Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells

Abstract

Background: Extracellular histones exert part of their prothrombotic activity through the stimulation of blood cells. Besides platelets, histones can bind to red blood cells (RBCs), which are important contributors to thrombogenesis, but little is known about the functional consequences of this interaction.

Objectives: To evaluate the effect of histones on the procoagulant potential of human RBCs with particular regard to the expression of surface phosphatidylserine (PS).

Methods: PS exposure on human RBCs treated with a natural mixture of histones or recombinant individual histones was evaluated with fluorescein isothiocyanate-annexin-V binding and measured with flow cytometry. Calcium influx in RBCs loaded with the calcium-sensitive fluorophore Fluo-4 AM was assessed with flow cytometry. The procoagulant potential of histone-treated RBCs was evaluated with a purified prothrombinase assay and a one-stage plasma recalcification clotting test.

Results: Natural histones induced PS exposure on RBCs in a dose-dependent manner, and neutralization or cleavage of histones by heparin or activated protein C, respectively, abolished PS externalization. H4 was mainly responsible for the stimulating activity of histones, whereas the other subtypes were almost ineffective. Similarly, natural histones and H4 induced influx of calcium into RBCs, whereas the other individual histones did not. Histone-induced exposure of PS on RBCs translated into increased prothrombinase complex-mediated prothrombin activation and accelerated fibrin formation in plasma.

Conclusions: Histones induce RBCs to express a procoagulant phenotype through the externalization of PS. This finding provides new insights into the prothrombotic activity of extracellular histones.

Keywords: blood coagulation; erythrocytes; histones; inflammation; phosphatidylserine.

Figure 1. Effect of natural histones on the exposure of phosphatidylserine (PS) on red blood cells (RBCs)

A) Typical dot plot analysis of FITC-annexin-V binding to RBCs upon treatment with buffer, ionophore A23187 (4 μM) or calf thymus histones (20 μg mL^-1); histone-stimulated RBCs were stained with FITC-Annexin-V either in the presence or in the absence of calcium, a condition achieved by chelation with EDTA (Histones/EDTA); the percentage of PS-expressing RBCs is indicated next to the drawn region. B) Percentage of PS-expressing RBCs upon stimulation with buffer, A23187 or histones (plus or minus EDTA). Results are the mean ± SEM, n=5; *, p<0.01 vs control; **, p<0.01 vs histones by paired t-test. C) PS exposure induced by increasing concentrations of calf thymus histones on RBCs; results are the mean ± SEM of 3 independent experiments; *, p<0.05 versus control by paired t-test. D) Representative dot plot analysis of FITC-annexin-V binding to RBCs incubated with histones (20 μg mL^-1) pretreated with heparin (1 IU mL^-1) or APC (6 μg mL^-1).

Figure 2. Effect of individual histones on the exposure of PS on RBCs

A) Representative dot plot analysis showing FITC-annexin-V binding to RBCs treated with 10 μg mL^-1 individual recombinant histones. B) Summary of PS externalization on RBCs induced by 10 μg mL^-1 recombinant histone subtypes; mean ± SEM, n=3; *, p<0.01 versus control by paired t-test.

Figure 3. Effect of natural histones on the expression of a procoagulant phenotype in RBCs

A) Prothrombinase activity of RBCs treated with increasing concentrations of calf thymus histones evaluated by chromogenic assay and expressed as mOD₄₀₅ min^-1 (mean ± SEM, n=3). *, p<0.05 versus control by paired t-test. B) Recalcification clotting test in plasma enriched with untreated (control) RBCs or cells stimulated by histones (40 μg mL^-1). Results are the mean ± SEM, n=4; **, p<0.01 by paired t-test.