TPEN (20 mM), a chelator for Zn2þ, Fe2þ and Mn2þ, abolished the effects of Zn2þ (Fig. 1A).
The Zn2þ (20 mM)-induced expression of PS on erythrocytes, which was inhibited by TPEN, was not observed by other divalent cations, including Mg2þ and Ca2þ (Fig. 2A).
TPEN also inhibited erythrocyte adhesion, but the effect at 20 mM was weaker compared to that in the HRG (100 mg/mL)- treatment group.
Eryptosis is the apoptotic-like suicidal process of erythrocytes, and is characterized by cell shrinkage and phosphatidylserine (PS) expression on the outer membrane.
Stimulation with 20 mM Zn2þ resulted in aggregation of washed human erythrocytes 1 h after incubation.
Thus focusing on Zn2þ-stimulated erythrocytes might be an important new approach that could ultimately reveal the mechanism of immunothrombus formation in sepsis.
We found that a considerable number of erythrocytes were attached to the EA.hy926 cells after Zn2þ-stimulation (Fig. 4A).
In addition, we observed that Zn2þ induced the adhesion of erythrocytes to endothelial cells, and this effect was not inhibited by 100 mg/mL of HSA (Fig. 4), suggesting a specific interaction between erythrocytes and HRG.
Although neutrophils, platelets and vascular endothelial cells are the main effector cells involved in immunothrombosis, abnormal aggregation of erythrocytes under septic conditions may also contribute to immunothrombosis in sepsis.
The cascade of immunothrombosis includes platelet activation and secretion, which may result in a local increase in Zn2þ concentration because of the presence of Zn2þ in platelet dense granules.
In 2007, Kempe10 et al. reported that erythrocytes showed an apoptotic tendency in septic patients.
HRG strongly inhibited the aggregation at a concentration of 1 mg/mL, whereas the same concentration of HSA showed no effect (Fig. 1B).
HRG and HSA both strongly inhibited aggregation when the concentration was increased to 100 mg/mL (Supplementary Fig. 1).
HRG and HSA both inhibited PS expression when their concentrations were increased to 10 and 100 mg/mL (Fig. 2B).
HRG treatment significantly inhibited the attachment of erythrocytes to the vascular endothelial cell monolayer in a concentration dependent manner (Fig. 4A and B), while HSA did not affect the adhesion.
To determine the role of PS in the Zn2þ-induced aggregation of erythrocytes, we used Annexin V to block PS expression on the erythrocyte membrane.
Annexin V (100 nM) completely inhibited the Zn2þ-induced aggregation (Fig. 1E).
The decrease was due to the reduction in HRG gene expression, deposition of HRG on immunothrombi and degradation of HRG by thrombin.
Surprisingly, HRG at high concentration (100 mg/mL) significantly decreased PS expression on erythrocytes.
Although a significant difference in PS expressionwas not observed among the CLP groups treated with PBS, HSA or HRG, incubation of erythrocytes for 4 h enabled the observation of beneficial effects of HRG on PS expression on erythrocytes (Fig. 5B).
Hemin-induced hemolysis was also inhibited by HRG and HRGderived peptide (amino acid sequence in the Histidine-rich domain of HRG: HHPHGHHPHG) (Fig. 6A and B).
Supplementary treatment of septic mice with purified HRG from human plasma remarkably suppressed immunothrombosis in the lungs as well as the associated inflammation.
BEL Commons is developed and maintained in an academic capacity by Charles Tapley Hoyt and Daniel Domingo-Fernández at the Fraunhofer SCAI Department of Bioinformatics with support from the IMI project, AETIONOMY. It is built on top of PyBEL, an open source project. Please feel free to contact us here to give us feedback or report any issues. Also, see our Publishing Notes and Data Protection information.
If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.