Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation

Supplemental Digital Content is available in the text.


Transient focal middle cerebral artery occlusion with reperfusion (tMCAo/R)
As a cerebral ischemia reperfusion model, transient middle cerebral artery occlusion with reperfusion (tMCAo/R) was performed as previously reported. 3 Briefly, mice were anaesthetized with i.p. injection of ketamine (150 mg/kg) and xylazine (7.5 mg/kg) and MCA was occluded for 60 minutes (min) using a 6-nylon intraluminal filament (Doccol Corporation, Massachusetts, USA), followed by 4 h and 24 h of reperfusion. Sham-operated mice were subject to anesthesia and other surgical procedures without MCA occlusion.

Platelet and leukocyte labeling
Blood from donor mice was withdrawn in syringes prefilled with 85 mM sodium citrate, 62.2 mM citric acid, and 110 mM glucose (ACD buffer, Sigma-Aldrich, St Louis, MO). Platelet-rich plasma was obtained by centrifugation (118 × g, 8 min) before platelet isolation by centrifugation at 735 g for 10 min. Platelets were gently resuspended, counted, and labeled by using carboxyfluorescein succinimidyl ester (CFSE, 90 µM, 10 min, Sigma-Aldrich). After confirming absence of aggregates by light microscopy, 100 × 10 6 platelets in 120 µL saline were injected through the jugular vein of a recipient mouse over 5-min infusion as previously described. 3 This was followed by the continuous infusion of 0.02 % rhodamine 6G, which fluorescently labeled circulating leukocytes.

Neutrophil depletion
In some experiments, neutropenia was induced using mouse anti-neutrophil serum (ANS; 1A8; BioXCell, West Lebanon, NH, USA). ANS was administered (150 µg/mouse) i.p. 24 h before the experiment. 4 A reduction in circulating neutrophils was verified by counting neutrophils in the 5 peripheral blood before ANS injection and immediately before the experiment. ANS did not alter the number of circulating lymphocytes, monocytes, or platelets.

Neutrophil isolation and adoptive transfer
Mouse bone marrow cells were harvested by flushing marrow from femurs and tibias with RPMI-1640 medium. Neutrophils were isolated from bone marrow cells by density centrifugation with Histopaque-1077 and Histopaque-1119. The purity of isolated neutrophils was routinely > 95% as assessed by light microscopic analysis of the cells stained with Diff-Quick (Wako Pure Chemical Industries, Osaka, Japan), and > 98% viable as assessed by a trypan blue exclusion test. The neutrophils were labelled with CellTracker dye (ThermoFisher, Waltham, MA) at a final concentration of 5 µM, 5 and then treated with WRW4 (10µM. Tocris) 6 for 10 min prior to injection (5 x 10 6 ) through the jugular vein of a recipient neutropenic MCAo/R mouse. Mice were then treated with AnxA1 (1 µg/mouse) 30 min prior to intravital microscopy (IVM).
injection of ketamine (150 mg/kg) and xylazine (7.5 mg/kg). The jugular vein was cannulated for i.v. administration of rhodamine 6G and ex vivo labeled platelets. The carotid artery was cannulated with PE-10 tubing and mean arterial blood pressure (MABP) and heart rate (HR) were continuously recorded by a MacLab system (AD Instruments, Chalgrove, UK). The head of each mouse was then fixed in a plastic frame in sphinx position. The left parietal bone was exposed by a midline skin incision, followed by a craniectomy (diameter: 2.5 mm) with a drill at 1 mm posterior from the bregma and 4 mm lateral from the midline. The dura mater was not cut because 6 the fluorescent-labeled leukocytes were readily visualized. A 12 mm glass coverslip was placed over the craniectomy and the space between the glass and dura mater was filled with artificial cerebrospinal fluid (aCSF; Na + 147.8 mEq/L, K + 3.0 mEq/L, Mg 2+ 2.3 mEq/L, Ca 2+ 2.3 mEq/L, Cl − 135.2 mEq/L, HCO3 − 19.6 mEq/L, lactate − 1.67 mEq/L, phosphate 1.1 mM, and glucose 3.9 mM; all Sigma-Aldrich). A Zeiss Axioskop microscope (Zeiss, New York, USA) with a mercury lamp was used to observe the pial venules in the cerebral cortex. A Hitachi charge-coupled device color camera (model KPC571; Tokyo, Japan) acquired images that were recorded for offline analysis. One to five randomly selected vessels, 30-70 µm in diameter and 100 µm long, were observed for each mouse after treatment. Adherent leukocytes and platelets were defined as cells remaining stationary within venules for ≥30 seconds (s) or ≥2 s respectively. Platelet-leukocyte aggregates (PLAs), were defined as platelets interacting directly with adherent leukocytes on the endothelium. These parameters were expressed as the number of cells per square millimeter of the vessel surface and calculated from diameter and length, assuming cylindrical shape. Estimates of pseudoshear rate were obtained using measurements of vessel diameter (Dv) and the maximal velocity of flowing red blood cells (Vcell), according to the following formulation: pseudo-shear rate = (Vcell/1.6)/Dv × 8. 4
Slidebook software (3i) was used to drive the confocal system and capture images. 3

Systemic PNA assessment
Murine blood obtained by tail-vein bleed before and after tMCAo was mixed with heparin (20U/mL), as described previously. 8 The samples were treated with rat anti-mouse CD16/CD32 antibody to reduce non-specific antibody staining. The samples were diluted with phosphate buffered saline and then stained, followed by lysis of the red blood cells. A four-color flow cytometry assay was used to separate the PNAs: Leukocytes were labeled with rat anti-mouse CD45.2-FITC, Gr-1-PE, and F4/80-eFluor450 and isotype controls (eBioscience, San Diego, CA) while platelets were labeled with CD41-APC. CD45.2 and CD41 double-positive events were identified as a percentage of 10,000 gated leukocyte events. The percentage of neutrophil forming aggregates was calculated from the number of platelet leukocyte aggregates/µl blood divided by the number of neutrophils/µl blood x 100.

Assessment of activated murine platelets by flow cytometry
0.9 mL of whole blood was collected via carotid artery into syringe containing 0.1mL ACD buffer, transferred to an eppendorf tube, and centrifuged at 1200 rpm for 8 min. The platelet-rich plasma layer was transferred to a new eppendorf tube and centrifuged at 1200 rpm for 3 min. The Platelet rich plasma (PRP) was transferred and centrifuged at 3000 rpm for 10 min. The supernatant was discarded and the platelet pellet was resuspended in Tyrodes buffer (with 1 mM Ca 2+ ) buffer along 8 with the appropriate antibody and allowed to incubate for 15 min at 37 0 C.The reaction was stopped by the addition of 450 µL of 1% paraformaldehyde. Two-color staining of αIIbβ3 (Integrin αIIbβ3-PE (clone JON/A. BD Pharmingen, San Jose, CA, USA)) and P-selectin FITC (Emfret Analytics) were used to measure levels of activated αIIbβ3 and P-selectin expression on platelets stimulated with either with thrombin 0.1 U/1x10 6 platelets or ADP 1 µM using flow cytometry (FACS Calibur). AnxAV FITC (BD Pharmingen, San Jose, CA, USA) was also used to measure phosphatidylserine (PS) externalization. IgG isotype antibodies were used as controls. Blood samples were stimulated and stained with antibody for 15 min at RT. Platelets and platelet aggregates were identified by their light scattering using an LSRII flow cytometer and Diva8 software by assessing at least 20,000 events that were collected per sample.

Assessment of activated human platelets by flow cytometry
5 ml of whole blood obtained from healthy volunteers was collected into a 10 ml syringe containing 1mL of ACD buffer. Blood was transferred to a 15 ml conical tube and centrifuged at 300 g for 5 min. The PRP layer was transferred to a new eppendorf tube and centrifuged at 3000 rpm for 10 min. The supernatant was discarded and the platelet pellet was resuspended in Tyrode`s buffer  reaction was stopped by the addition of 450 µL of 1% paraformaldehyde. Platelets were identified by their light scattering using an LSRII flow cytometer and Diva8 software by assessing at least 20,000 events that were collected per sample.

Platelet aggregation assay
Arterial blood was freshly collected from the carotid artery of tMCAo/R-WT mice treated with either vehicle (saline) or AnxA1 into 1 ml syringe loaded with anticoagulant citrate dextrose (ACD) with 0.2 u/ml of apyrase (final concentration) (apyrase, grade VII, Sigma-Aldrich) in a 1/7 ratio. PRP was obtained immediately by centrifugation at 140 g for 8 min, and then top layer of PRP with RBC was harvested into new Eppendorf tube and centrifuged again at 60 g for 4 min to remove RBC contamination (Thermo Scientific Legend Micro 17 centrifuge). Platelets 8-10 x 10 6 /ml were used to monitor platelet aggregation velocity after agonist exposure using a laserparticle analyzer (LasCa-1C, Lumex Ltd., St. Petersburg, Russia) by low angle light scattering method as previously described 9 in 6 ml of platelet media (140 mM NaCl. 10 mM HEPES, 10 mM NaHCO3, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM D-glucose, pH 7.4). Platelets were stimulated with either 0.1 u/ml of thrombin (Sigma-Aldrich a) or ADP 1 µM (Sigma-Aldrich) and normalized velocity of aggregation was calculated using original software LasCa_32.
Thrombosis 10mg/kg of 5% FITC dextran (150,000 mol wt, Sigma-Aldrich a) was injected intravenously, allowed to circulate for 10 min before photoactivation. Photoactivation was initiated (excitation, 495 nm; emission, 519 nm) by exposing 100µm of vessel length to epi-illumination, with a 175-W xenon lamp (Lamda LS, Sutter) and a fluorescein filter cube (HQ-FITC, Chroma). 6 The excitation power density was measured daily (ILT 1700 Radiometer, SED033 detector; International Light, MA) and maintained within 1% of 0.77 W/cm 2 , as previously described. 6 Epiillumination was applied continuously, and time of blood flow cessation (≥60s duration) was recorded in both venules and arterioles (30-70µm). Epi-illumination was discontinued once blood flow ceased in the vessel under study. Typically, 2-4 thrombi were induced in each mouse, and the results of each vessel type (venules, arterioles) were averaged. 6 prepared for western blotting to detect Active Rap1 using the using rabbit-anti-Rap1 antibody (Cell Signaling). Data were quantified as Rap1-GTP/Rap1 using ImageJ. The ratio of Active Rap1/Rap1 was calculated to get the fold change in reference to the untreated control.

Western blotting
Freshly prepared platelets (1x10 7 cells) were incubated with AnxA1 (100ng/1x10 6 ) for 15 min were used as primary antibodies. Secondary antibodies used were HRP-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG. Bands were visualized by incubating antibody-labeled membranes with chemiluminescence reagent (PerkinElmer Life Sciences, Rigaweg, Groningen, Netherlands) and exposed for autoradiography. Bands were quantified using ImageJ analysis. Analysis of total Akt and p-Akt1 was quantified as a ratio of the protein of interest to a loading control to get the fold change in reference to the untreated control.

Measurement of intracellular Ca 2+ levels in Fluo-3-acetoxymethyl ester (Fluo-3 AM) loaded
platelets 5 ml of whole blood obtained from healthy volunteers was collected into a 10 ml syringe containing 1 ml of ACD buffer and PRP was obtained by centrifugation 300 x g for 5 min. Next, a platelet pellet was obtained by centrifugation at 3000 rpm for 10 min and resuspended with PBS. Washed platelets (1 x 10 6 ) were stained with Fluo-3 AM (5µM) for 30 min at 37 0 C as previously described 10 and some samples were incubated with vehicle (saline) or AnxA1 (100 ng) prior to thrombin stimulation. Fluorescent intensity of Fluo-3 AM loaded platelets was immediately recorded using BD LSR II (as previously described) 10 to determine a baseline of Fluo-3 AM loaded platelets and immediately following stimulation with human alpha thrombin (0.1 U/1x10 6 platelets). The mean fluorescent intensities of samples was analyzed using BD FACSDiva Software v 8.0.1 ) and data plotted as fluorescent arbitrary units (FAU).

Cytokines in Plasma and Brain Tissue
24 h after reperfusion, blood was collected into heparin-coated syringe, and plasma was obtained.

Infarct volume (IV)
After a 24 h reperfusion period, mice were euthanized with an overdose of anesthetic and brains were immediately removed and placed into 4°C phosphate-buffered saline (PBS, Sigma-Aldrich) for 15 min; 2 mm coronal sections were then cut with a tissue cutter. The brain sections were stained with 2 % 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate buffer at 37°C for 15 min and fixed by immersion in 10% formaldehyde. 3 The stained sections were photographed and the digitized images of each brain section (and the infarcted area) were quantified using a computerized image analysis program (NIH 1.57 Image Software).

Neurological score
The functional consequences of cerebral I/RI were evaluated by using a five-point neurological deficit score (0, no deficit; 1, failure to extend right paw; 2, circling to the right; 3, falling to the right; and 4, unable to walk spontaneously) 3 and were assessed in a blinded fashion. A maximal score of 4 could be assigned to each experimental animal.

Blood-Brain Barrier (BBB) Permeability
BBB permeability was assessed using the Evans blue (EB) extravasation, as described previously. 3 Briefly, a 2 % solution of EB (Sigma-Aldrich) was injected (4 mL/kg) i.v. 24 h later, blood was obtained for plasma collection and brains were harvested after transcardial perfusion with PBS for 5 min. The cerebral hemispheres and the plasma were homogenized in 50 % trichloroacetic acid, sonicated, and then centrifuged. The supernatant was diluted with ethanol and the concentrations of Evans blue in brain tissue and plasma were measured using a fluorescence spectrophotometer (FLUOstar Optima, BMG LABTECH, Inc. Ortenberg, Germany). BBB permeability was normalized by dividing tissue EB concentration (µg/g brain weight) by the plasma concentration (µg/mL).

Thrombin-antithrombin (TAT) complex measurement
Plasma was collected from WT and AnxA 1-/mice and TAT was measured using standard TAT complex ELISAs (Assay Pro, St. Charles, Missouri, USA). 11

Statistical Analysis
Results from IVM experiments were confirmed to follow a normal distribution using Kolmogorov-Smirnov test of normality with Dallal-Wilkinson-Lillie for corrected p value. Data that passed the WT = Wild-type mice. AnxA1 -/-= Annexin A1 knockout mice. I/R = 60 min ischemia + 24 h reperfusion. g = grams. µm = micrometer. per s = per second mmHg = millimeter of mercury.

Supplemental Figure 1. AnxA1 -/mice display an increase in stroke severity
Wild type (WT) and AnxA1 -/mice were subjected to tMCAo for 60 min, followed by 4h or 24 h reperfusion. A) Intravital fluorescence microscopy was performed to assess leukocyte adhesion in the cerebral microcirculation (pial vessels) of mice subjected to cerebral ischemia reperfusion injury (I/RI). B) Neurological score (NS) was assessed. Brains were removed to quantify C) infarct volume (IV) (representative brains slices shown from WT mice. Scale bar = 5mm) and D) blood brain barrier (BBB) dysfunction was measured in right and left hemispheres from mice using  Wild type (WT) mice were subjected to tMCAo for 60 min, followed 24 h reperfusion. Vehicle