FIG 6 .

Hirugen inhibits endothelial barrier dysfunction but not IT4var19 cleavage induced by thrombin. (A) Flow cytometry analysis of PfEMP1 expression on IRBC of IT4var19 at the trophozoite stage treated with 10 nM thrombin with or without 1 and 5 µM hirugen for 30 min at 37°C. Results are representative of 2 experiments. (B) Immunofluorescence microscopy of IRBC from panel A. Images are representative of 10 randomly selected fields examined at ×600 magnification in each of 3 independent experiments. (C) Hirugen did not inhibit the effect of thrombin on IRBC adhesion under flow conditions. IRBC of IT4var19 were treated with thrombin with or without 1 µM hirugen for 30 min at 37°C before being used in a flow chamber assay. Results are expressed as mean ± SEM and were analyzed by ANOVA followed by post hoc multiple comparisons using Tukey’s test (n = 3). (D) Changes in endothelial resistance as monitored by ECIS of 3-day postconfluence HLMEC treated with 10 nM thrombin with or without 0.5 to 5 µM hirugen. The results shown are representative of 3 experiments. (E) Confocal immunofluorescence microscopy of HLMEC monolayers showing expression of VE-cadherin (green) and DNA (blue) in response to thrombin ± hirugen as in panel D. Images are representative of 10 random fields examined at 600× in each of 2 independent experiments.