Platelet-RBC interaction mediated by FasL/FasR induces procoagulant activity important for thrombosis
Christoph Klatt, Irena Krüger, Saskia Zey, Kim-Jürgen Krott, Martina Spelleken, Nina Sarah Gowert, Alexander Oberhuber, Lena Pfaff, Wiebke Lückstädt, Kerstin Jurk, Martin Schaller, Hadi Al-Hasani, Jürgen Schrader, Steffen Massberg, Konstantin Stark, Hubert Schelzig, Malte Kelm, Margitta Elvers
Christoph Klatt, Irena Krüger, Saskia Zey, Kim-Jürgen Krott, Martina Spelleken, Nina Sarah Gowert, Alexander Oberhuber, Lena Pfaff, Wiebke Lückstädt, Kerstin Jurk, Martin Schaller, Hadi Al-Hasani, Jürgen Schrader, Steffen Massberg, Konstantin Stark, Hubert Schelzig, Malte Kelm, Margitta Elvers
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Research Article Cell biology Vascular biology

Platelet-RBC interaction mediated by FasL/FasR induces procoagulant activity important for thrombosis

Abstract

Red blood cells (RBCs) influence rheology, and release ADP, ATP, and nitric oxide, suggesting a role for RBCs in hemostasis and thrombosis. Here, we provide evidence for a significant contribution of RBCs to thrombus formation. Anemic mice showed enhanced occlusion times upon injury of the carotid artery. A small population of RBCs was located to platelet thrombi and enhanced platelet activation by a direct cell contact via the FasL/FasR (CD95) pathway known to induce apoptosis. Activation of platelets in the presence of RBCs led to platelet FasL exposure that activated FasR on RBCs responsible for externalization of phosphatidylserine (PS) on the RBC membrane. Inhibition or genetic deletion of either FasL or FasR resulted in reduced PS exposure of RBCs and platelets, decreased thrombin generation, and reduced thrombus formation in vitro and protection against arterial thrombosis in vivo. Direct cell contacts between platelets and RBCs via FasL/FasR were shown after ligation of the inferior vena cava (IVC) and in surgical specimens of patients after thrombectomy. In a flow restriction model of the IVC, reduced thrombus formation was observed in FasL–/– mice. Taken together, our data reveal a significant contribution of RBCs to thrombosis by the FasL/FasR pathway.

Authors

Christoph Klatt, Irena Krüger, Saskia Zey, Kim-Jürgen Krott, Martina Spelleken, Nina Sarah Gowert, Alexander Oberhuber, Lena Pfaff, Wiebke Lückstädt, Kerstin Jurk, Martin Schaller, Hadi Al-Hasani, Jürgen Schrader, Steffen Massberg, Konstantin Stark, Hubert Schelzig, Malte Kelm, Margitta Elvers

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Figure 7

FasL serves as ligand for FasR to mediate platelet-induced activation of RBCs.

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FasL serves as ligand for FasR to mediate platelet-induced activation of...
(A) Exposure of FasL at the platelet membrane after incubation of platelets with ADP (10 μM) and CRP (5 μg/ml) in the absence and presence of RBCs was determined by flow cytometry (n = 4–10). MFI, mean fluorescence intensity. (B and C) Platelets stimulated with ADP (10 μM) were allowed to adhere to immobilized FasR (50 μg/ml). Representative images (B) and quantification (C) of adherent platelets in the presence of hDcR3 or control IgG-Fc (10 μg/ml). hDcR3, human decoy receptor 3. Scale bar: 50 μm. (D) No effect of hDcR3 on platelet adhesion on collagen (200 μg/ml) was observed. (n = 5). (E) Platelets were activated with ADP (10 μM), pretreated with hDcR3 (10 μg/ml), and incubated with RBCs. Effect of hDcR3 treatment on PS exposure of RBCs was determined (n = 10). (F–I) Aggregate formation of hDcR3-treated (10 μg/ml) platelets on collagen under flow using a shear rate of 1,000 s–1. Controls received IgG-Fc. (F) Representative phase-contrast (left panel) and fluorescence images with mepacrine-labeled platelets (middle panel, overlay right panel) at the end of the perfusion period. Scale bar: 50 μm. (G and H) Mean surface coverage and relative platelet deposition after treatment with hDcR3, as measured by integrated fluorescence intensity (IFI) per visual field is shown (n = 6). (I) Cells of thrombi were isolated by Accutase treatment and number of RBCs was quantified by flow cytometry (n = 6). (J) PS exposure of RBCs treated with hDcR3 or control IgG-Fc was determined by flow cytometry (n = 6). Controls were treated with IgG-Fc protein (n = 8). Data are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test (D and G–J) or 1-way ANOVA with Tukey’s multiple-comparisons test (A, C, and E). Plts, platelets.