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Science 338 (6112): 1348-1351

Copyright © 2012 by the American Association for the Advancement of Science

Platelet Factor 4 and Duffy Antigen Required for Platelet Killing of Plasmodium falciparum

Brendan J. McMorran1,2,*, Laura Wieczorski2, Karen E. Drysdale2, Jo-Anne Chan3, Hong Ming Huang1, Clare Smith2, Chalachew Mitiku2, James G. Beeson3, Gaetan Burgio1,2, and Simon J. Foote1,2

1 Australian School of Advanced Medicine, Macquarie University, Sydney, NSW 2109, Australia.
2 Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000, Australia.
3 Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria 3004, Australia.


Figure 1
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Fig. 1. Interaction of platelets and PF4 with P. falciparum (iRBC). (A and C) Immunofluorescent assay images of iRBC treated with platelets for 24 hours. Cells were stained with Hoechst S769121 (blue), antibody to PF4 (red) and labeled using the TUNEL assay [green; (C) only]. Arrowheads indicate platelets, small arrows indicate uninfected RBC, and large arrows indicate platelet-bound and anti-PF4 stained [and, in (C), TUNEL-labeled] iRBC. (B) Percentage of anti-PF4–stained iRBC, and (D) TUNEL and anti-PF4 colabeled iRBC, after platelet treatment. Error bars represent mean proportions (±SEM) determined in three independent experiments. **P = 0.005 compared with 28-hour platelet treatment.

 

Figure 2
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Fig. 2. The P. falciparum killing function of PF4. (A) Growth inhibition of P. falciparum by rhPF4. Data represent the mean of at least two independent assays, each performed in duplicate (±SEM) for three parasite strains. **P < 0.01 compared with nonspecific rabbit immunoglobulin G (IgG) control antibody. (B) Growth inhibition of P. falciparum 3D7 by rhPF4 after treatment for 4 hours and washout, or continuous treatment. Synchronized ring or trophozoite-stage parasites were used. Error bars represent the mean of at least two independent assays, each performed in triplicate (±SEM). ***P < 0.001 compared with continuous treatment of ring-stage parasites. (C) Percentage of TUNEL-labeled P. falciparum 3D7 iRBC after treatment with rhPF4. The predominant developmental parasite stages at each time point were either immature rings (ring), pigmented trophozoites (troph) or schizonts (schiz). Bars represent mean proportions (±SEM) determined in two independent experiments. *P < 0.05 compared with untreated control cultures at each respective time point.

 

Figure 3
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Fig. 3. Requirement of Fy for platelet- and PF4-mediated parasite killing. (A) Growth inhibition of P. falciparum 3D7 by platelets or rhPF4 and the effect of antibodies to Fy (anti-Fy and anti-Fy6) and Fy-deficient erythrocytes (Fy–/–). Error bars represent the mean (±SEM) of at least three independent experiments, each performed in duplicate. The blood from seven different Fy–/– donors was tested. *P < 0.05 compared with isotype control IgG antibody; ***P < 0.001 compared with respectively treated parasites grown in Fy+/+ cells. (B) Effect of chemokine Fy ligands on P. falciparum growth inhibition by rhPF4. Data represent the mean (±SEM) of at least two independent experiments, each performed in duplicate. ***P < 0.001 compared with no chemokine. (C) Percentage of TUNEL-labeled P. falciparum 3D7 parasites grown in Fy-sufficient (Fy+/+) or Fy-deficient (Fy–/–) red cells after platelet treatment for 24 hours. Error bars represent mean proportions (±SEM) determined in three independent experiments. The blood from three different Fy-negative donors was tested. *P = 0.02 compared with platelet-treated Fy+/+ cells.

 

Figure 4
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Fig. 4. Requirement of Duffy antigen for PF4 binding and internalization by iRBC. (A) Percentage of anti-PF4 stained P. falciparum 3D7 iRBC grown in Fy-expressing (Fy+/+) or Fy-deficient (Fy–/–) cells after treatment with human platelets for 24 hours. Error bars represent mean proportions (±SEM) determined in three independent experiments. The blood from three different Fy-negative donors was tested. **P = 0.002 compared with Fy+/+ cells. (B) Immunofluorescent assay images of PF4-treated iRBC stained with Hoechst (blue), antibody to PF4 (red), and antibody to Fy (green). rhPF4 was added to synchronized ring-stage parasites for 24 hours. Arrowheads indicate uninfected cells, and arrows indicate iRBC (PF4 and Fy costained). (C) Immunoblot analysis of PF4 in rhPF4-treated P. falciparum 3D7 parasites. Immunoblots (using antibodies to PF4 or EXP2) of saponin-purified trophozoites, treated before purification with or without rhPF4 (0.5 μM) for 2 hours. Parasites were grown in Fy-deficient (Fy–/–) or sufficient (Fy+/+) red cells. (D) Quantification of the PF4 immunoblot analysis. Error bars represent the mean pixel intensity (±SEM) of the PF4 immunoreactive bands relative to EXP2 [shown in (C)]. ***P < 0.001 compared with Fy+/+ cells.

 


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