Hepatic sinusoidal endothelium avidly binds platelets in an integrin-dependent manner, leading to platelet and endothelial activation and leukocyte recruitment

Abstract

Platelets have recently been shown to drive liver injury in murine models of viral hepatitis and promote liver regeneration through the release of serotonin. Despite their emerging role in inflammatory liver disease, little is known about the mechanisms by which platelets bind to the hepatic vasculature. Therefore, we referenced public expression data to determine the profile of potential adhesive receptors expressed by hepatic endothelium. We then used a combination of tissue-binding and flow-based endothelial-binding adhesion assays to show that resting platelets bind to human hepatic sinusoidal endothelial cells and that the magnitude of adhesion is greatly enhanced by thrombin-induced platelet activation. Adhesion was mediated by the integrins Gp1b, αIIbβIII, and αvβ3, as well as immobilized fibrinogen. Platelet binding to hepatic endothelial cells resulted in NF-κB activation and increased chemokine secretion. The functional relevance of platelet binding was confirmed by experiments that showed markedly increased binding of neutrophils and lymphocytes to hepatic endothelial cells under shear conditions replicating those found in the hepatic sinusoid, which was in part dependent on P-selectin expression. Thus the ability of platelets to activate endothelium and promote leukocyte adhesion may reflect an additional mechanism through which they promote liver injury.

Fig. 1.

Resting platelets bind to vessels in human liver tissue sections. A and B: data from Stamper-Woodruff adhesion assays used to detect binding of exogenously added platelets to tissue. Adherent platelets were fluorescently labeled with antibody raised against CD41 to visualize binding. 2 representative fields of view captured using fluorescent microscopy with Axiovision software (Zeiss) are shown. Original magnification ×400. C: representative images from Stamper-Woodruff adhesion assays using normal and primary biliary cirrhosis (PBC) liver specimens. Binding of platelets was visualized by indirect immunohistochemical labeling of antibody raised against CD41 and diaminobenzidine substrate. Sections were counterstained with hematoxylin, and adherent platelets are stained brown. 2 representative fields of view from each donor captured using bright-field microscopy with Axiovision software (Zeiss) are shown. Original magnification ×400.

Fig. 2.

Adherent platelets localize to vascular structures in liver and bind to normal and diseased tissue. A: data from Stamper-Woodruff adhesion assays used to detect binding of exogenously added platelets to tissue. Adherent platelets were fluorescently labeled with antibody raised against CD41 to visualize binding. The figure shows representative fluorescent microscopic images from 3 experiments performed using indicated tissue samples from different donors. B: 6 random nonoverlapping views per sample centered on vascular structures at ×200 magnification were quantified using threshold analysis and calculation of % area occupied by platelets with ImageJ software (rsbweb.nih.gov/ij/). Data represent means ± SE % area coverage with platelets on indicated liver types per high-powered field (HPF). ANOVA indicated no significant difference in amount of platelet binding to normal and PBC tissue, whereas adhesion to regrafted tissue was significantly reduced (**P < 0.01 for both).

Fig. 3.

Platelet adhesion to liver tissue sections is integrin dependent and is inhibited by pretreatment with fibrinogen-binding peptide. A and B: representative fields of platelet binding to normal liver in the absence (control) or presence of indicated concentrations of either arg-gly-asp (RGD) peptide (A) or Fb-binding peptide (B). C: quantitative data generated using ImageJ to calculate % area of HPF occupied by platelets in 4 fields of view from 3 different normal liver samples. Data represent means ± SE % area coverage, and ANOVA indicated significant inhibition in the presence of RGD peptide and the highest dose of FB-binding peptide (*P < 0.05, **P < 0.01).

Fig. 4.

Resting and activated platelets bind to isolated, cultured endothelial cells under static conditions. Platelet binding was assessed using phase-contrast microscopy. At least 10 fields of view were counted to calculate the average number of platelets binding per endothelial cell (Platelet/EC ratio). A: data generated experiments where resting platelets were allowed to bind to hepatic sinusoidal endothelial cells (HSEC) or human umbilical vein endothelial cells (HUVEC) for up to 75 min. ANOVA revealed significantly increased binding to HSEC vs. HUVEC (**P < 0.001). B: adhesion of both resting and thrombin activated (0.5 U/ml thrombin pretreatment) platelets to HSEC and ANOVA indicated a significant stimulatory effect (***P < 0.0001). All data are means ± SE of 3 replicate experiments.

Fig. 5.

Integrin-dependent binding of platelets bind to resting endothelial cells under conditions of shear stress. Platelets were perfused over endothelial cells for 5 min at a wall shear stress of 0.1 Pa. Nonadherent platelets were then washed free of the system before assessment of the platelet/EC ratio as before. A: binding of resting platelets from matched donors under static or flow conditions. Data represent means ± SD of 2 replicate experiments with different platelet donors. B: binding of platelets per endothelial cell expressed as a percentage of control binding. Platelets were perfused over endothelial cells for 5 min at a wall shear stress of 0.1 Pa in the absence or presence of fibrinogen-binding peptide (100 μg/ml, Fg peptide) or blocking monoclonal antibodies raised against αIIbβ3 integrin (10 μg/ml), Gp1b (10 μg/ml), or αvβ3 integrin (av Block, 10 μg/ml). Data represent means ± SE of 4 replicate experiments. All reagents significantly inhibited adhesion compared with untreated controls (ANOVA, *P < 0.05 or **P < 0.01).

Fig. 6.

Binding of platelets to HSEC induces activation of NF-κB and release of CCL2 and CXCL8 and promotes P-selectin-dependent adhesion of neutrophils and lymphocytes. A: colorimetric quantification of NF-κB activation was performed using a commercially available ELISA (NF-κB p50 Transfactor Kit, Clontech) according to manufacturer's instructions. Nuclear extracts were generated from endothelial cells (2 independent donors) following stimulation with platelets (1 × 108/ml, 10 min) or TNF-α (10 ng/ml, 30 min) or unstimulated controls. Binding of NF-κB p50 to consensus or scrambled (mut) DNA was quantified. A competitive inhibitor supplied in the kit (platelets+inhibitor) was used to confirm specificity of signal. Data represent means ± SD absorbance at 655 nm. Statistical analysis was not performed, as samples from only 2 donors were tested. B: production of CXCL8 and CCL2 was determined using commercially available ELISA kits (R+D Systems HS800 and DCP00, respectively) according to manufacturer's instructions. Supernatants were collected from unstimulated EC or cells incubated with indicated numbers of platelets for 4 h. Data represent means ± SE chemokine production (pg/ml) in triplicate samples with 2 HSEC donors. *P < 0.05, **P < 0.01. C and D: platelets were perfused over endothelial cells for 5 min at a wall shear stress of 0.1 Pa. Nonadherent platelets were then washed free of the system before perfusion of 1 × 10⁶/ml freshly isolated neutrophils (PMN) or peripheral blood lymphocytes (PBL) for 5 min (C). Leukocyte adhesion was counted and expressed as adherent cells/mm²/10⁶ cells perfused on EC alone (EC+) or EC with preimmobilized platelets (EC+Plat). Data represent means ± SE from 4 replicate experiments with independent blood donors, and paired t-test indicated significant enhancement of neutrophil adhesion to HSEC in the presence of platelets **P < 0.01. D: binding of neutrophils to endothelium alone (control) endothelium and immobilized platelets (platelets) or endothelium and immobilized platelets pretreated with P-selectin inhibitor (P-selectin block, KF 38789, 10 μM). Paired t-test indicated significant enhancement of neutrophil adhesion to HSEC in the presence of platelets and significant inhibition by KF38789, ***P < 0.001 for both.