Histones Induce Endothelial Von Willebrand Factor Release and Subsequent Platelet Capture in In Vitro and In Vivo Models

Abstract Thrombosis is the leading cause of mortality worldwide and can result from uncontrolled immune activation in inflammatory conditions. Von Willebrand factor (VWF) is an acute phase glycoprotein that mediates primary hemostasis by binding to platelets. It is stored in platelets and in Weibel-Palade bodies (WPB) in endothelial cells. Cell-free DNA (CF-DNA) and histones are released during cell damage and have been shown to stimulate innate immune and pro-thrombotic responses including, in the case of histones, thrombocytopenia in mice and VWF release due to platelet activation/aggregation. We recently demonstrated the correlation between VWF and CF-DNA plasma levels in human and mouse models of chronic inflammation associated with aging. In these studies we investigate the influence of CF-DNA and histones on VWF release and platelet-binding in murine and endothelial cell models. C57BL6 mice received a retro-orbital injection of unfractionated (UFH), lysine-rich (H1) or arginine-rich (H3/H4) calf thymus histone (20 mg/kg), calf thymus DNA (4 mg/kg) or saline and blood was collected 1 hr post-injection from the inferior vena cava into buffered citrate. Platelet count was quantified from whole blood via a complete blood count. Blood-outgrowth endothelial cells (BOEC) from normal individuals were stimulated with UFH, H1, H3/H4, DNA and DNA/histone combinations (25 µg/mL) for 75 min. VWF and ANG-2 (stored exclusively in WPB) was quantified from plasma and BOEC media by ELISA and results expressed as fold-change ± SD. To visualize the influence of histone on platelet binding to BOEC surface associated VWF, BOEC were seeded onto collagen coated flow chamber slides. BOEC were stimulated for 20 min. with UFH (50 µg/mL) or histamine (25µM). Washed platelets were labelled with DiOC6 and flowed at a shear rate of 500s-1 over BOEC for 10 min. VWF-platelet strings (≥ 3 consecutive platelets) were quantified from still images obtained post-flow. In C57BL6 mice, VWF:Ag levels were significantly increased by UFH infusion (1.31±0.17, p=0.0015, n=8) but not DNA (1.09±0.19, p=0.20, n=10) relative to saline controls (1.00±0.16, n=14). H1 (1.64±0.20, n=5) mediated a greater VWF release than H3/H4 (1.21±0.20, p=0.01, n=6) and UFH (p=0.006). As previously described, we observed that UFH (0.74±0.08, p=0.0005), H1 (0.73±0.09, p=0.0011) and H3/H4 (0.87±0.14, p=0.08) induced thrombocytopenia in normal mice. ANG-2 levels were also significantly higher in H3/H4 (1.38±0.20, p=0.004) than saline (1.00±0.20) treated mice. Mice receiving UFH (1.25±0.31, p=0.13) and H1 (1.36±0.33, p=0.07) also had elevated ANG-2 levels although not statistically significant. Taken together this data suggests that both platelet and endothelial cells may contribute to VWF release upon histone-treatment. In vitro treatment of BOEC with H1 induced greater VWF (2.13±0.61, p=0.004) and ANG-2 (2.66±0.60, p=0.004) release as compared to the unstimulated condition (1.00±0.36). Interestingly, when BOEC were stimulated with UFH and DNA together we observed an increase in ANG-2 release (1.39±0.23, n=4, p=0.02) compared to unstimulated BOEC, but not in VWF:Ag when the cells were treated with each stimulus alone. As histones have been shown to bind to VWF, we hypothesize that these results could be explained by an impaired detection of VWF that is partially mitigated by the addition of DNA to the system. Using the in vitro flow chamber model, we observed significantly more VWF-platelet strings formed when cells were pre-incubated with UFH (5.51±0.97 strings, p=0.002) than when not stimulated (0.72±0.77 strings) (Figure 1). The ability of histones to activate platelets may also contribute to VWF-platelet string formation. Histones, but not DNA, stimulate the release of VWF from endothelial cells and platelets and facilitate platelet capture by endothelial VWF in a flow chamber model. As CF-DNA may serve as a surrogate marker for plasma histone levels, the correlation between CF-DNA and VWF:Ag we have previously observed in chronic inflammatory conditions, may be related in part to histone-induced VWF release from platelet and endothelial cell stores. These experiments demonstrate that histones induce VWF release in vivo and suggest a novel connection between the innate immune and hemostatic systems. Figure 1 Representative images taken after flow experiment. Arrows indicate VWF-platelet strings. Graph is expressed in mean ± SD (**p=0.0025). Figure 1. Representative images taken after flow experiment. Arrows indicate VWF-platelet strings. Graph is expressed in mean ± SD (**p=0.0025). Disclosures No relevant conflicts of interest to declare.

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