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Platelets kill circulating Plasmodium parasites in human malaria: greater killing of circulating P. vivax than P. falciparum or P. malariae

Steven Kho 1 ; Benediktus Andries 2 ; Jeanne Rini Poespoprodjo 3 ; Enny Kenangalem 4 ; Yati Soenarto 5 ; Richard N Price 6 ; Tonia Woodberry 7 ; Gabriela Minigo 1 ; Tsin W. Yeo 7 ; Brendan McMorran 8 ; Nicholas M. Anstey 7
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Background: Platelets kill Plasmodium chabaudi in murine models and P. falciparum in vitro via platelet factor-4 (PF4) activity, but their role in parasite killing in human malaria is not known. The aim of this study was to determine the role of platelets in red cell (RBC) binding and killing of Plasmodium parasites in human malaria. Materials and Methods: Peripheral blood was collected from 99 patients (≥16 years) with malaria attending hospital in Timika, Papua, Indonesia, including P. falciparum (Pf; n=51), P. vivax (Pv; n=31) and P. malariae (Pm; n=11), and 17 healthy controls. Using fluorescent microscopy, platelet-associated killing of infected RBC (iRBC) was counted by TUNEL staining of degraded/sheared parasite DNA and intraerythrocytic PF4 release. Platelet binding to iRBC and uninfected RBC (uRBC) was quantified by fresh whole blood flow cytometry. Results: Platelet binding to iRBC was higher than to uRBC in Pv (12-fold higher; median 1.44% of iRBC [IQR 0.63-2.03] vs median 0.11% of uRBC [IQR 0.08-0.17]; p<0.0001), Pf (12-fold higher; median 1.37% [IQR 0.42-2.14] and median 0.11% [IQR 0.07-0.15]; p<0.0001) and Pm (7-fold higher; median 1.05% [IQR 0.46-2.51] and median 0.13% [IQR 0.10-0.18]; p=0.004). Platelet-associated killing of circulating Plasmodium was evident across all species causing malaria, with 59.7% [IQR 44.8-81.4] of Pv-iRBCs showing evidence of platelet-associated killing (PF4+TUNEL+), 5.6% [IQR 1.8-11.8] of Pf-iRBCs, and 14.3% [13.6-18.2] of Pm-iRBCs. In both Pv and Pm, there was no significant correlation between %ring-stage parasites and platelet-iRBC binding or platelet-associated killing, suggesting killing may not be restricted to mature stages only. A higher proportion of dying Pv parasites (95.8%) had intraerythrocytic PF4 than dying Pf (54.6%; p<0.0001) and Pm (68.8%; p=0.04). Platelet binding to uninfected erythrocytes was lower in malaria compared to controls, but not after controlling for platelet count. Platelet binding to iRBC correlated inversely with parasitaemia in Pv (r=-0.60; p=0.002; n=25) and Pf (r=-0.62; p=0.001; n=24), though not significantly in Pm (r=-0.20; p=0.61; n=9). Conclusions: Platelets target and kill circulating Plasmodium parasites in vivax, falciparum and malariae malaria, with binding inversely proportional to parasitaemia. Findings support an important role for platelets in innate host response and parasite killing in clinical infection, particularly in vivax malaria.