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Keywords:

  • Gas6;
  • platelets;
  • thrombosis;
  • tyro-3

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Summary.  Gas6 (encoded by growth arrest-specific gene 6) is a vitamin-K dependent protein highly homologous to coagulation protein S that is secreted from platelet α-granules and has recently been demonstrated to participate in platelet thrombus formation. The current study evaluated the contribution of each of the three known Gas6 receptors (Axl, Sky and Mer) in human and mouse platelet function. Flow cytometry analyses confirmed that all three receptors are present on both human and mouse platelets. Pre-incubation of human platelets with either an anti-Gas6 antibody or blocking antibodies to Sky or Mer inhibited platelet aggregation and degranulation responses to both ADP and the PAR-1 activating peptide, SFLLRN, by more than 80%. In contrast, a stimulatory anti-Axl antibody increased activation responses to these agonists, suggesting a potentiating role for Gas6 in platelet activation. Moreover, in a mouse model of thrombosis, administration of Gas6 or Sky blocking antibodies resulted in a decrease in thrombus weight similar to clopidogrel but, unlike clopidogrel, produced no increase in template bleeding. Thus, Gas6 enhances platelet degranulation and aggregation responses through its known receptors, promoting platelet activation and mediating thrombus formation such that its inhibition prevents thrombosis without increasing bleeding.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Platelet activation involves a complex myriad of responses at the site of vascular injury that include platelet adhesion to the exposed constituents of the subendothelial matrix. These interactions result in platelet aggregation and formation of a platelet plug leading to the cessation of bleeding. Platelet activation results from the stimulation of one or more transmembrane receptors present on the platelet surface. Receptor activity is propagated via several overlapping signaling pathways culminating in the engagement of integrin αIIbβ3 and the manifestation of numerous other platelet activation responses. Recently, the product of the growth-arrest specific gene 6 (Gas6) has been implicated in platelet activation and thrombus development [1,2]. Gas6 knockout mice are protected from thrombotic challenge, and in vitro assays have identified Gas6 as a potential mediator of platelet function. Gas6 is a member of the vitamin-K dependent family of proteins, all of which contain several γ-carboxylated Glu residues located within the first 46 residues in the N terminus [3]. The presence of this Gla-domain in Gas6 suggests a role in cell–cell interactions or responses that require Gas6 binding to phospholipid membranes. Gas6 has been found in many cellular locations including a prominent presence in platelet α-granules [1,4] from which Gas6 is released to the extracellular milieu following platelet activation. Gas6 displays 44% sequence homology to anticoagulant protein S, but does not express similar anticoagulant activity. The ability of protein S to activate Gas6 receptors, however, remains in question [5,6].

Receptors for Gas6 belong to the Tyro3 receptor subfamily of single transmembrane tyrosine kinase receptors. These include Axl (Ufo, Ark, Tyro7), Sky (Rse, Brt, Tif, Dtk, Etk2, Tyro3) and Mer (c-Eyk, Nyk, Tyro12). These receptors share significant domain similarity, all containing two extracellular N-terminal immunoglobulin-like domains as well as two fibronectin-III-like domains followed by a tyrosine kinase domain that lies at the C-terminal cytoplasmic end of the receptors [7,8]. The relative affinities of these receptors for Gas6 vary over 100-fold, with Axl demonstrating the greatest affinity with a reported Kd = 1.0 nmol L−1[9]. Reverse transcription–polymerase chain reaction analyses indicate that human platelets contain message for all three receptors [1], suggesting that each of these may be available to signal in response to Gas6 while similar analyses using washed murine platelets indicated that only Mer was present [10]. Receptor signaling may involve the messenger proteins PI3-kinase and Akt [11,12], both of which have been shown to be responsible for mediating platelet activation responses to a variety of platelet agonists [13].

While the role of Gas6 in mouse platelet-dependent thrombus formation has been reported [1], the contributions of each of the individual Gas6 receptors to platelet activation have not been identified. This study describes both human in vitro and mouse in vivo characterization of the inhibition of Gas6 receptors. Both Sky and Mer were found to mediate Gas6-enhanced human platelet responses to ADP and to the PAR-1 activating receptor peptide, SFLLRN, and preactivation of Axl led to an enhancement of platelet activation in response to these agonists. Furthermore, the administration of antibodies to Gas6 or Sky led to a decrease in thrombus weight in a mouse model of thrombosis. However, blockade of Gas6 or Sky activation in mice did not result in an increase in bleeding, suggesting that specific inhibition of Gas6-mediated responses through Gas6 receptors represents an attractive target for antithrombotic therapy.

Reagents

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Anti-human Gas6 (sc-1935), anti-human Mer (sc-6872), and anti-human Sky (sc-1094) antibodies were used as supplied (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-human Axl (AF154), anti-human Sky (Dtk, FAB859P), isotype-matched control–phycoerythrin (PE) antibody (IC002P), and normal goat immunoglobulin G (IgG; AB-108-C) antibodies were purchased from R & D Systems (Minneapolis, MN). For the detection of murine Tyro3 family receptors, the antibodies anti-mouse Axl (AF854), anti-mouse Mer (AF591), anti-mouse Sky (AF759), and goat IgG (AB-108-C) antibodies were purchased from R & D Systems. Anti-mCD61-PE (553347) was purchased from BD Biosciences (San Jose, CA). Fluorescein isothiocyanate (FITC) -conjugated rabbit anti-goat antibodies (F0250) were used as supplied from DakoCytomation (Carpinteria, CA). Both 2-methylthio-ADP (2-MeS-ADP, Sigma, St Louis, MO) and TRAP (SFLLRN) (#H-8365, Bachem AG, King of Prussia, PA) were reconstituted following the manufacturer's directions with phosphate-buffered saline or Hepes buffer saline (HBS), respectively. ADP and Chrono-Lume® were purchased from Chrono-Log Corporation (Havertown, PA). Cell line IV.3 was purchased from the American Type Culture Collection (Manassas, VA) and the antibody IV.3 was purified using a MAbTrap kit purchased from Amersham Biosciences (Piscataway, NJ). F(ab′)2 fragments of the anti-Axl antibody (AF154) were produced using an ImmunoPure F(ab′)2 preparation kit from Pierce (Rockford, IL).

Platelet preparation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Blood was collected from healthy, drug-free adult volunteers into 5-mL vacutainer tubes containing sodium citrate as the anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifugation of whole anticoagulated blood at 190 × g for 15 min at ambient temperature. Platelet-poor plasma (PPP) was prepared by centrifugation of the remaining sample at 2100 × g for 15 min at ambient temperature. The platelet counts in PRP and PPP were determined using a Coulter counter (Model Z2, Beckman Coulter, Miami, FL). PRP was adjusted to a final platelet concentration of 3 × 108 platelets mL−1 using autologous PPP.

Platelet aggregation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Platelet aggregation was performed using PRP (3 × 108 platelets mL−1) in a siliconized cuvette with a stir bar. Aggregation was detected using a PAP-4 aggregometer (Bio/Data, Horsham, PA). Control antibody or functional antibody (20 μg mL−1 final) was added to 250 μL PRP and the sample was incubated for 5 min at 37 °C followed by the addition of ADP or TRAP at the indicated concentrations. The samples were monitored for the extent of aggregation measured at 5 min. Agonist concentrations were chosen to be either submaximal or super-maximal as determined by a dose–response curve for each agonist.

Platelet degranulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Platelet degranulation was performed as previously described [14]. Briefly, antibodies (20 μg mL−1 final) and PRP (200 μL, 3 × 108 platelets mL−1) were added to flat-bottom white polystyrene 96-well plates (Corning Life Sciences, Acton, MA). The plates were shaken on a Titer Plate Shaker (Model #4625, Laboratory-Line Instruments Inc, Melrose Park, IL) for 1 min at room temperature at speed 8 (∼1108 r.p.m). Plates were then incubated for 10 min at 37 °C followed by the addition of either 2-MeS-ADP (150 nm final) or TRAP (10 μm final) to each plate. The 2-MeS-ADP was used in these assays as commercial preparations of ADP often contain significant amounts of ATP that contaminate the assay. Plates were shaken again at speed 8 for an additional 2 min at room temperature and immediately read in an Lmax Luminescence Reader (Molecular Devices Corporation, Sunnyvale, CA) with the following specifications: 50 μL Chrono-Lume® injection, 1 second postinjection delay, and 1 second integration. Data from the functional antibodies were compared to samples treated with IgG control antibody and percentage inhibition determined.

Flow cytometry

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Whole anticoagulated blood was diluted 1 : 10 in Hanks' balanced salt solution without Ca2+ and Mg2+. For the detection of human Gas6 receptors, unlabeled goat anti-human Axl (40 μg mL−1) or goat antihuman Mer (40 μg mL−1) antibody was added and incubated for 15 min followed by the addition of anti-goat IgG-FITC (50 μg mL−1) and anti-CD42a-PE for an additional 15-minute incubation. For controls, anti-mouse IgG-FITC (50 μg mL−1) or anti-goat IgG-FITC (50 μg mL−1) was added to the sample with 20 μL of anti-CD42a-PE and incubated for 15 min at ambient temperature. For the detection of Sky, anti-Sky-PE (30 μg mL−1) was added to diluted whole blood and incubated for 15 min followed by the addition of anti-CD42a-FITC and an additional 15 min incubation. For the detection of mouse platelets, PRP was used. The antibodies anti-mouse Axl (AF854), anti-mouse Mer (AF591), anti-mouse Sky (AF759) and goat IgG (AB-108-C) were used at 30 μg mL−1. FITC-conjugated rabbit anti-goat secondary antibodies (F0250) were used as supplied. Anti-mouse-CD61-PE was used for the specific detection of mouse platelets. Both human and mouse platelets were fixed with 1 mL of 1% formaldehyde and directly analyzed on a FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA). Acquisition and processing of data from 5000 platelets were carried out with cellquest software (Becton-Dickinson, San Jose, CA).

Mouse thrombosis model

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

All methods were approved by the Pfizer Institutional Animal Care and Use Committee according to the NIH Guide for the Care and Use of Laboratory Animals. Male CD-1 mice, weighing 30–40 g (Charles River Laboratories, Portage, MI) were anesthetized with sodium pentobarbital (60 mg kg−1 induction, and 20 mg kg−1 every 20 min, intraperitoneally). Body temperature was maintained at 37 °C. Thrombus formation was induced by placing a ligature around the inferior vena cava just below the level of the kidneys for 60 min. The thrombus was then harvested, blotted on filter paper and weighed.

Doses were selected following initial experiments titrating each inhibitor, and equally efficacious doses of each agent were chosen. Vehicle control (saline, 2 mL kg−1), goat IgG control antibody (500 μg kg−1), anti-Gas6 antibody (100 μg kg−1), or anti-Sky antibody (500 μg kg−1) was given intravenously 15 min before the vena cava was ligated. Vehicle (1% methylcellulose and 0.2% Tween 80, 5 mL kg−1), or clopidogrel (20 mg kg−1 in 5 mL kg−1 of vehicle) was dosed orally 24 h before the vena cava was ligated. Each treatment group consisted of four to six animals. At the end of the study, template bleeding time was determined by making a 3-mm long by 1-mm deep incision in the lip margin with a #15 scalpel blade. The site of the incision was blotted every 15 s until no blood was observed on #2 filter paper as described previously [15].

Inhibition of platelet aggregation by anti-Gas6 antibodies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Antibodies directed against the C-terminal portion of Gas6, which is responsible for Gas6 binding to its receptors [16], were utilized to block Gas6 function. In contrast to isotype matched control antibodies, Gas6 neutralizing antibodies were found to inhibit human platelet aggregation in response to low doses of the agonists ADP and TRAP (37 ± 5% inhibition, P < 0.0001, n = 9, measured at 5 min) (Fig. 1A,B) resulting in only reversible aggregation being observed. While the initial rate of aggregation remained unaltered, the presence of anti-Gas6 antibody reduced the activation response such that reversible aggregation was observed. However, the inhibitory effect observed with Gas6 antibodies was overcome by the addition of super-maximal concentrations (75 μmol L−1 ADP or 100 μmol L−1 TRAP) of agonist (Fig. 1C,D). These data suggest a role for Gas6 in platelet activation responses at low levels of known agonists.

image

Figure 1. Effect of anti-Gas6 antibodies on human platelet aggregation. Human platelets were incubated with either isotype matched control IgG or anti-Gas6 antibodies (20 μg mL−1 each) for 5 min at 37 °C prior to stimulation with agonist. Platelet aggregation was inhibited at low doses of agonist (A) 5 μmol L−1 ADP or (B) 20 μmol L−1 TRAP (SFLLRN), whereas the inhibition was overcome with higher concentrations of agonist (C) 75 μmol L−1 ADP, or (D) 100 μmol L−1 TRAP. The extent of platelet aggregation was measured at 5 min. Data are representative of n = 9 experiments. Each label indicates duplicate tracings.

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Detection of Axl, Sky and Mer on human and mouse platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Flow cytometry was used to confirm the expression of Gas6 receptors on the surface of human platelets (Fig. 2A–C). Antibodies specific for Axl, Sky and Mer detected the presence of all three receptors on human platelets. Axl appeared to be the most abundant, however, this may be the result of a higher affinity for this antibody for its receptor. To date, conflicting data have been published by other investigators regarding the presence of Axl, Sky and Mer on murine platelets [1,10]. Therefore, we also analyzed murine platelets for the presence of these receptors. All three receptors were readily detected on the surface of murine platelets by flow cytometry (Fig. 2D–F).

image

Figure 2. Human and mouse platelets express all three Gas6 receptors Axl, Sky and Mer. Flow cytometry was used to identify the expression of Gas6 receptors on human and mouse platelets. Human whole blood, or mouse platelet-rich plasma, was incubated with anti-Axl, anti-Sky, anti-Mer, or isotype-matched control antibody and analyzed by flow cytometry. Histograms are presented for human (A) Axl (40 μg mL−1), (B) Sky (30 μg mL−1), and (C) Mer (40 μg mL−1), and mouse (D) Axl (E) Sky, and (F) Mer (30 μg mL−1 each). Data are representative of n = 3 human platelet donors, or n = 3 individual mice.

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Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

To define the role of the three receptors in platelet function, platelets were incubated with each of the receptor-specific antibodies and then stimulated with agonist. Similar to the results obtained for Gas6 blockade, antibodies directed against the receptors Sky and Mer also inhibited irreversible human platelet aggregation to ADP and TRAP [59 ± 12% inhibition (P = 0.0013) n = 6, and 58 ± 12% inhibited (P = 0.0007) n = 8, for Sky and Mer, respectively, measured at 5 min], while the initial rate of aggregation remained unchanged (Fig. 3A,B). This level of inhibition resulting from receptor blockade was overcome with higher doses of agonist. Interestingly, the level of platelet inhibition observed with anti-Sky or anti-Mer antibodies was comparable to that seen with the same concentration of anti-Gas6 antibodies (Fig. 3c), despite the receptor-directed antibodies possessing less than 2% cross-reactivity as judged by Western blotting analyses. In addition, a combination of Sky and Mer antibodies did not inhibit platelet aggregation to any greater extent than either one alone (data not shown). Thus, it appears that the receptors do not function as a redundant system and that each of the receptors may play a unique role in normal platelet function.

image

Figure 3. Inhibition of platelet aggregation by antibodies specific for Sky and Mer. Human platelets were incubated with either isotype-matched control IgG, anti-Sky, or anti-Mer antibodies (20 μg mL−1 each) for 5 min at 37 °C prior to stimulation with agonist. Inhibition of platelet aggregation induced by 5 μmol L−1 ADP was observed with both (A) anti-Sky and (B) anti-Mer antibodies. Each label indicates duplicate tracings. The level of inhibition seen with antibodies against Sky and Mer was similar to that observed with the anti-Gas6 antibody (20 μmol L−1) as shown in (C) with 20 μmol L−1 TRAP. The extent of aggregation was measured at 5 min. Data are representative of n = 6 anti-Sky and n = 8 anti-Mer experiments.

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In contrast to Sky and Mer, the antibody directed against the Gas6 receptor Axl was found to enhance, rather than inhibit, platelet aggregation [25 ± 6% stimulation (P = 0.0026), n = 6](Fig. 4A,B). The highly specific anti-Axl antibody (< 2% cross-reactivity for Sky and Mer) was stimulatory and, at a concentration of 20 μg mL−1, increased the aggregation responses induced by submaximal doses of both ADP and TRAP.

image

Figure 4. Anti-Axl antibody enhances platelet aggregation. Human platelets were incubated with either isotype-matched control IgG or anti-Axl antibody (20 μg mL−1 each) for 5 min at 37 °C prior to stimulating with agonist. Pre-treating platelets with an anti-Axl antibody increased platelet aggregation approximately 20% in response to (A) 5 μmol L−1 ADP or (B) 20 μmol L−1 TRAP. Data are representative of n = 6 experiments. The extent of aggregation was measured at 5 min. Each label indicates duplicate tracings.

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Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

ATP release from platelet dense granules was quantified to evaluate platelet secretion following treatment with antibodies directed against Gas6 and its receptors. It has been previously reported that Gas6 deficiency can inhibit ATP release from platelet dense granules induced by various agonists (ADP, collagen, U46619, thrombin) [1]. In the current study, we compared the inhibition of human platelet degranulation by Gas6 blockade with that of its receptors Axl, Sky and Mer (Fig. 5A,B). In a similar manner to the observations for platelet aggregation, antibodies directed against Sky and Mer inhibited degranulation in response to 2-MeS-ADP and TRAP. The level of inhibition was comparable to that seen with anti-Gas6 antibodies. Stimulation with super-maximal agonist concentrations (100 μmol L−1 SFLLRN or 75 μmol L−1 2-MeS-ADP) yielded similar results to control (data not shown).

image

Figure 5. The role of Gas6 and its receptors in platelet degranulation. Platelets were incubated with antibodies against Gas6, Sky, Mer, or Axl prior to stimulation with either 2-MeS-ADP or TRAP and the results compared to isotype-matched control IgG. Platelet dense granule secretion was quantified by measuring ATP release. Antibodies directed against Gas6, Sky, and Mer (20 μg mL−1 each) demonstrated similar inhibition of ATP release when stimulated by (A) 150 μmol L−1 2-MeS-ADP or (B) 10 μmol L−1 TRAP. In the presence of the anti-Axl antibody (20 μg mL−1), there was an increase in ATP release from stimulated platelets. Data include n = 3 platelet donors.

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The anti-Axl antibody enhanced platelet degranulation to these agonists, as reflected by the quantity of ATP released. However, the magnitude of stimulation was much greater than was observed in aggregation experiments. To control for the possibility that the anti-Axl antibody was enhancing platelet activation by stimulating the Fc-receptors present on the platelet surface, experiments were performed using the IV.3 antibody to block Fc-receptors. The presence of the IV.3 antibody did not significantly affect the stimulation of platelet degranulation in response to the anti-Axl antibody (data not shown). Additionally, anti-Axl F(ab′)2 fragments were generated which also showed stimulation of platelet degranulation (data not shown) indicating that the enhancing effect of the anti-Axl antibody is specific to activation of the Axl receptor present on human platelets.

Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

Mice were treated with either the antiplatelet agent clopidogrel (20 mg kg−1), an anti-Gas6 antibody (100 μg kg−1) or an antibody directed against Sky (500 μg kg−1) prior to induction of thrombus. As shown in Fig. 6(A), all three treatments significantly reduced thrombus weight. The 100 μg kg−1 dose of the anti-Gas6 antibody had an efficacy similar to that of the 20 mg kg−1 dose of clopidogrel. However, the clopidogrel treated animals had a 4-fold increase in bleeding time, whereas there was no significant difference in bleeding time between Gas6 antibody-treated mice and control mice (Fig. 6B). Mice receiving the anti-Sky antibody also exhibited a decrease in thrombus weight, but not to the same extent as that observed with the anti-Gas6 antibody. The anti-Sky antibody utilized for this study was directed against human Sky and the exact cross-reactivity with mouse Sky is unknown. In vitro experiments using mouse platelets produced only 30% of the reactivity of the anti-Sky antibody compared to human platelets (data not shown), suggesting that the decreased activity observed in vivo may be the result of low cross-reactivity of the blocking antibody used. However, it should be noted that administration of the anti-Sky antibody resulted in a statistically significant decrease in thrombus weight but did not increase bleeding time compared to control animals. There are currently no commercially available blocking antibodies for Axl and Mer that react with the mouse receptors. Thus, we were unable to evaluate the role of these two receptors in the in vivo thrombosis model.

image

Figure 6. Inhibition of Gas6 or Sky is protective in a mouse model of thrombosis without increasing bleeding. Mice were treated with either control goat IgG (500 μg kg−1, intravenous), clopidogrel (20 mg kg−1, per os), an anti-Gas6 antibody (100 μg kg−1, intravenous) or an anti-Sky antibody (500 μg kg−1, intravenous). Thrombosis was induced by ligating the inferior vena cava for 60 min, and the resulting thrombus was weighed and bleeding time was assessed. (A) Inhibition of Gas6 or Sky significantly decreased thrombus weight (*P < 0.05) similar to the results obtained with clopidogrel-treated animals. (B) Clopidogrel-treated animals exhibited significantly prolonged bleeding times (*P < 0.05) when compared to similarly efficacious doses of antibodies against Gas6 or Sky. Data are representative of n = 4 animals for each condition.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References

This study is the first demonstration that selective blockade of either of the Gas6 receptors Sky or Mer with a neutralizing antibody inhibits human platelet aggregation and degranulation. The magnitude of inhibition of each receptor individually was as great as that observed by sequestering Gas6 itself. In addition, the combination of Sky and Mer antibodies did not inhibit platelet aggregation to a greater extent than either one alone. Thus, it appears that the receptors do not function as a redundant system and that each of the receptors may play a unique role in normal platelet function. However, the role of the Gas6 receptor system in platelets may not be absolutely essential for platelet activation in that inhibition of both platelet aggregation and degranulation could be overcome by high concentrations of ADP and TRAP. Treatment with a stimulatory anti-Axl antibody enhanced human platelet aggregation and degranulation with all the tested agonists. Thus, these data suggest that the Gas6 pathway serves to amplify platelet activation signals that may lead to granule secretion and engagement of integrin αIIbβ3.

The Gas6 receptor Axl contains putative binding sites for three intracellular modulators; PI3-kinase p85 subunit, PLCγ, and Grb2 [17,18]. Each of these has been previously identified as supporting platelet degranulation and integrin engagement [19–24]. Therefore, we hypothesize that the Gas6 system supports and enhances platelet activation responses to low levels of agonist by increasing the active levels of these intracellular mediators. This would allow lower levels of known agonists to produce more robust platelet responses, whereas super-maximal concentrations of agonist would overcome any requirement of Axl, Sky, or Mer. This hypothesis is supported by the stimulatory effect of the anti-Axl antibody in both aggregation and degranulation assays of platelet activation. This antibody appears to cause the engagement of Axl specifically (< 2% cross-reactivity with Sky and Mer; data not shown). As Axl engagement will lead to the stimulation of intracellular signaling molecules, this antibody is most likely functioning to lessen the amounts of signaling mediators activated by known agonists required to elicit an activation response in platelets. The signaling mechanisms of Axl, Sky and Mer, and the means by which each participates in human platelet function, are currently being investigated in our laboratory. Although several specific antibodies were tested, we have been unable to identify an Axl-specific antibody that effectively blocks platelet activation responses. We are currently attempting to identify a negative modulator of Axl so that the specific role of this receptor can be further characterized.

The relative numbers of Gas6 receptors on human platelets are currently unknown, although the data presented in this study indicate that all three receptors are present on platelets. Axl appeared to be the most prominent of the Tyro3 receptor family on human platelets; however, the observed differences in expression could be the result of differing affinities of the various antibodies for their respective receptors. Studies to decipher the number of these receptors are currently underway. A recent report evaluating Gas6 receptors on mouse platelets indicated that Axl and Sky were absent, with only Mer detected [10]. Those results are in contrast to this study in which all three were detected, as well as to data previously published [1]. The prior studies of mouse receptor expression only reported reverse transcription–polymerase chain reaction analyses of platelet mRNA and did not analyze protein expression [10]. In addition to the detection of mouse Axl and Sky as well as Mer protein on mouse platelets by flow cytometry, our results indicate that specifically blocking the function of Sky in mice decreases thrombus weight, providing further evidence that Sky is indeed present on mouse platelets.

In this study, administration of an anti-Gas6 antibody reduced thrombus formation in mice without increasing template bleeding time. In contrast, comparable thrombus weight reduction via P2Y12 antagonism with clopidogrel was associated with a significant increase in template bleeding time. Treatment with the anti-Sky antibody also significantly reduced thrombus weight in mice, but not to the same magnitude as the anti-Gas6 antibody treatment. The smaller antithrombotic effect of Sky blockade in vivo may be the result of reduced reactivity of the anti-human Sky antibody to mouse Sky. PRP prepared from mice treated with the anti-human Sky antibody also showed a reduction in platelet aggregation to 5 μmol L−1 ADP that paralleled the in vivo antithrombotic response (30% vs. 33%, respectively, data not shown).

Further evidence that Gas6 may be involved in atherothromobotic disease was suggested by a recent report linking human Gas6 allelic variation with the incidence of stroke. In a small trial, the variance in the frequency of a single nucleotide polymorphism, c.834 + 7G[RIGHTWARDS ARROW]A in intron 8, of the Gas6 gene was associated with stroke incidence [25]. Analysis of the genotypic frequencies showed a statistically significant difference between the stroke patients and controls, with a lower prevalence of the AA homozygous genotype in patients than in controls. These studies provide further evidence to support the theory that modulation of Gas6 activity, possibly through its receptors, may indeed provide an important intervention point for future antithrombotic therapies.

Platelet-selective inhibition of Gas6 signaling may prove problematic given the wide distribution of Gas6 receptors across brain [26], testis [27], kidney [28], vasculature [29] and hemopoietic [30] tissues. However, an attractive property of the Gas6 system from a pharmacological standpoint is that platelets from Gas6-knockout mice are reported to exhibit normal expression of the Gas6 receptors Axl and Sky. This suggests that chronic inhibition should not produce a compensatory up-regulation of these receptors in platelets.

In conclusion, our results show that inhibition of Sky or Mer is sufficient to inhibit platelet degranulation and aggregation. In addition, the enhancement of platelet activation to known agonists by Axl stimulation further suggests a distinct role for Gas6 in platelet function. Inhibition of the Gas6 pathway using blocking antibodies to either Gas6 or Sky confers protection against thrombosis in a mouse model, but does not significantly increase template bleeding. This lack of increased bleeding in the treated mouse model suggests that blockade of the Gas6 system may be both an efficacious and safe means of anticoagulant therapy. These results demonstrate that the Gas6 pathway may be a promising new pharmacological target for the treatment of thrombosis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Reagents
  6. Platelet preparation
  7. Platelet aggregation
  8. Platelet degranulation
  9. Flow cytometry
  10. Mouse thrombosis model
  11. Statistical analyses
  12. Results
  13. Inhibition of platelet aggregation by anti-Gas6 antibodies
  14. Detection of Axl, Sky and Mer on human and mouse platelets
  15. Effects of antibodies specific for the Gas6 receptors Axl, Sky and Mer on human platelet aggregation
  16. Effects of antibodies against the Gas6 receptors Axl, Sky and Mer on platelet degranulation
  17. Inhibition of Gas6 and Sky is protective against thrombosis without increasing bleeding
  18. Discussion
  19. References
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