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von Willebrand factor and ADAMTS13 in mouse models of severe malaria

Boek - Dissertatie

Malaria remains a devastating global health problem, leading to half a million deaths per year. More than 90% of malaria cases and deaths occur in African regions, mainly in children aged under 5 years. Cerebral malaria (CM) is the most common complication and cause of death in severe Plasmodium falciparum infection. In addition to CM, malaria-associated acute respiratory distress syndrome (MA-ARDS) is also common in adult patients, leading to a high degree of lethality despite antimalarial treatment. Regardless of the high degree of mortality, the pathogenic mechanisms of both CM and MA-ARDS remain elusive.Patient studies have shown that severe malaria is associated with elevated levels of plasma von Willebrand factor (VWF), a multimeric glycoprotein that is crucial for normal hemostasis. More specifically, malaria patients commonly present with accumulation of hyperactive forms of VWF, and reduced activity of its cleaving protease ADAMTS13 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motif, 13). VWF is released upon endothelial cell (EC) activation and mediates local thrombo-inflammatory responses. Therefore, the increased activity of VWF observed in patients with severe malaria raises an intriguing possibility that VWF may not only be a marker of EC activation but could also directly modulate the pathogenesis of severe malaria. Since ADAMTS13 is an important regulator of VWF activity, reduction in ADAMTS13 activity might also be involved in the development of severe malaria. In this PhD project, we aimed to unravel the contribution of VWF and ADAMTS13 in the pathogenesis of severe malaria by using mouse models of MA-ARDS and CM.To induce experimental MA-ARDS and experimental CM (ECM), C57BL/6 mice were infected with Plasmodium berghei NK65-E (PbNK65) and P. berghei ANKA (PbANKA) parasites, respectively. During the infection, alterations of plasma VWF antigen (VWF:Ag) levels, VWF multimer composition, ADAMTS13 antigen (ADAMTS13:Ag), and ADAMTS13 activity, were measured in both mouse models (chapter 3-5). In agreement with clinical studies, plasma VWF:Ag levels were also elevated in our mouse models. These data suggest that increased VWF concentration is a consistent hallmark of malaria infection in both mouse and human malaria. In addition, we observed an ADAMTS13- and plasmin-independent reduction of high molecular weight (HMW) VWF multimers at the end-stage of disease in both experimental MA-ARDS and ECM models. This phenomenon might be indicative of severe consumptive coagulopathy during the final phase of malaria infection. Reduced activity of ADAMTS13 was observed only in experimental MA-ARDS, but not in ECM, suggesting that alteration of ADAMTS13 in malaria infection differs among parasite strains.To investigate whether the increased levels of VWF:Ag are simply a marker of EC activation or VWF directly modulate the pathogenesis of MA-ARDS, we studied PbNK65 infection in VWF-deficient(Vwf-/-) mice compared with wild type (Vwf+/+) animals (chapter 3). Pathological parameters including thrombocytopenia, parasitemia, pulmonary edema, and mortality rate were assessed in our MA-ARDS model. Given its prothrombotic role, VWF has been proposed to contribute to thrombocytopenia in malaria. Interestingly, our data reveal that malaria-associated thrombocytopenia occurred through a VWF-independent mechanism. In addition, we have shown that VWF contributes to significantly increased alveolar leakage, potentially through its proinflammatory role. Although less severe pulmonary edema was developed in Vwf-/- mice, their survival time was shorter than that observed in Vwf+/+ animals. This rapid mortality might be explained by the significant increase in parasite load together with severe malarial anemia seen in PbNK65-infected Vwf-/- mice. Finally, we have also demonstrated that VWF deficiency is associated with early reticulocytosis following PbNK65 infection, which potentially accounts for the increase in parasite accumulation in Vwf-/- mice.In addition to experimental MA-ARDS, the effect of VWF on the pathogenesis of ECM was also investigated (chapter 4). Pathological parameters following PbANKA infection were compared between Vwf-/- and Vwf+/+ mice. In this study, we observed no major difference in parasitemia between Vwf-/- and Vwf+/+ mice. Increased reticulocyte numbers were seen in Vwf-/- mice following PbANKA infection, which is consistent to what was observed in experimental MA-ARDS. Regardless of its prothrombotic and proinflammatory roles, VWF is not a major player mediating neither thrombocytopenia, brain leukocyte infiltration, nor intravascular platelet accumulation in the brain. In line with those observations, Vwf-/- mice also developed ECM features and reached a humane endpoint similarly to those observed in Vwf+/+ controls. Altogether, it seems likely that increased plasma VWF levels observed in ECM is a consequence of malaria infection rather than a major cause of disease development. However, a subsequent risk for thrombosis in malaria patients from the elevation of VWF levels should still be taken into account.Given its role in regulating VWF activity, alteration of ADAMTS13 activity during severe malaria may directly modulate the disease pathogenesis. Here, we also investigated the role of ADAMTS13 in experimental MA-ARDS and ECM (chapter 5). Following PbNK65 infection, no significant differences in either parasitemia, alveolar leakage, or overall survival were observed between ADAMTS13 knockout (Adamts13-/-) and wild-type (Adamts13+/+) mice. These data suggest that the reduction of ADAMTS13 activity observed in our MA-ARDS model (chapter 3) could be a consequence of PbNK65 infection rather than an active contributor of MA-ARDS progression. Likewise, PbANKA infected-Adamts13-/- mice also developed parasitemia levels, ECM progression and mortality rate, similarly to those observed in Adamts13+/+ mice. Therefore, based on our experiments, we do not see a major role for in ADAMTS13 in ECM pathogenesis.To conclude, we have studied the potential roles of VWF and ADAMTS13 in the pathogenesis of severe malaria using experimental MA-ARDS and ECM. Our data have shown that increase in plasma VWF concentration is a consistent hallmark upon malaria infection. Thrombocytopenia occurs independently of the presence of VWF. Furthermore, VWF promotes increased pulmonary edema in experimental MA-ARDS, whereas the elevated plasma VWF levels do not have any major influence on ECM pathogenesis. Finally, alteration of ADAMTS13 has no direct effect on the pathogenesis of both experimental MA-ARDS and ECM.
Jaar van publicatie:2019