Platelet Activation in End-Stage Renal Disease Is Mediated By Extracellular Nucleosomes That Originate from White Blood Cells
Authors: Phan Trung , Ryan McMillan , Skiadopoulos Leonidas , Walborn Amanda , Hoppensteadt Debra , Bansal Vinod , Fareed Jawed 
Published: October 2019

Abstract
Introduction
Extracellular nucleosomes in plasma (PNs) are complexes of DNA and histones that are released during cell death and inflammatory responses. End-stage renal disease (ESRD) represents a complex syndrome where inflammation, endothelial dysfunction, and hemostatic aberrations contribute to the observed vascular manifestations. The purpose of this investigation is to profile PNs in patients with ESRD and to demonstrate their relevance to blood cells and platelet activation products.
Methods
Pre-dialysis plasma samples from patients undergoing maintenance hemodialysis (n = 90) at Loyola University outpatient dialysis unit were collected under an approved IRB protocol. Plasma samples from healthy individuals (n = 50) were purchased from a biobank as a control (George King Biomedical, Overland Park, Kansas). Complete blood count profiles, including white blood cells (WBCs), red blood cells (RBCs), and platelets were obtained from the patients' medical records. The levels of PNs in ESRD patients and healthy volunteers were measured using the Cell Death Detection ELISA PLUS assay (Roche Diagnostics, Mannheim, Germany). MP-TF levels were measured using the Zymuphen MP-TF kit (Hyphen BioMed, Neuville-sur-Oise, France). PDGF levels were measured using the Human PDGF-BB Quantikine ELISA Kit (R&D Systems, Minneapolis, Minnesota). Human PF4 levels were measured using the Zymutest PF4 ELISA Kit (Hyphen BioMed, Neuville-sur-Oise, France). All individual results were tabulated and analyzed using the statistical software GraphPad Prism 7. The Mann-Whitney test for non-parametric data was utilized in comparing ESRD to control groups. PN levels were correlated with cell counts and platelet activation factors using the non-parametric Spearman correlation. Individual cell counts were also correlated with platelet activating factors using the same method.
Results
In the ESRD patients, the average hematocrit was 31.7 ± 4.4 %, the average WBC count was 6.5 ± 4.0 K/uL, and the average platelet count was 179.4 ± 66.3 K/uL. The levels of PNs in the ESRD patients (15.5 ± 14.1 Arbitrary Units (AU)) were markedly higher in comparison to that of the controls (6.74 ± 13.7 AU; p < 0.0001). Similarly, MP-TF levels were significantly elevated in ESRD patients (3.00 ± 1.42 pg/mL) compared to normal (0.363 ± 0.263 pg/mL; p < 0.0001). PF4 levels were also significantly elevated in ESRD patients (95.3 ± 35.3 ng/mL) compared to normal (27.4 ± 19.8 ng/mL; p < 0.0001). While PDGF levels were higher in the ESRD group (116.0 ± 172.5 pg/mL) in comparison to the controls (82.7 ± 113.5 pg/mL), this increase was not statistically significant (p = 0.405). A positive correlation was observed between PNs and WBCs (p = 0.024; r = 0.244). PN levels did not show a correlation with RBC (p = 0.448; r = 0.083) and platelet levels (p = 0.545; r = 0.066). Furthermore, there was no correlation between PNs and MP-TF (p = 0.501; r = 0.077), PDGF (p = 0.314; r = 0.110) and PF4 (p = 0.524; r = -0.070) in the ESRD patients. However, the platelet count showed a positive correlation with PDGF (p = 0.044; r = 0.218) and MP-TF (p = 0.042; r = 0.237). Similarly, the WBC count showed a positive correlation with the platelet count (p < 0.0001; r = 0.476) and PDGF (p = 0.016; r = 0.260).
Conclusion
This study clearly demonstrates that extracellular nucleosomes are elevated in the plasma of patients with ESRD. The fact that the PN levels correlated with the number of circulating white blood cells suggest that the PNs originate from these cells. Since the ESRD patients exhibited platelet activation, as evident by the observed increase in PDGF, MP-TF and PF4, it is plausible that this activation is mediated by PNs originating from the WBCs. As observed by the positive correlation between WBCs, PDGF, and platelet count, this study underscores the potential role of nucleosomes originating from WBCs in the activation of platelets. These results are consistent with previously reported observations that extracellular histones can induce platelet activation in a TLR2 and TLR4 dependent manner (http://www.ncbi.nlm.nih.gov/pubmed/21673343).

Disclosures
No relevant conflicts of interest to declare.