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 3

RBCs support occlusive thrombus formation ex vivo and in vivo.

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RBCs support occlusive thrombus formation ex vivo and in vivo. (A) Flow ...
(A) Flow chamber system T-TAS. Whole blood and PRP were loaded into the flow chamber at a shear rate of 600 s–1 at 37°C. Bar graphs show full occlusion of the capillary (n = 6). (B) Representative images of whole blood and PRP are shown. Blood flow direction was from left to right. (C) Whole blood and PRP were measured until full occlusion of the capillary. When flow pressure increased to 80 kPa, blood perfusion stopped and the capillary was almost completely occluded by thrombi. (D) Human PRP and RBCs (4 × 106) were perfused through the flow chamber. Where indicated, RBCs were substituted with fixed RBCs and occlusion of the capillary was monitored. (E) Bleeding times of control and anemic mice after amputating the tail tip. The time from the incision to the cessation of bleeding was recorded (no blood flow for 1 minute). (F) Time to initiation of thrombus formation (left) and time to occlusion (right) were measured with an ultrasonic flow probe in mice where the carotid artery was injured by topical application of FeCl3 (n = 5–6). (G) After injury of the carotid artery, thrombi were isolated and stained with hematoxylin and eosin to visualize RBCs within the thrombus. Representative images of control mice (upper panel, same control sample but different detail as shown in Figure 2D) and anemic mice (middle and lower panel) are shown. Scale bars: 50 μm (upper and middle panel) and 20 μm (lower panel). (H) Bleeding times of mice that received RBC injections to increase the hematocrit were analyzed. (I) Time to occlusion in mice with increased hematocrit was determined after injury of the carotid artery with FeCl3 (n = 9–10). HCT+, mice with increased hematocrit. Data are the mean ± SEM. *P < 0.05, **P < 0.01 by Student’s t test.