P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries (original) (raw)
Generation of P2Y12–/– mice. The P2Y12 targeting vector was prepared from genomic clones containing the mouse P2Y12 gene. A bacterial artificial chromosome (BAC) clone containing the murine P2Y12 (m_P2Y12_) sequence was amplified by PCR screening of a mouse 129/Sv library (Incyte Genomics Inc., St. Louis, Missouri, USA) using primers derived from the coding sequence of m_P2Y12_. A 15-kb HindIII fragment was identified from the mouse BAC clone, which contained the m_P2Y12_ locus. Using PCR primers, a 5′ HindIII site and a 3′ Xba site were introduced to generate a 4-kb fragment just upstream of the ATG initiation codon for mP2Y12. Similarly, a XhoI site and NotI site were introduced by PCR to generate a 6-kb 3′ arm. Both of these fragments were subcloned into the pLNL vector such that they flanked a neomycin resistance gene (neo) (Figure 1a). The vector was linearized with PvuI and introduced into the ES cell line E14Tg2a (derived from 129/Ola) by electroporation, and neomycin-resistant clones were selected in media containing G418. DNA was isolated from surviving colonies, digested with XbaI, and assessed by Southern blot analysis using a probe corresponding to a DNA sequence located downstream of the targeting construct. Chimeric mice were generated with P2Y12-targeted ES cell lines and were bred to C57BL/6 mice. Mice with the targeted mutation were identified by Southern blot analysis or PCR and intercrossed with siblings that inherited the WT 129 P2Y12 allele. Control and experimental mice were then obtained by intercross of the heterozygous mice derived from those sibling intercrosses.
Targeting the P2Y12 locus. (a) The neomycin resistance gene in pLNL is flanked by a 4-kb (5′) and 6-kb (3′) fragment from the P2Y12 genomic locus. HindIII, XbaI, XhoI, and NotI sites were introduced by PCR. The WT P2Y12 coding exon is denoted as ORF. ORF, open reading frame.(b) Southern hybridizations of XbaI-digested mouse genomic DNA from progeny of breeding pairs heterozygous at the mutant allele. The XbaI fragment detected by the indicated probe (gray oval in Figure 1a) is reduced from 9.1 to 8.3 kb due to introduction of a new XbaI site in the targeted locus. Intro, introduction.
Hemostatic parameters. Aggregation using platelet-rich plasma (PRP) was measured turbidimetrically using a Chronolog aggregation analyzer (Chronolog Corp., Havertown, Pennsylvania, USA) following calibration with platelet-poor plasma, at a stirring speed of 800 rpm. Aliquots (250 μl) of PRP were placed in cuvettes containing magnetic stirrer bars, warmed at 37°C, and stirred for 1 minute to obtain a stable baseline. Aggregation was induced by either ADP (0.5 or 10 μM>), collagen (1 or 20 μg/ml), or murine TRAP (mTRAP) (AYPGKF; 1 or 2.5 mM), and change in light transmittance was recorded for 6 minutes. Inhibition of cyclooxygenase by ASA was confirmed by obtaining mouse PRP 2 hours after ASA treatment and measuring aggregation induced by 500 μM arachidonic acid (Cayman Chemical Inc., Ann Arbor, Michigan, USA).
Mice used for bleeding time were sex-matched littermates of heterozygous breeding pairs of a mixed 129/Sv and C57BL/6J background. Genotyping was performed after the bleeding time study by an investigator blinded to the experiment. Male and female progeny (10–13 weeks) were anesthetized (by subcutaneous injection) with ketamine cocktail (ketamine [75 mg/kg; Fort Dodge Animal Health, Fort Dodge, Iowa, USA)], xylazine [8 mg/kg; Vetus, Burns Veterinary Supply Inc., Westbury,New York, USA], and acepromazine [1.5 mg/kg; Vetus]) 6 minutes prior to tail transection. Mice were laid in lateral recumbency on a firm surface with the tail straight out. Tails were transected 2 mm from the tip with a number 10 scalpel blade (Bard-Parker; Becton Dickinson, Franklin Lakes, New Jersey, USA) and immediately immersed into a 20-ml scintillation vial filled with normal saline held at 37°C. A stopwatch was started immediately upon transection to determine time to cessation of bleeding or 15 minutes, whichever occurred first. If bleeding did not reoccur within 60 seconds of cessation, bleeding was considered stopped.
Determination of cAMP. Platelet cAMP levels were measured using the cAMP-Screen ELISA System (Perkin-Elmer Applied Biosystems, Foster City, California, USA). Following incubation with 500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, Missouri, USA) for 10 minutes at 37°C, mouse washed platelets (2.5 × 108/ml, prepared as previously described) (13) were treated with 10 μM ADP (Sigma-Aldrich) or 0.001, 0.1, or 10 μM epinephrine (Burns Veterinary Supply, Farmers Branch, Texas, USA) in the presence of 50 μM forskolin (Sigma-Aldrich) for 10 minutes. Reactions were terminated, processed, and quantitated according to manufacturer’s instructions.
Intravital microscopy. Blood sampling and platelet preparation were performed as described previously with minor modifications (14). Blood (approximately 800 μl per mouse) was collected in polypropylene tubes containing 140 μl of acid-citrate-dextrose (38 mM citric acid, 75 mM trisodium citrate, and 100 mM dextrose), 400 μl of washing buffer (129 mM NaCl, 13.6 mM trisodium citrate, 11.1 mM dextrose, 1.6 mM KH2PO4, 0.02 U/ml apyrase, pH 6.8), and 1 μM PGE1 (Sigma-Aldrich). PRP was obtained by centrifugation at 220 g for 7 minutes. The plasma and buffy coat were gently transferred to a fresh polypropylene tube containing 0.7 ml of the washing buffer and centrifuged at 160 g for 5 minutes. The same operation was repeated once in order to pellet contaminating leukocytes and red blood cells. The supernatant was transferred to two polypropylene tubes containing 4 ml of washing buffer. After centrifugation at 2,000 g for 12 minutes, the platelet pellet was resuspended in resuspension buffer (137 mM NaCl, 4 mM KCL, 0.5 mM MgCl2, 0.5 mM sodium phosphate, 11.1 mM dextrose, 0.1% BSA, 10 mM HEPES, pH 7.4). Platelets were fluorescently labeled with calcein AM (0.25 μg/ml; Molecular Probes Inc., Eugene, Oregon, USA) for 20 minutes at RT and infused (5 × 109 platelets/kg) through the tail vein.
In some experiments, ASA was administered in WT and P2Y12+/– mice 2 hours before FeCl3-induced injury. Epinephrine (blood volume of the recipient mouse [13–17 g] estimated at 1 ml), was injected 15 minutes before injury at 1, 10, and 50 μM in WT and P2Y12–/– mice. In another experiment, 30 minutes before 50 μM epinephrine infusion, WT and P2Y12–/– mice were administered the GP IIb-IIIa inhibitor CT51464 (15) by oral gavage (50 mg/kg). Immediately after infusion of fluorescent platelets into mice of matching genotype, mice were anesthetized and mesenteric arteries (85–150 μm diameter) were studied. Vessels with a shear rate of 1,000–1,400 per second were selected using an Optical Doppler Velocimeter (Texas A&M University, System Health Science Center, Cardiovascular Research Institute, College Station, Texas, USA). Arteries were visualized using a Carl Zeiss (Oberkochen, Germany) Axiovert S100 inverted microscope (objective ×20) equipped with a 250-W HBO fluorescent lamp source (Opti Quip, Highland Mills, New York, USA) with a narrow-band FITC filter set (Chroma Technology Corp., Brattleboro, Vermont, USA) and a Hamamatsu (Tokyo, Japan) chilled CCD C5985 intensified camera connected to a VHS video recorder (Sony SVO-9500MD). One artery per mouse was filmed for 2 minutes before vessel wall injury. Vessel wall injury was generated using a modified FeCl3-induced thrombosis protocol. A 2 × 1–mm filter paper was immersed into a 12.5% FeCl3 solution for 2 minutes and then placed over the artery for 7 minutes. After 7 minutes, the paper was removed and the vessel covered with NaCl at 37°C. Platelet vessel wall interactions were then recorded for 33 minutes or until full occlusion occurred and lasted for more than 20 seconds.
Video analysis. Platelet vessel wall interactions and thrombus formation were analyzed in real time using Simple PCI software (Compix Inc., Imaging System, Cranberry Township, Pennsylvania, USA). In summary, the mean fluorescent intensity (which reflects the kinetics of the thrombotic process) at the site of vessel wall injury was recorded on 2 frames per second for 40 minutes and plotted automatically versus time. Each experiment was simultaneously recorded on videotape and analyzed by the computer. Hence, this software allows continuous monitoring of the thrombotic process for each animal.
Perfusion chamber experiments. The murine ex vivo perfusion chamber protocol has been described previously (16). Briefly, non-anticoagulated blood was collected from the vena cava of anesthetized mice and perfused for 2.5 minutes through either fibrinogen-coated (500 μg/ml in saline solution incubated for 2 hours at RT; Sigma-Aldrich) or human type III collagen-coated (Sigma-Aldrich) capillary chambers. Capillary chambers with a diameter of 345 μm were used to establish a shear rate of 871/s (flow rate of 212 μl/min) (17). To study binding of murine GP Ibα to human vWF (18, 19), washed platelets (3 × 108/ml supplemented with 1 mM CaCl2) from WT or P2Y12-deficient mice were incubated with 6 μg/ml botrocetin (19), with or without 10 μM epinephrine, and perfused through human vWF-coated (100 μg/ml incubated at 4°C overnight; kind gift of Zaverio Ruggeri, The Scripps Research Institute, La Jolla, California, USA) capillary chambers at 871/s for 4 minutes. For perfusion over fibrinogen and vWF, the number of platelets present in a 300 × 150–μm window was determined using Simple PCI software. For perfusion over collagen, P2Y12–/– mice were infused intravenously with 10 mg/kg ASA (Aspegic; Sanofi-Synthelabo Inc., Malvern, Pennsylvania, USA) 2 hours before blood perfusion. Epinephrine (50 μM) was injected in ASA-treated P2Y12–/– mice 10 minutes before ex vivo blood perfusion. Mean thrombus volume (micrometer cubed per micrometer squared, corresponding to the mean area of all thrombi divided by their mean length of adhesion) was quantified on semithin cross sections and by mean gray level measurements at 5 mm from the proximal part of the capillary using Simple PCI software.
Immunohistological staining of WT and P2Y12–/– murine thrombi formed ex vivo. Rectangular capillaries (0.3 × 0.3 mm; VitroCom, Mountain Lakes, New Jersey, USA) were coated with human type III collagen and perfused with non-anticoagulated blood (collected from the vena cava) for 2.5 minutes at 220 μl/min (840/s). Immediately after, capillaries were rinsed for 15 seconds, fixed in 2% paraformaldehyde at 4°C for 1 hour, rinsed three times with PBS/0.5% BSA, then incubated for 45 minutes either with FITC-conjugated anti-mouse P-selectin (1:25 dilution; PharMingen, San Diego, California, USA) or goat anti-human Gas6 (100 μg/ml; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), followed by TRITC-conjugated rabbit anti-goat Ab (1:1,000 dilution; Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA). In some experiments, thrombi were fixed for 30 minutes, incubated for 1 hour with 1 mg/ml Alexa 488-fibrinogen (Molecular Probes Inc.), and rinsed four times with PBS/0.5% BSA.
FACS study of the level of GP IIb-IIIa activation. Washed platelets (± 1 mM CaCl2) from WT and P2Y12–/– mice were incubated for 1 hour at 37°C with either saline, 0.6 mM murine TRAP (SynPep Corp., Dublin, California, USA), or 5 μM ADP, in the presence or absence of 1, 10, or 50 μM epinephrine and 200 μg/ml Alexa 488-fibrinogen (Molecular Probes Inc.). Analysis of platelet-bound Alexa 488-fibrinogen was performed using a FACSort flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, California, USA).
All procedures conformed to institutional guidelines and to the Guide for the Care and Use of Laboratory Animals (The NIH, Bethesda, Maryland, USA).
Statistical analysis. Bleeding time values are expressed as mean plus or minus SEM. The effect of various concentrations of epinephrine in vivo as well as the bleeding time study were tested by one-way ANOVA and comparison analyzed using Dunnett multiple comparison tests. Statistical significance of the difference between two groups of data was tested by student t tests.