Binding of human serum proteins to Plasmodium falciparum-infected erythrocytes and its association with malaria clinical presentation

Abstract Background The pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite‑infected erythrocytes (IEs) to adhere to the vascular endothelium (cytoadhesion/sequestration) or to surrounding uninfected erythrocytes (rosetting). Both processes are mediated by the expression of members of the clonally variant PfEMP1 parasite protein family on the surface of the IEs. Recent evidence obtained with laboratory-adapted clones indicates that P. falciparum can exploit human serum factors, such as IgM and α2-macroglobulin (α2M), to increase the avidity of PfEMP1-mediated binding to erythrocyte receptors, as well as to evade host PfEMP1-specific immune responses. It has remained unclear whether PfEMP1 variants present in field isolates share these characteristics, and whether they are associated with clinical malaria severity. These issues were investigated here. Methods Children 1–12 years reporting with P. falciparum malaria to Hohoe Municipal Hospital, Ghana were enrolled in the study. Parasites from children with uncomplicated (UM) and severe malaria (SM) were collected. Binding of α2M and IgM from non-immune individuals to erythrocytes infected by P. falciparum isolates from 34 children (UM and SM) were analysed by flow cytometry. Rosetting in the presence of IgM or α2M was also evaluated. Experimental results were analysed according to the clinical presentation of the patients. Results Clinical data from 108 children classified as UM (n = 54) and SM cases (n = 54) were analysed. Prostration, severe malaria anaemia, and hyperparasitaemia were the most frequent complications. Three children were diagnosed with cerebral malaria, and one child died. Parasite isolates from UM (n = 14) and SM (n = 20) children were analysed. Most of the field isolates bound non-immune IgM (33/34), whereas the α2M-binding was less common (23/34). Binding of both non-immune IgM and α2M was higher but not significant in IEs from children with SM than from children with UM. In combination, IgM and α2M supported rosette formation at levels similar to that observed in the presence of 10% human serum. Conclusions The results support the hypothesis that binding of non-immune IgM and/or α2M to IEs facilitates rosette formation and perhaps contributes to P. falciparum malaria severity.

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Binding of human serum proteins to Plasmodium falciparum-infected erythrocytes and its association with malaria clinical presentation

10.21203/rs.3.rs-48813/v3 ◽

2020 ◽

Author(s):

Mary Lopez-Perez ◽

William van der Puije ◽

Filip C. Castberg ◽

Michael F. Ofori ◽

Lars Hviid

Keyword(s):

Human Serum ◽

Severe Malaria ◽

Clinical Presentation ◽

Serum Proteins ◽

Clinical Malaria ◽

Rosette Formation ◽

Parasite Protein ◽

Serum Factors ◽

Field Isolates ◽

Malaria Severity

Abstract Background The pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite‑infected erythrocytes (IEs) to adhere to the vascular endothelium (cytoadhesion/sequestration) or to surrounding uninfected erythrocytes (rosetting). Both processes are mediated by the expression of members of the clonally variant PfEMP1 parasite protein family on the surface of the IEs. Recent evidence obtained with laboratory-adapted clones indicates that P. falciparum can exploit human serum factors, such as IgM and α2‑macroglobulin (α2M), to increase the avidity of PfEMP1-mediated binding to erythrocyte receptors, as well as to evade host PfEMP1-specific immune responses. It has remained unclear whether PfEMP1 variants present in field isolates share these characteristics, and whether they are associated with clinical malaria severity. These issues were investigated here.Methods Children 1-12 years reporting with P. falciparum malaria to Hohoe Municipal Hospital, Ghana were enrolled in the study. Parasites from children with uncomplicated (UM) and severe malaria (SM) were collected. Binding of α2M and IgM from non-immune individuals to erythrocytes infected by P. falciparum isolates from 34 children (UM and SM) were analysed by flow cytometry. Rosetting in the presence of IgM or α2M was also evaluated. Experimental results were analysed according to the clinical presentation of the patients.Results Clinical data from 108 children classified as UM (n=54) and SM cases (n=54) were analysed. Prostration, severe malaria anaemia, and hyperparasitaemia were the most frequent complications. Three children were diagnosed with cerebral malaria, and one child died. Parasite isolates from UM (n=14) and SM (n=20) children were analysed. Most of the field isolates bound non‑immune IgM (33/34), whereas the α2M-binding was less common (23/34). Binding of both non-immune IgM and α2M was higher but not significant in IEs from children with SM than from children with UM. In combination, IgM and α2M supported rosette formation at levels similar to that observed in the presence of 10% human serum. Conclusions The results support the hypothesis that binding of non-immune IgM and/or α2M to IEs facilitates rosette formation and perhaps contributes to P. falciparum malaria severity.

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Binding of human serum proteins to P. falciparum-infected erythrocytes and its association with malaria clinical presentation

10.21203/rs.3.rs-48813/v2 ◽

2020 ◽

Author(s):

Mary Lopez-Perez ◽

William van der Puije ◽

Filip C. Castberg ◽

Michael F. Ofori ◽

Lars Hviid

Keyword(s):

Human Serum ◽

Severe Malaria ◽

Clinical Presentation ◽

Serum Proteins ◽

Clinical Malaria ◽

Rosette Formation ◽

Parasite Protein ◽

Serum Factors ◽

Field Isolates ◽

Malaria Severity

Abstract BackgroundThe pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite‑infected erythrocytes (IEs) to adhere to the vascular endothelium (cytoadhesion/sequestration) or to surrounding uninfected erythrocytes (rosetting). Both processes are mediated by the expression of members of the clonally variant PfEMP1 parasite protein family on the surface of the IEs. Recent evidence obtained with laboratory-adapted clones indicates that P. falciparum can exploit human serum factors, such as IgM and α2‑macroglobulin (α2M), to increase the avidity of PfEMP1-mediated binding to erythrocyte receptors, as well as to evade host PfEMP1-specific immune responses. It has remained unclear whether PfEMP1 variants present in field isolates share these characteristics, and whether they are associated with clinical malaria severity. These issues were investigated here.MethodsChildren 1-12 years reporting with P. falciparum malaria to Hohoe Municipal Hospital, Ghana were enrolled in the study. Parasites from children with uncomplicated (UM) and severe malaria (SM) were collected. Binding of α2M and IgM from non-immune individuals to erythrocytes infected by P. falciparum isolates from 34 children (UM and SM) were analysed by flow cytometry. Rosetting in the presence of IgM or α2M was also evaluated. Experimental results were analysed according to the clinical presentation of the patients.ResultsClinical data from 108 children classified as UM (n=54) and SM cases (n=54) were analysed. Prostration, severe malaria anaemia, and hyperparasitaemia were the most frequent complications. Three children were diagnosed with cerebral malaria, and one child died. Parasite isolates from UM (n=14) and SM (n=20) children were analysed. Most of the field isolates bound non‑immune IgM (33/34), whereas the α2M-binding was less common (23/34). Binding of both non-immune IgM and α2M was higher but not significant in IEs from children with SM than from children with UM. In combination, IgM and α2M supported rosette formation at levels similar to that observed in the presence of 10% human serum.ConclusionsThe results support the hypothesis that binding of non-immune IgM and/or α2M to IEs facilitates rosette formation and perhaps contributes to P. falciparum malaria severity.

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Binding of Human Serum Proteins to P. Falciparum Infected Erythrocytes and Its Association With Malaria Clinical Presentation

10.21203/rs.3.rs-48813/v1 ◽

2020 ◽

Author(s):

Mary Lopez-Perez ◽

William van der Puije ◽

Filip C. Castberg ◽

Michael F. Ofori ◽

Lars Hviid

Keyword(s):

Human Serum ◽

Severe Malaria ◽

Falciparum Malaria ◽

Clinical Presentation ◽

Serum Proteins ◽

Rosette Formation ◽

Parasite Protein ◽

Serum Factors ◽

Field Isolates ◽

Malaria Severity

Abstract Background The pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite‑infected erythrocytes (IEs) to adhere to the vascular endothelium (cytoadhesion/sequestration) or to surrounding uninfected erythrocytes (rosetting). Both processes are mediated by the expression of members of the clonally variant PfEMP1 parasite protein family on the surface of the IEs. Recent evidence obtained with laboratory-adapted clones indicates that P. falciparum can exploit human serum factors, such as IgM and α 2 ‑macroglobulin (α 2 M), to increase the avidity of PfEMP1-mediated binding to host receptors, as well as to evade host PfEMP1-specific immune responses. It has remained unclear whether PfEMP1 variants present in field isolates share these characteristics, and whether they are associated with clinical malaria severity. These issues were investigated here. Methods Children between 1-12 years reporting with P. falciparum malaria to Hohoe Municipal Hospital, Ghana were enrolled in the study. Parasites from children with uncomplicated (UM) and severe malaria (SM) were collected. Binding of α 2 M and IgM from non-immune individuals to erythrocytes infected by P. falciparum isolates from 34 children (UM and SM) were analysed by flow cytometry. Rosetting in the presence of IgM or α 2 M was also evaluated. Experimental results were related to the clinical presentation of the patients. Results Clinical data from 108 children classified as UM (n=54) and SM cases (n=54) were analysed. Prostration, severe malaria anaemia, and hyperparasitaemia were the most frequent complications. Three children were diagnosed with cerebral malaria, and one child died. Parasite isolates from 34 children were analysed. Most of the field isolates bound non‑immune IgM (33/34), whereas the α 2 M-binding was less common (23/34) and mostly low. Binding of both non-immune IgM and α 2 M was higher in IEs from children with SM than from UM. In combination, IgM and α 2 M supported rosette formation at levels similar to that observed in the presence of 10% human serum. Conclusions The results support the hypothesis that binding of non-immune IgM and/or α 2 M to IEs facilitates rosette formation and contributes to P. falciparum malaria severity.

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Identifying targets of protective antibodies against severe malaria in Papua, Indonesia using locally expressed domains of Plasmodium falciparum Erythrocyte Membrane Protein 1.

Infection and Immunity ◽

10.1128/iai.00435-21 ◽

2021 ◽

Author(s):

Janavi S Rambhatla ◽

Gerry Q Tonkin-Hill ◽

Eizo Takashima ◽

Takafumi Tsuboi ◽

Rintis Noviyanti ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Membrane Protein ◽

Severe Malaria ◽

Erythrocyte Membrane ◽

Uncomplicated Malaria ◽

Igg Antibodies ◽

Erythrocyte Membrane Protein ◽

African Children ◽

Genes Encoding ◽

Malaria Severity

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multi-domain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal components analysis, antibodies to three of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLβ13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults. Importance Severe Plasmodium falciparum malaria kills many African children, and lack of antibody immunity predisposes to severe disease. A critical antibody target is the P. falciparum erythrocyte membrane 1 (PfEMP1) family of multidomain proteins, which are expressed on the infected erythrocyte surface and mediate parasite sequestration in deep organs. We previously identified var genes encoding PfEMP1 that were differentially expressed between severe and uncomplicated malaria in Papua, Indonesia. Here, we have expressed domains from 32 of these PfEMP1s and measured IgG antibody responses to them in Papuan adults and children. Using Principal Component Analysis, IgG antibodies to three domains distinguished between severe and uncomplicated malaria and were higher in uncomplicated malaria. Domains included CIDRα1.6, implicated in severe malaria; a DBLβ13 domain; and a DBLδ domain of unknown function. Immunity to locally relevant PfEMP1 domains may protect from severe malaria. Targets of immunity show important overlap between Asian adults and African children.

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Invasion Pathways and Malaria Severity in Kenyan Plasmodium falciparum Clinical Isolates

Infection and Immunity ◽

10.1128/iai.00249-07 ◽

2007 ◽

Vol 75 (6) ◽

pp. 3014-3020 ◽

Cited By ~ 35

Author(s):

Anne-Marie Deans ◽

Susana Nery ◽

David J. Conway ◽

Oscar Kai ◽

Kevin Marsh ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Severe Malaria ◽

Uncomplicated Malaria ◽

Molecular Basis ◽

Glycophorin A ◽

Invasion Pathways ◽

Significant Difference ◽

Erythrocyte Binding ◽

Malaria Severity

ABSTRACT The invasion of erythrocytes by Plasmodium falciparum occurs through multiple pathways that can be studied in vitro by examining the invasion of erythrocytes treated with enzymes such as neuraminidase, trypsin, and chymotrypsin. We have studied the invasion pathways used by 31 Kenyan P. falciparum isolates from children with uncomplicated or severe malaria. Six distinct invasion profiles were detected, out of eight possible profiles. The majority of isolates (23 of 31) showed neuraminidase-resistant, trypsin-sensitive invasion, characteristic of the pathway mediated by an unknown parasite ligand and erythrocyte receptor “X.” The neuraminidase-sensitive, trypsin-sensitive phenotype consistent with invasion mediated by the binding of parasite ligand erythrocyte binding antigen 175 to glycophorin A, the most common invasion profile in a previous study of Gambian field isolates, was seen in only 3 of 31 Kenyan isolates. No particular invasion profile was associated with severe P. falciparum malaria, and there was no significant difference in the levels of inhibition by the various enzyme treatments between isolates from children with severe malaria and those from children with uncomplicated malaria (P, >0.1 for all enzymes; Mann-Whitney U test). These results do not support the hypothesis that differences in invasion phenotypes play an important role in malaria virulence and indicate that considerable gaps remain in our knowledge of the molecular basis of invasion pathways in natural P. falciparum infections.

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Genetic diversity of Plasmodium falciparum isolates from patients with uncomplicated and severe malaria based on msp-1 and msp-2 genes in Gublak, North West Ethiopia

Malaria Journal ◽

10.1186/s12936-019-3039-9 ◽

2019 ◽

Vol 18 (1) ◽

Author(s):

Hussein Mohammed ◽

Kedir Hassen ◽

Ashenafi Assefa ◽

Kalkidan Mekete ◽

Gemechu Tadesse ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

Severe Malaria ◽

Severe Disease ◽

Northwest Ethiopia ◽

Multiplicity Of Infection ◽

Cross Sectional ◽

North West ◽

Severe Infections ◽

Malaria Severity

Abstract Background Malaria infection can present with a wide variety of symptoms, ranging from mild to severe. Plasmodium falciparum isolates in uncomplicated and severe malaria infections may have different parasite genetic profiles. This study was conducted to assess differences in genetic diversity and allelic frequencies in P. falciparum isolates according to malaria severity and age of patients in the Gublack area, northwest Ethiopia. Methods Cross-sectional health facility-based study conducted in Gublak, Ethiopia between July, 2017 and October, 2017. Symptomatic P. falciparum malaria patients with microscopically-confirmed infection were enrolled. Parasite DNA was extracted from filter paper blood spots and the polymorphic regions of the msp-1 and msp-2 genes were genotyped using allele-specific nested-PCR with fragment analysis by gel electrophoresis. Results A total of 118 patients were enrolled including 95 (80.5%) with uncomplicated infection and 23 (19.5%) with severe disease. In msp-1, the K1 allelic family was similarly prevalent in uncomplicated 42 (44.2%) and severe disease 12 (52.2%). In msp-2, FC27 was detected in 55 (57.9%) of uncomplicated infections and IC/3D7 in 14 (60.9%) of severe infections. 76 (64.4%) of the 118 isolates contained multiple genotypes; 56 (58.9%) in uncomplicated infections and 19 (82.6%) in severe infections. The overall of multiplicity of infection was 2.2 (95% CI 1.98–2.42) with 1.4 (95% CI 1.23–1.55) and 1.7 (95% CI 1.49–1.86) for msp-1 and msp-2, respectively. Multiplicity of infection was significantly higher in severe than uncomplicated infections (3.0 (95% CI 2.61–3.47) versus 2.0 (95% CI 1.83–2.23), respectively, p = 0.001). There was no difference in multiplicity of infection across age groups (p = 0.104). Conclusion Patients with severe malaria were more likely to have multiclonal infections. Further studies are needed to describe the association between P. falciparum genotypes and malaria severity in different malaria transmission areas.

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Inhibition of Plasmodium falciparum Field Isolates-Mediated Endothelial Cell Apoptosis by Fasudil: Therapeutic Implications for Severe Malaria

PLoS ONE ◽

10.1371/journal.pone.0013221 ◽

2010 ◽

Vol 5 (10) ◽

pp. e13221 ◽

Cited By ~ 14

Author(s):

Estelle S. Zang-Edou ◽

Ulrick Bisvigou ◽

Zacharie Taoufiq ◽

Faustin Lékoulou ◽

Jean Bernard Lékana-Douki ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Endothelial Cell ◽

Severe Malaria ◽

Cell Apoptosis ◽

Endothelial Cell Apoptosis ◽

Therapeutic Implications ◽

Field Isolates

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Enhanced Basophil Reactivities during Severe Malaria and Their Relationship with the Plasmodium falciparum Histamine-Releasing Factor Translationally Controlled Tumor Protein

Infection and Immunity ◽

10.1128/iai.00072-12 ◽

2012 ◽

Vol 80 (8) ◽

pp. 2963-2970 ◽

Cited By ~ 13

Author(s):

Stéphane Pelleau ◽

Sylvie Diop ◽

Méry Dia Badiane ◽

Joana Vitte ◽

Pierre Beguin ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Severe Malaria ◽

Ex Vivo ◽

Statistical Significance ◽

Basophil Activation ◽

Content Type ◽

Translationally Controlled Tumor Protein ◽

Mild Malaria ◽

Excessive Activation ◽

Malaria Severity

ABSTRACTRecent studies suggest shared pathogenic pathways during malaria and allergy. Indeed, IgE, histamine, and the parasite-derivedPlasmodium falciparumhistamine-releasing factor translationally controlled tumor protein (PfTCTP) can be found at high levels in serum from patients experiencing malaria, but their relationship with basophil activation remains unknown. We recruitedP. falciparum-infected patients in Senegal with mild malaria (MM;n= 19) or severe malaria (SM;n= 9) symptoms and healthy controls (HC;n= 38). Levels of serum IgE, PfTCTP, and IgG antibodies against PfTCTP were determined by enzyme-linked immunosorbent assays (ELISA). Basophil reactivities to IgE-dependent and -independent stimulations were measuredex vivousing fresh blood by looking at the expression level of the basophil activation marker CD203c with flow cytometry. Unstimulated basophils from MM had significantly lower levels of CD203c expression compared to those from HC and SM. After normalization on this baseline level, basophils from SM showed an enhanced reactivity to calcimycin (A23187) and hemozoin. Although SM reached higher median levels of activation after anti-IgE stimulation, great interindividual differences did not allow the results to reach statistical significance. When primed with recombinant TCTP before anti-IgE, qualitative differences in terms of a better ability to control excessive activation could be described for SM. IgE levels were very high in malaria patients, but concentrations in MM and SM were similar and were not associated with basophil responses, which demonstrates that the presence of IgE alone cannot explain the various basophil reactivities. Indeed, PfTCTP could be detected in 32% of patients, with higher concentrations for SM. These PfTCTP-positive patients displayed significantly higher basophil reactivities to any stimulus. Moreover, the absence of anti-PfTCTP IgG was associated with higher responses in SM but not MM. Our results show an association between basophil reactivity and malaria severity and suggest a pathogenic role for plasmodial PfTCTP in the induction of this allergy-like mechanism.

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Multiple human serum components act as bridging molecules in rosette formation by Plasmodium falciparum-infected erythrocytes

Blood ◽

10.1182/blood.v95.2.674 ◽

2000 ◽

Vol 95 (2) ◽

pp. 674-682 ◽

Cited By ~ 24

Author(s):

Elizabeth A. Somner ◽

Julie Black ◽

Geoffrey Pasvol

Keyword(s):

Plasmodium Falciparum ◽

Human Serum ◽

Molecular Mechanisms ◽

Gradient Centrifugation ◽

Immunoglobulin M ◽

Host Susceptibility ◽

Rosette Formation ◽

Cellular Interaction ◽

Percoll Density Gradient Centrifugation ◽

Serum Components

Rosetting, the binding of parasitized erythrocytes to 2 or more uninfected erythrocytes, is an in vitro correlate of disease severity in Plasmodium falciparum malaria. Although cell ligands and receptors have been identified and a role for immunoglobulin M has been suggested, the molecular mechanisms of rosette formation are unknown. The authors demonstrate unequivocally that rosette formation by P falciparum-infected erythrocytes is specifically dependent on human serum, and they propose that serum components act as bridging molecules between the cell populations. Using heparin treatment and Percoll density gradient centrifugation, they have developed an assay in which parasitized erythrocytes grown in serum-containing medium and optimally forming rosettes are stripped of serum components. These infected cells were no longer able to form rosettes when mixed with erythrocytes and incubated in serum-free medium. Rosette formation was restored by the addition of serum or certain serum fractions obtained by concanavalin A (conA) affinity, anti-IgM affinity, anion exchange, and gel filtration chromatography. The authors clearly demonstrate that multiple serum components—IgM and at least 2 others—are involved in rosette formation. Those others consist of 1 or more acidic components of high-molecular mass that binds to conA (but that is not thrombospondin, fibronectin, or von Willebrand's factor) and of at least 1 more basic, smaller component that does not bind to conA. Data on the size and number of rosettes formed support the authors' hypothesis that multiple bridges are involved in this complex cellular interaction. These findings have important implications for the understanding of pathogenic adhesive interactions of P falciparum and host susceptibility to severe malaria.

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Thrombin Cleavage ofPlasmodium falciparumErythrocyte Membrane Protein 1 Inhibits Cytoadherence

mBio ◽

10.1128/mbio.01120-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 7

Author(s):

Mark R. Gillrie ◽

Bernard Renaux ◽

Eleanor Russell-Goldman ◽

Marion Avril ◽

Andrew J. Brazier ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Membrane Protein ◽

Severe Malaria ◽

Coagulation Cascade ◽

Thrombin Inhibitor ◽

Parasite Line ◽

Barrier Dysfunction ◽

Microvascular Endothelium ◽

Parasite Protein ◽

Thrombin Cleavage

ABSTRACTPlasmodium falciparummalaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium byPlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy.IMPORTANCEPlasmodium falciparummalaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion ofPlasmodium falciparum-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria. While the recent implementation of artemisinin-based antimalarial therapy for severe malaria improves patient survival by targeting all parasite stages, antiparasite drugs alone may not immediately reverse pathophysiological processes in occluded vessels. Here we show that thrombin, an enzyme intimately involved in the clotting process, cleaves the main parasite adhesin expressed on the surface of IRBC, thereby preventing and reversing the binding of IRBC to endothelial cells. This beneficial effect of thrombin can be achieved by modified thrombins that cause significantly less clotting and vessel leakage while preserving the ability to cleave the parasite protein. Our results provide the basis for using modified thrombins as adjunctive therapy in severe malaria.

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