Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function

Background:

Sequestration and cytoadherence of Plasmodium falciparum

-infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria.

Methods:

We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with P. falciparum material, recombinant PfEMP1 or lysates from IE, and measured barrier function by transendothelial electrical resistance (TEER). A selection of PAR1 inhibitors was tested for their ability to reverse the P. falciparum and thrombin induced decrease in barrier function.

Results:

An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, in the IE lysate co-culture system we identified a echanism that slowly reduces barrier function and which is insensitive to PAR1 inhibitors.

Conclusions:

The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism. These findings have implications for the

design of adjunct therapies to reduce brain swelling in cerebral malaria.