Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy

Abstract

Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing "outside-in" signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the beta3 integrin, thereby amplifying outside-in signaling via alphaIIbbeta3. Blocking the Gas6-R-alphaIIbbeta3 integrin cross-talk might be a novel approach to the reduction of thrombosis.

Figure 1

Effect of the lack of Tyro3, Axl, or Mer on hemostasis. After 2-mm tail-tip transection, the cut end of the tail was immersed in saline at 37–C. Aliquots of saline solution containing blood were sampled every minute for 10 minutes and then every 5 minutes until 30 minutes. The amount of blood loss was determined by measurement of the hemoglobin content of the aliquots of blood collected in saline (mean ± SEM, n = 10). In WT mice, blood loss reached a plateau within 10 minutes, indicating cessation of bleeding. In contrast, Tyro3^–/–, Axl^–/–, and Mer^–/– mice had a tendency to repetitively rebleed after transient hemostasis.

Figure 2

Loss of Tyro3, Axl, or Mer protects mice against thrombosis. (A) Stasis-induced thrombosis in the inferior vena cava (mean ± SEM, n = 10). *P < 0.005. (B) Thromboembolism induced by collagen/epinephrine injection in WT (n = 15) and in Tyro3^–/– (n = 15), Axl^–/– (n = 10), and Mer^–/– (n = 12) mice. *P < 0.005. (C and D) Light microscopy (H&E staining) of the lungs after collagen/epinephrine injection, revealing extensive platelet thromboemboli (arrows) in WT mice (C) but not in surviving Axl^–/– mice (D) or Tyro3^–/– or Mer^–/– mice (not shown). Scale bars: 100 μm.

Figure 3

Effect of the lack of Tyro3, Axl, or Mer on aggregation of WT, Tyro3^–/–, Axl^–/–, or Mer^–/– PRP. (A) Response of WT or Tyro3^–/– platelets to 5 μM and 50 μM ADP. (Similar results were obtained with Axl^–/– and Mer^–/– platelets; data not shown.) (B) Response of WT or Axl^–/– platelets to 2 μg/ml and 10 μg/ml collagen. (Similar results were obtained with Tyro3^–/– and Mer^–/– platelets; data not shown.) (C) Response of WT or Mer^–/– platelets to 1 μM and 7 μM TXA₂ analogue U46619. (Similar results were obtained with Tyro3^–/– and Axl^–/– platelets; data not shown.) (D) Aggregation response to ADP (5 μM) of washed human platelets after preincubation with human Axl extracellular domain (hAxl-EC-Fc) or control IgG, revealing that hAxl-EC-Fc reduces platelet aggregation. A representative example of 3 independent experiments is shown. Arrows in A–D indicate addition of the platelet agonists.

Figure 4

Effect of the lack of Tyro3, Axl, or Mer on clot retraction. Photographs show the degree of clot retraction after 60, 90, and 120 minutes in WT, Tyro3^–/–, Axl^–/–, and Mer^–/– PRP samples treated with 10 IU/ml thrombin. Tyro3^–/–, Axl^–/–, and Mer^–/– PRP was slower than WT PRP in retracting clots (n = 3). Clots are surrounded by dotted lines.

Figure 5

Effect of the lack of Tyro3, Axl, or Mer on fibrinogen binding. Loss of 1 Gas6-R (results shown for Axl^–/– mice) does not inhibit fibrinogen binding to α_IIbβ₃ integrin. The binding of labeled fibrinogen to platelets activated with ADP (1.25–20 μM) was measured by flow cytometry. Black lines denote resting platelets; red shading denotes ADP-stimulated platelets. A representative example of 4 independent experiments is shown.

Figure 6

Delayed Tyro3^–/–, Axl^–/–, or Mer^–/– platelet spreading on immobilized fibrinogen. WT or Mer^–/– platelets (similar results were obtained with Tyro3^–/– and Axl^–/– platelets) were incubated on a fibrinogen-coated surface for 15 minutes (A and B), 30 minutes (C and D), and 60 minutes (E and F) without agonist and stained with rhodamine-phalloidin to visualize actin. A representative example of 3 independent experiments is shown. Field width: 50 μm.

Figure 7

Effect of Axl deficiency on platelet spreading time and spreading area. (A) Spreading time after adhesion, recorded by video microscopy, was longer in Axl^–/– than in WT platelets. Field width: 6 μm. (B) Platelet area measured using Openlab 3.0.6 software (Improvision) was smaller in Axl^–/– than in WT mice.

Figure 8

Effect of the absence of Tyro3, Axl, or Mer on P-selectin expression, a marker of α granule secretion. Analysis by flow cytometry revealed that P-selectin levels on the platelet surface were significantly reduced in Tyro3^–/–, Axl^–/–, or Mer^–/– platelets, after activation with ADP. Black lines denote resting platelets; red shading denotes ADP-stimulated platelets. A representative example of 3 independent experiments is shown.

Figure 9

Gas6 signaling through its receptors Tyro3, Axl, and Mer. (A) Gas6 promotes phosphorylation of its receptors (shown for Axl), PI3K, Akt, and β₃ integrin in WT but not in Tyro3^–/–, Axl^–/– (not shown), or Mer^–/– (not shown) platelets. Platelets were incubated with 400 ng/ml human recombinant Gas6 (hrGas6) for 3 minutes. For detection of Axl or PI3K phosphorylation, platelets were lysed and phosphotyrosine-containing proteins were immunoprecipitated. The precipitates were then separated by SDS-PAGE and Western-blotted (WB) with anti-Axl or anti-PI3K antibodies. For Akt and β₃ integrin phosphorylation studies, lysed platelets in sample buffer were subjected to SDS-PAGE and Western-blotted with anti–phospho-Akt antibody, anti–total Akt antibody, anti–β₃ integrin [pY⁷⁷³] phosphospecific antibody, or anti–β₃ integrin antibody. (B) β₃ Integrin levels measured by flow cytometry were comparable on the surface of WT and Tyro3^–/–, Axl^–/–, or Mer^–/– platelets (only Tyro3^–/– data are shown). Black lines denote controls; red shading denotes platelets stained with PE-conjugated anti–β₃ integrin antibody. A representative example of 3 independent experiments is shown. (C) β₃ Integrin tyrosine phosphorylation in response to thrombin in WT and Tyro3^–/– platelets. Platelets were stimulated with increasing concentrations of thrombin for 3 minutes. A representative example of 3 independent experiments is shown.

Figure 10

Effect of the absence of Tyro3, Axl, or Mer on Gas6 binding. Analysis by flow cytometry revealed that binding of myc-tagged recombinant Gas6 to resting platelets was significantly lower in Tyro3^–/–, Axl^–/–, or Mer^–/– than in WT platelets. Black lines denote negative control (PBS); red shading denotes binding of myc-tagged Gas6, detected by FITC-conjugated anti–myc-tag antibody. A representative example of 3 independent experiments is shown.

Figure 11

Axl expression on the surface of WT, Tyro3^–/–, Axl^–/–, or Mer^–/– platelets, activated by the PAR4-activating peptide AYPGKF (0.25 mM). Flow cytometry revealed that surface expression of Axl was dramatically reduced in platelets lacking any 1 of the Gas6-Rs as compared with WT platelets. Axl expression was not detected on the surface of activated Axl^–/– platelets. Black lines denote isotype-matched control; red shading denotes Axl. A representative example of 3 independent experiments is shown.

Figure 12

Akt phosphorylation in response to thrombin in WT and Mer^–/– platelets. (Similar results were obtained with Tyro3^–/– and Axl^–/– platelets; data not shown.) (A) Platelets were stimulated with increasing concentrations of thrombin for 3 minutes. (B) Time course of 1 IU/ml thrombin. A representative example of 3 independent experiments is shown.