Heme and hemozoin induce platelet activation and platelet-monocyte aggregation in vivax malaria

Malaria is a worldwide infectious disease and Brazilian cases are mostly caused by P. vivax and P. falciparum species. During the infection, the parasite digests hemoglobin and the heme is transformed into hemozoin crystals (Hz), a less toxic form. Heme and Hz are both released in the circulation during hemolysis. Anemia and thrombocytopenia are major hematological alterations in vivax malaria patients. Once platelets are altered during malaria, we hypothesized that heme and Hz, provenient from hemolysis induce platelet activation. We observed that vivax patients have more activated platelets than healthy donors, exposing CD62p and releasing PF4 and RANTES. When healthy donor platelets’ are incubated with plasma from vivax and mixed patients (infected with both vivax and falciparum species), they expose CD62p and CD63. To explore the role of heme and Hz on platelet activation, we stimulated platelets with heme and Hz and we observed that both induce CD62p and CD63 exposure and PAC-1 activation. Interestingly, only Hz induced CD40 suppression. To further understand the granule secretion upon the activation, we analysed the inflammatory mediators on platelet’s supernatant. We observed that heme and Hz release them in a differential kinetic. Both release PF4, RANTES, TGF-β and VEGF. Only Hz induce MMP2 secretion and, interestingly, suppresses PDGF secretion. Moreover, we wanted to understand how platelets recognise the stimuli. We verified that platelets recognise heme and Hz through CLEC2 and TLR4 receptors, independently of endossomal TLRs, even on the presence of plasmodial DNA on natural hemozoin. As consequence of platelet activation, we sought if platelets interact with monocytes. We observed that heme and Hz induce platelet-monocyte aggregates formation in healthy donors' whole blood ex vivo. Finally, we observed that heme and hemozoin induce platelet activation and platelet-monocyte aggregation, which may contribute to thrombocytopenia seen in patients.