Platelet disturbances correlate with endothelial cell activation in uncomplicated Plasmodium vivax malaria

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson’s correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406–651] vs. 1029 [732–1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.

selectin; endothelial cell activation. unclear. Thrombocytopenia is the most common hematological disturbance in P. 53 vivax-infected patients, and platelets have been implicated in parasitemia control. 54 In this study, we studied the activation of platelets in association with endothelial 55 cell activation in vivax malaria. Platelets retrieved from infected peripheral blood 56 were non-activated when analyzed by flow cytometry; however, they displayed 57 higher mean volume and significantly reduced counts. We also found higher levels   Thrombocytopenia is the most common hematological alteration in malaria, 68 although there is no definitive mechanistic explanation to its occurrence (1, 2). In 69 a retrospective cohort, patients who died from malaria had lower platelet counts in 70 comparison to those with less severe disease (3). Moreover, P. vivax malaria 71 patients with thrombocytopenia showed higher levels of markers of endothelial 72 cell (EC) activation compared to those with normal platelet counts (4) and some 73 grade of platelet activation has been reported during the disease (5). Together, 74 these reports point out to a significant role of platelets in the pathophysiology of 75 malaria.

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In cerebral malaria, the most severe presentation of the disease, platelets 77 have been shown to accumulate in the brain microvessels of affected children (6).

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In mice, the adhesion of platelets to brain endothelial cells was crucial for the 79 development of the syndrome (7). In addition, platelets lead to a deleterious 80 inflammatory response in the disease through platelet-factor 4 (PF4), with PF4-81 KO mice surviving the infection (8). However, PF4 also plays a protective role in 82 the disease, as it mediates P. falciparum killing by platelets in vitro (9) and in vivo, 83 as shown in patients from Southeast Asia, and this was correlated with reduced 84 parasitemias (10).

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EC activation is present in all malaria species, occurring in both mild and 86 severe cases (11). In terms of pathogenesis, EC activation is important for P. 87 falciparum-infected erythrocytes adhesion to microvasculature, avoiding 88 immunological clearance and leading to severe disease, while inducing more EC 89 damage (12). P. vivax-infected erythrocytes also adhere to EC (13), but the 90 magnitude of the phenomenon is smaller and if this has a role in endothelium 91 pathology and disease severity is not clear (14).

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While the role of platelets in P. falciparum malaria has been extensively 93 studied, its role in the pathogenesis of EC dysfunction during P. vivax malaria 94 remains to be investigated. In this study, we show that platelet counts were reduced 95 in P. vivax malaria patients, while circulating markers of platelet activation were 96 higher. Importantly, platelet activation markers correlated with those related to    were named Control, Vivax low and Vivax high . In addition, the same software was 147 used to determine pair-wise Pearson's correlation coefficients to generate 148 correlation networks and the p value to test for non-correlation was evaluated using 149 p ≤ 0.05 as a cut-off. In order to analyze the structure of the networks, the graphics 150 for the network analysis were customized in the Cytoscape software (v 3.5.1) using 151 the prefuse force-directed layout, which in the equilibrium state for the system of  Fisher's exact test was used for categorical data. Student's t-test was used 157 to compare means between groups with normally distributed data, and data sets 158 with non-normal distributions were compared using the Mann-Whitney test, with 159 p<0.05 considered significant. Data are presented as means and SD unless 160 otherwise stated. Analysis were performed, and the graphs generated in GraphPad 161 Prism5 and R software.     In the current study, we aimed to assess platelet activation and its 212 relationship with endothelial cell activation in the context of P. vivax malaria.

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Thrombocytopenia was the most frequent hematological alteration in our cohort 214 and, as previously reported for malaria (1), was correlated to an increase in MPV.