Inhibition of platelet adherence to brain microvasculature protects against severe Plasmodium berghei malaria

Abstract

Some patients with Plasmodium falciparum infections develop cerebral malaria, acute respiratory distress, and shock and ultimately die even though drug therapy has eliminated the parasite from the blood, suggesting that a systemic inflammatory response contributes to malarial pathogenesis. Plasmodium berghei-infected mice are a well-recognized model of severe malaria (experimental severe malaria [ESM]), and infected mice exhibit a systemic inflammatory response. Because platelets are proposed to contribute to ESM and other systemic inflammatory responses, we determined whether platelet adherence contributes to experimental malarial pathogenesis. Indeed, a significant (P < 0.005) increase in the number of rolling and adherent platelets was observed by intravital microscopy in brain venules of P. berghei-infected mice compared with the number in uninfected controls. P-selectin- or ICAM-1-deficient mice exhibit increased survival after P. berghei infection. We observed a significant (P < 0.0001) reduction in the morbidity of mice injected with anti-CD41 (alpha(IIb) or gpIIb) monoclonal antibody on day 1 of P. berghei infection compared with the morbidity of infected controls injected with rat immunoglobulin G. Additionally, platelet rolling and adhesion in brain venules were reduced in P. berghei mice lacking either P-selectin or ICAM-1 or when the platelets were coated with anti-CD41 monoclonal antibody. Unlike other inflammatory conditions, we did not detect platelet-leukocyte interactions during P. berghei malaria. Because (i). leukocyte adhesion is not markedly altered in the absence of P-selectin or ICAM-1 and (ii). CD41 is not an adhesion molecule for parasitized erythrocytes, these findings support the hypothesis that inhibition of platelet adhesion to the brain microvasculature protects against development of malarial pathogenesis.

FIG. 1.

Rolling (A) and adhesion (B) of platelets obtained from an uninfected C57BL/6 source mouse in the brain microvasculature during the course of P. berghei malaria in C57BL/6 recipient mice. On each graph the y axis indicates the number of rolling or adherent cells in arterioles, small postcapillary venules, and large postcapillary venules, and the x axis indicates the day of infection. Significant (P ≤ 0.0005) differences between leukocyte rolling or adhesion in arterioles, small postcapillary venules, and large postcapillary venules during the course of P. berghei infection and leukocyte rolling or adhesion in uninfected C57BL/6 mice are indicated by an ampersand, asterisks, and number signs, respectively. Five vessels were analyzed in four recipient mice at each time point. R1, R2, and R3, rolling cells; A1 and A2, adherent cells.

FIG. 2.

Rolling (A) and adhesion (B) of platelets obtained from an uninfected donor (uninf source) or a day 6 infection donor (infected source) in the brain microvasculature in selected recipient mice during the course of P. berghei malaria. The donor mouse for platelets and the recipient mice are indicated on the y axis, and the average number of rolling or adherent cells is plotted on the x axis. The numbers of recipients are indicated in Table 1. Asterisks indicate significant differences (P ≤ 0.0005) between donor platelet rolling or adhesion in the large postcapillary venules in recipient mice and platelet rolling or adhesion in infected C57BL/6 recipients that received platelets from uninfected donor mice. Psel−/−, P-selectin deficient.

FIG. 3.

Rolling (A) and adhesion (B) of platelets obtained from an uninfected donor mouse in the large postcapillary brain venules of selected recipient mice that were either not infected or infected with P. berghei (day 6). The groups of recipient mice are indicated on the y axis, and the average numbers of rolling or adherent cells are plotted on the x axis. anti-CD41 day-6 indicates platelets incubated with anti-CD41 MAb prior to injection (n = 4), and rat IgG day-6 indicates the control for the MAb (n = 3). C57BL/6 day-0 and C57BL/6 day-6 indicate uninfected and infected C57BL/6 mice, respectively; the results shown in Fig. 2 are repeated for comparison to ICAM-1-deficient recipients (ICAM-1-def). Five ICAM-1-deficient mice were analyzed. The asterisk (P < 0.05) and the number signs (P < 0.0005) indicate significant differences in platelet rolling or adhesion in the recipient mice compared with platelet rolling or adhesion in infected C57BL/6 recipients.

FIG. 4.

(A) Kaplan-Meier survival plots for C57BL/6 mice infected with P. berghei and injected intraperitoneally with anti-CD41 MAb or rat IgG. (B) Average numbers of CD41⁺ cells detected by flow cytometry during the course of P. berghei infection. (C) Average levels of P. berghei parasitemia in the two groups of mice.

FIG. 5.

Summary of the proposed molecular mechanisms of platelet adhesion to the pial microvasculature during cerebral malaria.