peripheries), and respiratory distress (respiratory rate >30/min AND O2 saturation <92%).

c

Median parasitemia in severe (283,000 parasites/μL) versus non-severe P. falciparum (10,800 parasites/μL; Mann-Whitney test p=0.0009).

d

Median platelet count in severe (18,500/μL) versus non-severe P. falciparum (74,000/μL; Mann-Whitney test p<0.0001).

Table 2. Baseline characteristics of n=233 malaria patients and healthy controls from Sabah.

Controls
n =28
P. falciparum
n=14
P. vivax
n=85
P. knowlesi
n=106
Kruskal-Wallis test p-value
Age, yr 31.5 [26-36.8] 25 [18-37.8] 22 [12-34]** 34 [22-52.3] <0.0001
n aged ≤12 yr - - 22 6 -
Males, n (%) 10 (36%) 12 (86%) 62 (73%) 84 (79%) -
n with severe malaria a - 1 0 6 -
Parasitemia (parasites/μL) Not detected by PCR 9,850 [3,130-21,800] 4,640 [1,830-7,630] 2,650 [648-8,770] b 0.025
Hemoglobin (g/dL) 13.7 [12.9-14.8] 12.9 [11.1-14.8] 12.4** [10.5-13.9] 13.4 [11.9-14.6] 0.002
RBC count (x106/ μL) 5.1 [4.9-5.5] 4.9 [4.2-5.3] 4.6 [4.1-5.0]*** 5.1 [4.6-5.5] <0.0001
WBC count (x1000/μL) 7.8 [6.6-8.9] 5.7** [4.1-6.9] 6.4* [5.3-7.9] 6.2** [5.1-7.9] 0.002
Platelet count (x1000/μL) 348 [267-363] 86*** [56-156] 97*** [67-133] 72*** [52-108] c <0.0001

Footnotes:

All values are median [interquartile range] unless otherwise indicated.

Kruskal-Wallis with Dunn’s multiple comparisons test, significantly different to controls (***p<0.0005, **p<0.005, * p<0.05).

a

Severe malaria criteria encountered: acute kidney injury (creatinine >265 μmol/L), significant abnormal bleeding, severe anemia (hemoglobin <7 g/dL or hematocrit <20%), jaundice (visible jaundice OR creatinine >1.5 mg/dL OR bilirubin >3 mg/dL AND >100,000 parasites/μL [P. falciparum] or >20,000/μL [P. knowlesi]), hyperparasitemia (asexual parasitemia >10% [P. falciparum] OR >100,000/μL [P. knowlesi]), metabolic acidosis (HCO3 <15 mmol/L OR lactate >5 mmol/L).

b

Median parasitemia in severe (136,000 parasites/μL) versus non-severe P. knowlesi (2,450 parasites/μL; Mann-Whitney test p<0.0001).

c

Median platelet count in severe (52,500/μL) versus non-severe P. knowlesi (72,000/μL; Mann-Whitney test p=0.154).

Platelet-iRBC and platelet-uRBC complexes are formed in the circulation of malaria patients

Examination of patient thin blood smears revealed platelet binding to both iRBC and uRBC. Platelets were bound to all asexual parasite stages in each of the Plasmodium species, with evidence of platelet aggregates surrounding P. vivax-iRBC (Fig. 1A). Characteristic features of dying parasites within platelet-bound iRBC were observed, including spread of parasite pigment suggesting dissolution of the digestive vacuole.

Flow cytometry was used to quantify the proportions of uRBC and iRBC that were each bound to platelets, hereafter platelet-uRBC and platelet-iRBC complexes (Figs. 1B and S1A). Proportions of platelet-uRBC were similar amongst all the patient groups (median range: 0.10% – 0.19%) and were significantly lower than the controls (0.28% and 0.42%; Fig. 1, Ci and Cii, respectively). In contrast, greater proportions of platelet-iRBC were observed in all the patient groups (Fig. 1, Di and Dii, and Table S1). The greatest proportions were observed in P. knowlesi patients (median 18.1%), which was significantly higher than in Sabah P. falciparum patients (median 6.3%; p=0.002, Table S1); Overall complex magnitudes were lower in the Papua than the Sabah groups, which may be attributable to differences in sample preparation and staining. However, for all groups platelet-iRBC complexes were significantly greater than platelet-uRBC complexes. Compared as a ratio, the proportions of platelet-iRBC complexes were between eight and almost 100-fold greater than platelet-uRBC complexes (Table S1). We also separately analyzed the subgroup of 28 Sabah patients with P. vivax or P. knowlesi malaria who were children aged 12 or younger (Table 2); similar proportions of platelet-iRBC/uRBC complexes to adults, and significantly greater frequencies of platelet-iRBC versus platelet-uRBC were observed in these groups (Fig. S3, A–C).

Using the proportions of the complexes and the respective RBC count in each individual, we calculated concentrations of platelet-RBC complexes (iRBC and uRBC combined). The medians ranged between 3,100 and 13,600/μL blood (Table 3). Compared to respective circulating levels of free platelets (i.e. free platelet:platelet-RBC complex ratio; Table 3), the complexes comprise a substantial proportion of the total platelet pool. For example, in P. knowlesi-infected patients, on average, one complex was predicted to exist for every 7 non-complexed platelets. The highest ratio, in P. malariae, predicted one complex for every 19 platelets (Table 3). By comparison, free platelet:platelet-WBC ratios were much higher (≥118), indicating these complexes were only a minor component of the platelet pool (Table 3).

Table 3. Circulating platelet-RBC and platelet-WBC concentrations and ratios to free platelets.

Patient Cohort Plasmodium species n of samples analyzed Platelet-RBC complexes Platelet-WBC complexes
x103 per μL blood a free platelet : complex ratio b x103 per μL blood c free platelet : complex ratio d
Papua Controls 17 13.6 [10.1-20.9] 14.5 [10.3-18.8] 0.3 [0.2-0.4] 713 [425-1146]
P. falciparum 23 4.5 [3.0-7.2] 16.6 [13.2-22.5] 0.2 [0.1-0.4] 237 [147-451]***
P. vivax 26 4.8 [3.0-7.7] 17.2 [12.7-21.1] 0.2 [0.2-0.3] 393 [203-679]*
P. malariae 9 4.9 [3.6-6.5] 18.5 [16.2-22.1] 0.2 [0.1-0.3] 348 [199-674]
Mixed 7 3.1 [2.5-9.7] 17.9 [14.1-18.7] 0.3 [0.1-0.5] 287 [138-345]*
P-value e <0.0001 0.561 0.663 0.009
Sabah Controls 27 21.4 [11.2-27.6] 18.2 [11.8-35.2] 1.1 [0.8-1.4] 316 [216-448]
P. falciparum 12 6.0 [2.8-11.3]** 14.6 [7.5-34.5] 0.4 [0.1-0.6]**** 391 [203-998]
P. vivax 78 6.8 [4.4-13.9]**** 14.0 [6.1-26.2] 0.8 [0.5-1.4] 124 [66-209]****
P. knowlesi 92 10.5 [5.4-18.3]* 7.3 [4.0-13.2]**** 0.6 [0.3-1.0]** 118 [70-283]****
P-value e <0.0001 <0.0001 <0.0001 0.004

All values are median [interquartile range] unless otherwise indicated.

a

Formula = ([%platelet-uRBC from flow data] ÷ 102 x [RBC count from analyzer] x 106) + ([%platelet-iRBC from flow data] ÷ 102 x [parasites per μL])

b

Formula = (platelet count from analyzer x 103) ÷ (platelet-RBC per μLa)

c

Formula = ([%platelet-WBC from flow data] ÷ 102) x (WBC count from analyzer) x 103

d

Formula = (platelet count from analyzer x 103) ÷ (platelet-WBC per μLc)

e

Kruskal-Wallis with Dunn’s multiple comparisons test, significantly different to controls (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05).

Correlative analyses revealed inverse relationships between platelet-iRBC complexes and parasitemia; these were significant in malaria from each species, except P. malariae (Fig. 1, Ei, Eii and Fig. S2A). After adjusting for platelet count, these relationships remained significant for P. vivax and P. knowlesi (both p<0.0001). No significant relationships were observed between parasitemia and platelet-uRBC complexes. There was no consistent relationship between complexes and hemoglobin levels.

Taken together, these data show that platelets bind and form stable complexes with RBC, with a greater preference for iRBC than uRBC. The frequencies of these complexes in the circulation of patients and controls are substantial and compared to free platelets comprise a relatively large proportion of the total platelet pool. In addition, the proportions of platelet-iRBC complexes are higher in patients with low levels of parasites, and lower in patients with high parasite burden.

Platelets directly kill parasites in the circulation of malaria patients

We conducted TUNEL and PF4 immunostaining on patient blood to detect and quantify the frequencies of dead intraerythrocytic parasites and the co-occurrence of PF4 accumulation within the iRBC. With these methods, iRBC containing PF4 and TUNEL labeling (PF4+TUNEL+) are indicative of direct platelet contact with the cell (by uptake of platelet-produced PF4) and consequent PF4-mediated killing of the parasite.29,30 We observed PF4+TUNEL+ iRBC in all Plasmodium species in both patient cohorts (Fig. 2A). Quantification of these observations showed substantial variances in the proportions of PF4+TUNEL+ iRBC, although the medians calculated for each species were similar, except for P. vivax, which was significantly greater in both Papua and Sabah compared to the other species (Fig. 2B and Table 4). Notably, PF4 was present in more than half of the TUNEL+ (dead) parasites; in Papua, a median of 96% of TUNEL+ P. vivax-iRBC contained PF4, which was significantly greater than the 52% median observed in P. falciparum (p<0.0001) and 69% median in P. malariae (p=0.051) (Fig. 2C). No PF4 staining or TUNEL labeling was observed in enriched suspensions of uninfected reticulocytes. The proportion of PF4+TUNEL+ iRBC correlated inversely with parasitemia in Papuan patients infected with P. vivax (r=-0.42, p=0.020) and P. falciparum (r=-0.36, p=0.010); no significant relationships were observed in the other cohort groups, in which fewer samples were examined (Fig. 2D and Fig S2B).

Fig. 2. PF4-associated parasite killing in clinical malaria samples.

Fig. 2

A) Representative immunofluorescent images from Pf, Pv, Pk and Pm patient blood smears illustrating PF4-associated parasite killing (PF4+TUNEL+ iRBC). Scale bars = 5 μm. Arrows and arrowheads indicate platelets and parasites, respectively. Images were taken at 630x magnification on an Axio Scope A1 fluorescent microscope coupled to an Axiocam ICm-1 CCD camera, or an Axio Observer inverted fluorescence microscope coupled to an Axiocam 503 monochrome camera. ZEN 2 software was used for image acquisition and processing (all from Carl Zeiss, Germany). B) Percentage of PF4+TUNEL+ parasites in clinical samples with Pf (Papua n=50, Sabah n=14), Pv (Papua n=32, Sabah n=13), Pm (n=11), Pk (n=15) and mixed species infection (n=6). C) Comparison of intraerythrocytic PF4 (PF4+) parasites as a percentage of dying (TUNEL+) parasites in Pf, Pv, Pm, Pk and mixed species infection from Papua and Sabah (n as per panel B). D) Inverse correlation of PF4+TUNEL+ iRBC with parasitemia in Pf and Pv clinical samples (Spearman). E) Proportions of PF4+TUNEL+ rings versus mature stages in Pv (rings n=15, mature n=16), Pm (rings n=6, mature n=9) and Pk (rings n=8, mature n=5). F) Proportions of rings that were PF4+TUNEL+ and G) PF4+ in Pf (Papua n=50, Sabah n=14), Pv (n=15), Pm (n=6) and Pk clinical samples (n=8). Scatterplots indicate median ± interquartile range for each group. Parasitemia values are log transformed. Kruskal-Wallis or Mann-Whitney test used for statistical comparisons. Data presented in Table 4. Abbreviations: Pf, P. falciparum; Pv, P. vivax; Pm, P. malariae; Pk, P. knowlesi.

Table 4. Platelet-associated parasite killing (TUNEL+PF4+ iRBC) between species and stages.

Patient cohort Plasmodium species n of samples analyzed Median number parasites counted per sample [IQR] Median % TUNEL+PF4+ iRBC [IQR] P-value a P-value b
Papua P. falciparum 50 54 [52-59] 5.6 [1.8-11.3] <0.0001 -
P. vivax 32 54 [51-58] 59.3 [45.4-81.1] comparator -
P. malariae 11 45 [39-50] 14.3 [13.6-18.2] 0.009 -
Mixed infection 6 59 [42-66] 14.9 [7.8-51.5] 0.150 -
P. vivax (ring) 15 48 [28-61] 6.4 [1.1-16.1] - comparator
P. vivax (mature) 16 59 [44-88] 63.6 [52.9-74.1] - <0.0001
P. malariae (ring) 6 12 [16-18] 0 [0-3.5] - comparator
P. malariae (mature) 9 40 [63-80] 6.3 [4.4-8.8] - 0.007
Sabah P. falciparum 14 95 [76-131] 10.6 [5.4-14.6] 0.043 -
P. vivax 13 49 [21-104] 16.7 [13.5-39.1] comparator -
P. knowlesi 15 95 [78-101] 14.2 [19.8-26.9] 0.717 -
P. knowlesi (ring) 8 96 [92-103] 10.8 [8.0-24.3] - comparator
P. knowlesi (mature) 5 78 [72-98] 14.9 [8.6-51.5] - 0.354

Footnotes:

a

P-values from Kruskal-Wallis with Dunn’s multiple comparisons test for difference to P. vivax.

b

P-values from Mann-Whitney test between intra-species ring and mature stages.

Abbreviations: IQR, interquartile range; PF4, platelet factor-4.

We compared PF4+TUNEL+ frequencies in young (ring) versus mature (trophozoite and schizont) asexual stage parasites (Fig. 2E and Table 4). In the Papuan P. vivax samples, PF4+TUNEL+ mature-stage parasites outnumbered ring-stages almost ten times (63.6% vs 6.4%; p<0.0001); there was a similar significant difference in P. malariae (6.3% vs 0%; p=0.007), but not in P. knowlesi (14.9% vs 10.8%; p=0.354). Only ring-stage parasites were observed in the P. falciparum samples, consistent with the ability of mature stages to sequester in tissues. There were no significant differences in the proportions of PF4+TUNEL+ rings between patient location or parasite species (Fig. 2F). All ring-stage parasites (i.e. TUNEL+ and TUNEL-) were also categorized according to PF4 staining; the greatest proportions were observed in Papuan P. vivax samples (median 43.8%), which was significantly greater than in P. falciparum or P. malariae (median 12.0% and 9.1%, respectively; Fig. 2G).

Collectively these data show that, irrespective of species, a substantial proportion of Plasmodium accumulate platelet-derived PF4 and undergo intraerythrocytic death, most likely as a consequence of the known cytotoxic actions of PF4.

Platelet-derived PF4 accumulation in iRBC and parasite killing are not associated with systemic platelet activation

We observed that not all PF4-stained parasites were bound by platelets (Range 0-25%) and some platelet-bound cells were not stained for PF4 (Range 0-5.9%; Table S2). We determined if systemic platelet activation contributes to absorption of plasma PF4 into the iRBC. None of our markers for platelet activation, including plasma PF4 concentrations, platelet surface expression of CD62P (P-selectin) and PAC-1 (activated GPIIb/IIIa) on circulating platelets, were significantly elevated in any of the patient groups (Table S3). In addition, no relationships were observed that supported a role for systemic platelet activation in the iRBC accumulation of PF4 (Table S4).

Cultured P. knowlesi and P. falciparum are sensitive to human platelets and PF4

In vitro platelet killing of P. falciparum has been demonstrated previously12,2931 but not for other human Plasmodium species. We therefore co-cultured P. knowlesi with or without different concentrations of washed and rested human platelets and measured effects on parasite growth over 24 h. We observed a significant and platelet concentration-dependent reduction in parasite growth, maximal at 75 million platelets/mL (~60% reduction) (Fig. 3A). Implementation of platelet-to-iRBC ratios and parasitemias comparable to clinical settings, and strict control of platelet quality (Fig. S4) were critical components of our experimental system. A similar significant growth inhibition effect was also observed in P. falciparum cultures treated with the same platelet preparations (Fig. 3B). P. knowlesi was sensitive to recombinant human PF4 (IC50 ~ 0.5 μM), and to treatment with platelet lysates, which was blocked by inclusion of anti-PF4 antibodies (Fig. 3, C and D). Platelet inhibition of parasite growth was prevented when platelets and P. knowlesi-iRBC were physically separated in co-cultures using cell-impermeable transwells, but remained sensitive to platelet lysate and PF4 (Fig. 3E), indicating that direct platelet-cell contact and platelet-derived PF4 are responsible for the cytotoxic effects.

Fig. 3. In vitro cultures of P. knowlesi are sensitive to platelets and PF4.

Fig. 3

The growth of A) P. knowlesi (n=4) and B) P. falciparum (n=3) treated with different platelet concentrations or Tyrodes buffer for 48 h. C) The growth of P. knowlesi treated with platelet lysate, with and without anti-PF4 antibodies or IgG isotype control (n=2). D) P. knowlesi PF4 dose response curve (n=2). E) The growth of P. knowlesi treated with platelets (60 million/mL), platelet lysate or PF4 (0.5 μM), and co-cultured in standard wells or Transwells (n=2). F) Micrographs showing platelets bound to uninfected and P. knowlesi-infected cells. G) Percent platelet binding to uninfected, P. knowlesi (n=4) or P. falciparum iRBC (n=3), determined by flow cytometry. H) Percent TUNEL-labelled (TUNEL+) P. knowlesi parasites co-stained for PF4 (PF4+) or not PF4-stained (PF4-) (n=3). I) Micrographs showing a PF4+TUNEL+ P. knowlesi infected cell after platelet treatment. Scale bars = 5 μm. Images were taken at 630x magnification on an Axio Observer inverted fluorescence microscope coupled to an Axiocam 503 monochrome camera with ZEN 2 software (Carl Zeiss, Germany). Bars indicate means of replicate data points. Kruskal-Wallis test or one-way ANOVA used for statistical comparisons: *p<0.05 and **p<0.01. Abbreviation: DIC, differential interference contrast.

Platelets were observed physically bound to P. knowlesi-iRBC (Fig. 3F). The frequency of platelet-iRBC complexes was twice of platelet-uRBC complexes in both P. knowlesi and P. falciparum after 24 h incubation with platelets and was significant for P. knowlesi (p<0.05; Fig. 3G). Platelet-treated P. knowlesi cultures contained significantly greater proportions of TUNEL+ iRBC; the majority of these iRBC were also PF4+ (Fig. 3, H and I). PF4 was not detected in untreated iRBC (Fig. 3H). Collectively, human platelets can kill both P. falciparum and P. knowlesi asexual blood-stage parasites under well-defined culture conditions. The killing mechanism requires platelet-cell contact and PF4; intraerythrocytic PF4 is cytotoxic to P. knowlesi.

Discussion

Here we demonstrate for the first time in humans that platelets can directly kill a microbial pathogen, erythrocytic stage Plasmodium. Platelets were observed bound to Plasmodium-iRBC of all four of the major species that cause human disease, P. falciparum, P. vivax, P. malariae and P. knowlesi. Platelet binding to uRBC was also observed, although the proportions of these were significantly lower than platelet-iRBC complexes. In addition, the proportions of platelet-iRBC complexes were inversely related to parasite burden, suggesting a potential cause and effect relationship between cell-cell binding and parasite growth. We observed frequent intracellular accumulation of PF4 in these iRBC and death of the intraerythrocytic parasites. Further evidence of platelet’s cytotoxic mechanism of action against parasites was obtained using cultures of P. knowlesi and P. falciparum. The frequent occurrence of platelet-associated killing of parasites in the periphery of malaria patients, and its inverse correlation with parasite load, significant at least for P. falciparum and P. vivax, suggests the importance of platelets in the host control of parasites. Collectively, our data predict that platelets may kill as many as 5-20% of circulating blood-stage Plasmodium in clinical malaria, and in P. vivax this may be as high as 60%.

We have previously proposed a mechanism of PF4 accumulation and parasite killing involving direct platelet-iRBC contact followed by local release of PF4 and uptake into the parasite via the Duffy-antigen.30 Here, our in vitro studies using transwell filters to separate platelets and P. knowlesi parasites demonstrated that direct platelet-cell contact is necessary for parasite killing, which confirms previous studies.31 Our cumulative in vitro data contrast with recent findings reported by Gramaglia and colleagues32, who found no parasite growth inhibition by platelets in P. falciparum cultures. We predict this is due to differences in experimental design, noting especially that in vitro parasite killing by platelets is only observed in cultures containing physiologically relevant proportions of parasite-infected cells (<1%, in this study and previously 12,31). The aforementioned study also did not observe changes in parasite growth rates when platelet levels were altered in Plasmodium-infected mice, or evidence that platelets were required for survival, which conflicts the findings of others.12,13 This could be due to differences in the Plasmodium strains used amongst the studies, which can affect parasite virulence, growth and sequestration, and host response characteristics. In addition, recognizing the definitive parasite killing activity of platelets in the circulation requires distinguishing dead parasites from healthy parasites, which are greatest in platelet sufficient mice12 and in parasitized cells that contain platelet-derived PF4 (this study and 30). In the patient studies we also considered, and excluded, the possibility that PF4 is absorbed by circulating parasites from the plasma. Systemic platelet activation was negligible and plasma PF4 concentrations were approximately 1000-fold lower than in vitro concentrations required for Plasmodium killing. We also observed substantially higher frequencies of PF4 accumulation in mature-stage P. vivax-iRBC and greater rates of parasite death. Accumulation may be determined by the higher Duffy-antigen levels expressed on reticulocytes4244, for which P. vivax has an exclusive tropism.45 While circulating P. vivax parasitemia is intrinsically limited by the number of circulating reticulocytes, we speculate that the greater degree of platelet-associated killing observed in P. vivax infection may be a substantial additional contributor to the lower parasitemia generally observed in these patients compared to P. falciparum.18,46,47

Platelet cytoadherence to the endothelium and WBC are well-established pathological drivers of vascular and inflammatory diseases. However, the occurrence and consequences of platelets interacting with RBC in any disease setting have been under-reported.4850 Our unique ability to systematically characterize platelet-RBC interactions revealed that platelets have a greater capacity to bind Plasmodium-iRBC versus uRBC. This may be partly determined by parasite-expressed proteins present on the RBC surface, such as the P. falciparum cell adhesion molecule PfEMP1 shown previously to mediate platelet binding through CD36.51 Platelet binding to uRBC may be mediated through erythroid-expressed ICAM-4 and platelet GPIIb/IIIa.52 The identities and roles of other RBC and platelet molecules involved in platelet-RBC binding, especially in the other Plasmodium species, remain to be determined.

Platelet-RBC complexes may have a role in malaria-induced thrombocytopenia, which has been reported in many other clinical studies5355, and was also evident in our patient groups. The underlying causes of thrombocytopenia have been variously attributed to systemic platelet activation, immune-mediated clearance and vascular pooling.5661 Platelets complexed with RBC are not recognized by hematological analyzers, thus complex formation would lead to an apparent platelet loss. The high frequencies of these complexes relative to free platelets in our patients suggest they comprise a substantial proportion of the total platelet pool. If there is an accelerated turnover of these complexes, this would further enhance the contribution of complexes to platelet loss. Interestingly, host mechanisms that remove diseased and damaged circulating cells such as the spleen are upregulated in malaria62; the lifetime of platelet-RBC complexes in the circulation remains unknown. Further supporting this hypothesis, platelet-RBC complexes were greatest in our knowlesi malaria patients and this species causes the highest frequency of thrombocytopenia.35,53

Platelet activation and binding is considered a key mechanism in enabling sequestration of P. falciparum-iRBC to microvasculature and platelet-mediated iRBC sequestration is associated with fatal outcome in cerebral malaria studies.6366 Our data in predominantly non-severe P. falciparum, P. vivax, and P. malariae indicated negligible systemic platelet activation. Systemic platelet activation in severe and non-severe falciparum malaria were comparable, however, numbers were small and we cannot exclude greater systemic platelet activation in human severe malaria67,68, nor can we exclude tissue platelet activation not detected by the circulating measures used in this study.

Overall, our study demonstrates the direct pathogen-killing actions and host protective roles of platelets during human malarial infection. Given platelets show broad-spectrum anti-microbial activity, and the risks of infection associated with thrombocytopenia and platelet disorders are elevated, a general role for platelets in the innate host defence against microbial infection should be considered.

Supplementary Material

SupplementaryFile

Key points.

  • Platelets directly interact with and kill circulating Plasmodium parasites in malaria patients to help control parasitemia.

  • In vitro platelet anti-plasmodicidal activity against P. knowlesi involves platelet-cell binding and intracellular accumulation of PF4.

Acknowledgements

We thank Pak Prayoga, Leo Leonardo and Ruland Wandosa for their microscopy expertise, Bernardette Pedersen, Timothy Butler, Harpret Vohra and Michael Devoy for flow cytometry assistance, Guy Zimmerman for advice on measures of platelet activation, Grennady Wirjanata for assistance in the Timika laboratory and Prof Yati Soenarto for facilitating the Papua study. We thank study participants, Mitra Masyarakat Hospital Staff in Papua, the malaria research team, nursing and laboratory staff at Queen Elizabeth, Kudat, Kota Marudu and Pitas Hospitals in Sabah, and the Director-General of Health, Ministry of Health, Malaysia.

Funding

This work was supported by the Australian National Health and Medical Research Council (Grants #1037304, #1045156, #490037, #605524, # 1047082, #1047090, #1066502 and Fellowships to NMA [#1042072, #1135820], BEB [#1088738] and MJG [#1138860]), the Australian Research Council (grant #120100061), the Wellcome Trust (Fellowships to RNP [#200909] and JRP [#099875]), the Singapore National Medical Research Council (Award to TWY [CSA INV 15nov007]) and the Australian Department of Foreign Affairs and Trade. Funders had no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.

Footnotes

Author contributions

Conceptualization, S.K., B.E.B, Tonia W., S.F., G.M., T.W.Y., N.M.A., B.J.M.; Methodology, S.K., B.E.B, E.J., K.A.P, A.E., Tonia W., G.M., M.J.G., N.M.A., B.J.M.; Validation, S.K., B.E.B., E.J., K.A.P., A.E., B.J.M.; Formal Analysis, S.K., B.E.B., E.J., M.J.G., B.J.M.; Investigation, S.K., B.E.B., E.J., B.A., E.K., K.A.P., A.E., Timothy W., M.J.G., N.M.A., B.J.M.; Resources, S.K., B.E.B., J.R.P., E.K., K.A.P., Y.S., R.N.P., Timothy W., S.F., N.M.A., B.J.M.; Writing – Original Draft, S.K., B.E.B., M.J.G., N.M.A., B.J.M.; Writing – Review and Editing, J.R.P., E.K., R.N.P., Tonia W., S.F., G.M., T.W.Y.; Visualization, S.K., B.J.M.; Supervision, J.R.P., N.M.A., B.J.M.; Project Administration, S.K., B.E.B., K.A.P.; Funding Acquisition, R.N.P., N.M.A., B.J.M.

Competing interest

The authors declare that no competing interest exists.

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