Thrombin Cleavage of Plasmodium falciparum Erythrocyte Membrane Protein 1 Inhibits Cytoadherence.

Mark R Gillrie,Marion Avril,Enrico Di Cera,Koichiro Mihara,Andrew J Brazier,May Ho,Danny A Milner,Bernard Renaux,Joseph D Smith,Morley D Hollenberg,Eleanor Russell-Goldman,Louis H Miller

doi:10.1128/mbio.01120-16

Abstract

ABSTRACTPlasmodium falciparum malaria 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 by Plasmodium falciparum erythrocyte 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.

Highlights

Plasmodium falciparum malaria remains one of the most deadly infections worldwide
Sequestration results from the adhesion, or cytoadherence, of IRBC to vascular endothelial cells, and the process is mediated by the variant parasite ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and endothelial receptors, of which a number have been implicated in severe disease [7, 8]
To investigate if thrombin has any intrinsic effect on IRBC, the adhesion of diverse P. falciparum binding phenotypes to endothelial cells was studied in a parallelplate flow chamber using four previously characterized IT4 laboratory-adapted parasite lines that express a single predominant PfEMP1 [25, 26] (Fig. 1A)

Summary

Introduction

Plasmodium falciparum malaria 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 results from the adhesion, or cytoadherence, of IRBC to vascular endothelial cells, and the process is mediated by the variant parasite ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and endothelial receptors, of which a number have been implicated in severe disease [7, 8]. The secreted parasite product Plasmodium falciparum histidine-rich protein (PfHRP-2) contributes to a procoagulant environment by inhibiting the activity of antithrombin III [20], and the endothelial protein C receptor (EPCR)-binding cysteine-rich interdomain region ␣1.4 (CIDR␣1.4) domain of PfEMP1 inhibits the generation of activated protein C (APC) [21, 22], a key anticoagulant protein These in vitro observations and the demonstration of fibrin deposition in Ͼ85% of cerebral microvessels in children who died from cerebral malaria [16] make a strong case for the potential pathological significance of microvascular thrombosis in pediatric cerebral malaria

Methods

Results

Conclusion