Coumarin

Reagents—The α_IIbβ₃ antagonist lotrafiban was supplied by GlaxoSmithKline (King of Prussia, PA). The anti-Rac (23A8) monoclonal antibody was purchased from Upstate Biotechnology (TCS Biologicals, Bucks, UK). Anti-Rac2 polyclonal antibody and anti-Rac3 polyclonal antibody were generously provided from Gary Bokoch (Scripps Institute, La Jolla, CA) and Ivan de Curtis (San Rafaele Scientific Institute, Milan, Italy), respectively. The cDNA for the GST-CRIB domain of PAK1 prepared as described previously (21 (link)) and the active form of Rac (L61Rac) were the kind gifts from Dr. Doreen Cantrell (Imperial Cancer Research Fund, London, UK). D-Phenyl-alanyl-1-prolyl-1 arginine chloromethyl ketone was purchased from Calbiochem. Fibrinogen depleted of plasminogen, VWF, and fibronectin was from Kordia Laboratory Supplies, Leiden, Netherlands. VWF was a generous gift from Michael C. Berndt (Monash University, Clayton, Australia). All other reagents were from Sigma or previously named sources (22 (link), 23 (link)).
Preparation of Human Washed Platelets—Human venous blood was drawn by venipuncture from healthy volunteers into sodium citrate and acid/citrate/dextrose as described previously (23 (link)). Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 200 × g for 20 min. The platelets were then isolated from PRP by centrifugation at 1000 × g for 10 min in the presence of prostacyclin (0.1 μg/ml). The pellet was resuspended in modified HEPES/Tyrodes buffer (in mM: 129 NaCl, 0.34 Na₂HPO₄, 2.9 KCl, 12 NaHCO₃, 20 HEPES, 5 glucose, 1 MgCl₂; pH 7.3) containing 0.1 μg/ml prostacyclin. The platelets were washed once via centrifugation (1000 × g for 10 min) and resuspended at the desired concentration with HEPES/Tyrode buffer.
Preparation of Murine Washed Platelets—The generation of mice bearing a conditional loxP-flanked allele of Rac1, Rac1^flox, has been described previously (6 (link)). To induce expression of the Mx1-Cre trans-gene, the mice were given a 150-μl intraperitoneal injection of synthetic double-stranded RNA polyinosinic-polycytidylic acid (2 mg/ml) every other day for a total of three injections, and blood was taken at least 14 days after the last injection to ensure a complete turnover of platelets. The protein expression of Rac1 and Rac2 was verified for both control and Rac-deficient mice for each experiment (data not shown). The number of platelets in whole blood from Rac1^-/-, Rac2^-/-, or Rac1^-/-Rac2^-/- mice was no different from wild type (data not shown). Bleeding problems, such as the intraperitoneal hemorrhage seen in Syk- and SLP-76-deficient mice, were not observed for these mice.
Murine blood was drawn from CO₂terminally anesthetized mice by cardiac puncture and taken into 100 μl of acid/citrate/dextrose. PRP was obtained by centrifugation at 200 χ g for 6 min. Washed platelets were prepared via centrifugation of PRP at 1000 × g in the presence of prostacyclin (0.1 μg/ml) for 6 min. The pellet was resuspended in modified HEPES/Tyrode buffer to the desired platelet level. All animals were maintained using housing and husbandry in accordance with local and national legal regulations.
In separate experiments, human or murine platelet suspensions were treated with 10 μM cytochalasin D, 0.1-10 μg/ml CRP, 1-10 μM ADP, 0.04-1 units/ml thrombin, 10 μM lotrafiban or 1 mM adenosine 3′,5′-diphosphate (A3P5P), and 1 μM AR-C67085 for 10-30 min before use in the assays. Unless otherwise stated, all experiments were performed in the presence of 2 units/ml apyrase and 10 μM indomethacin and in the absence of exogenously added Ca²⁺.
Measurement of Rac Activity—Rac activity was measured essentially as described in Pearce et al. (18 (link)) using the CRIB domain of PAK1 (amino acids 67-150), which binds the GTP-bound form of Rac. Following stimulation of platelet suspensions (3 × 10⁸/ml), reactions were stopped with an equal volume of 2× lysis buffer (2% (v/v) Nonidet P-40, 2% (w/v) N-octyl glucoside, 300 mM NaCl, 20 mM Tris/HCl, 2 mM EGTA, 20 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, pH 7.4, and 2 mM orthovana-date). Alternatively, platelets (3 × 10⁸/ml) were incubated for 10-45 min in dishes coated with fibrinogen, collagen, laminin, or BSA²in the absence or presence of thrombin (1 units/ml) and apyrase (2 units/ml). Unbound platelets were removed by two washes with phosphate-buffered saline followed by aspiration, and adherent cells were solubilized with 1× lysis buffer. A sample of the suspension over BSA was taken and used as a control. Insoluble material was then removed by centrifugation (5 min, 13,000 rpm), and GST-PAK1, previously incubated with glutathione agarose beads, stored in glycerol at -80 °C, and washed with 1× lysis buffer, was added to the lysates and incubated for 1 h at 4 °C. Beads were then washed with 1× lysis buffer, and the bound protein was taken up into Laemmli buffer. The resulting samples were separated by 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with a Rac-specific antibody and horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). Protein was detected using ECL (Amersham Biosciences).
Measurement of Filamentous Actin Content—Filamentous actin content of washed platelets was measured using a modification of the method of Machesky and Hall (24 (link)). Basal or activated platelets (2 × 10⁸/ml) were fixed with an equal volume of 3.7% formaldehyde containing a saturating amount of FITC-phalloidin (20 mM KH₂PO₄, 10 mM Pipes, 5 mM EGTA, 2 mM MgCl₂, 0.1% Triton X-100, 3.7% formalde-hyde, 2 μM FITC-phalloidin) and incubated for1hat room temperature on a nutator. The platelets were then pelleted for 2 min in a microcentrifuge, and pellets were washed in 0.1% saponin, 20 mM KH₂PO₄, 10 mM Pipes, 5 mM EGTA, 2 mM MgCl₂. Pellets were then resuspended in methanol to extract the FITC-phalloidin and incubated for 1 h on a nutator at room temperature. FITC-phalloidin binding was measured for each sample with the fluorescence emission at 520 nm and excitation at 488 nm. Alternatively, F-actin levels were assessed via flow cytometry after fixation, permeabilization, and staining of platelets with FITCphalloidin as described previously (18 (link)). Filamentous actin content was expressed as a comparison with values obtained for untreated cells processed in parallel on the same day.
Platelet Aggregation and Shape Change—A quantity of 300 μl of PRP or washed platelets (2 × 10⁸/ml) was used for aggregation. Stimulation of platelets was performed in a PAP-4 aggregometer (Bio/Data Corp., Horsham, PA) with continuous stirring at 1200 rpm at 37 °C for the times shown. Aggregation of platelets was monitored by measuring changes in light transmission.
Static Adhesion Assays—Coverslips were incubated with a suspension of fibrinogen (100 μg/ml), collagen (100 μg/ml), or laminin (50 μg/ml) overnight at 4 °C. Surfaces were then blocked with denatured BSA (5 mg/ml) for 1 h at room temperature followed by subsequent washing with phosphate-buffered saline before use in spreading assays. Quiescent platelets failed to bind to surfaces coated with denatured BSA (data not shown).
Platelet spreading (2 × 10⁷/ml) was imaged in real time using Köhler illuminated Nomarski differential interference contrast optics with a Zeiss 63× oil immersion 1.40 NA plan-apochromat lens on a Zeiss Axiovert 200M microscope. Time-lapse events were captured by a Hamamatsu Orca 285 cooled digital camera (Cairn Research, Kent, UK) using Slidebook 4.0 (Intelligent Imaging Innovations, Inc., Denver, CO). To compute the length and thickness of filopodia and surface area of spreading platelets, images were manually outlined and quantitated by determining the number of pixels within each outline using a Java plugin for the Image J software package as described previously (23 (link)). Imaging a graticule under the same conditions allowed the conversion of pixels size to microns.
Flow Adhesion Studies—For flow adhesion studies using collagen, mouse blood was drawn into sodium heparin (10 IU/ml) and D-phenylalanyl-1-prolyl-1 arginine chloromethyl ketone (40 μM). Alternatively, mouse blood was drawn into sodium citrate (0.38% w/v) for immobilized VWF adhesion studies. Glass capillary tubes (Camlab, Cambridge, UK) were coated with 100 μg/ml type I collagen from equine tendon (Horm, Nycomed, Munich, Germany) or 100 μg/ml VWF, 100 units/ml thrombin for1hat room temperature. The capillaries were washed and blocked with phosphate-buffered saline containing 5 mg/ml BSA for 1 h at room temperature before being mounted on the stage of an inverted microscope (DM IRB; Leica, Milton Keynes, UK). Anticoagulated whole blood was perfused through the chamber for 4 min at a wall shear rate of 1000 s^-1, followed by washing for 3 min at the same shear rate with modified Tyrodes buffer before being fixed with 3.7% paraformaldehyde for 30 min and imaged using DIC microscopy. In selected experiments, fixed samples were incubated overnight with the fluorescent dye DiOC₆(2 μM; Molecular Probes Inc., Eugene, Oregon) before being imaged using confocal microscopy (DM IRE2; Leica, Milton Keynes, UK). In separate experiments, whole blood was fluorescently labeled with DiOC₆(2 μM, 10 min at 37 °C), and the accumulation of DiOC₆-labeled platelets was monitored in real time using fluorescence microscopy (CoolSnap ES, Photometrics, Huntington Beach, CA).
Image analysis was performed off-line using ImageJ. Platelet adhesion results are expressed as the percentage of surface area covered by platelets.
Laser-induced Vessel Wall Injury—All procedures were undertaken with approval from the United Kingdom Home Office in accordance with the Animals (Scientific Procedures) Act of 1986. Male mice were anesthetized with ketamine (100 mg/kg Vetalar; Amersham Biosciences and Upjohn Ltd., UK) and 2% xylazine (20 mg/kg; Millpledge Pharmaceuticals, UK). The cremaster, a transparent muscle surrounding the testicle, was exteriorized and continuously superfused with a bicarbonate-buffered saline (36 °C) aerated with 5% CO₂, 95% N₂. High speed intravital microscopy experiments were performed as described previously by Falati et al. (25 (link)). Arterioles with a diameter of 25-35 μm were selected for study, and endothelial injury was induced using a pulsed nitrogen dye laser (coumarin 440 nm) focused on the luminal surface. To label platelets fluorescently, 20 μl of Alexa Fluor 488 conjugated to goat anti-rat antibody (Molecular Probes, Eugene, OR) and 5 μl of rat anti-mouse CD41 antibody (Pharmingen) were added to 70 μl of saline and infused via the carotid cannula. Multiple thrombi in each cremaster preparation were generated upstream to previous injuries in the same or similar sized arterioles. The background fluorescence intensity, predominantly because of freely circulating platelets, was determined and subtracted from the fluorescence intensity of the developing thrombus. The resulting value was multiplied by the sum of all pixels above background to give a value for integrated intensity at each time point. This integrated intensity value was directly proportional to the size of the developing thrombus and when plotted against time provided a graph that illustrated the dynamic kinetics of platelet accumulation.
Analysis of Data—Experiments were carried out on at least three occasions, and images shown are representative data from one experiment. Where applicable, results are shown as mean ± S.E. Statistical significance of differences between the means was determined by analysis of variance. If the means were shown to be significantly different, multiple comparisons were performed by the Tukey test. Probability values of p < 0.01 were selected to be statistically significant.

The Zn²⁺-HDAC8 variants were used for crystallographic studies. A new crystal form of HDAC8 was discovered for the HDAC8-TSA and HDAC8-APHA complexes using minor modifications of previously published conditions (18 (link)). The inhibitors TSA and APHA were purchased from Sigma and used without further purification. Briefly, a 4 μL hanging drop of 5 mg/mL HDAC8 in 50 mM Tris (pH 8.0), 150 mM KCl, 5% glycerol, 1 mM dithiothreitol, and 2 mM inhibitor was mixed with a 4 μL drop of precipitant buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.3), 1–5 % polyethylene glycol (PEG) 6000, 2 mM Tris-2-carboxyethylphosphine (TCEP)) and a 0.4 μL drop of 0.3 M Gly-Gly-Gly, and was equilibrated against a 600 μL reservoir of precipitant buffer at room temperature. The final pH in the crystallization drop was 5.8. Large plate-like crystals appeared within 1 – 5 days and grew to typical dimensions of 300 × 150 × 50 μm³. Crystals were harvested and cryoprotected in 25 mM Tris-HCl, 50 mM MES (pH 5.8), 75 mM KCl, 0.5 mM TCEP, 50 μM inhibitor, and 8% or 20% polyethylene glycol (PEG) 6000 for the APHA and TSA complexes, respectively, or 10% or 30% glycerol for the TSA and APHA complexes, respectively.
The D101 HDAC8 variants were similarly crystallized and cryoprotected with slight modification of the precipitant buffer solution. Specifically, the D101A, D101L, D101N, and D101E variants complexed with M344 (purchased from Sigma) crystallized with 1–5% PEG 6000, PEG monomethyl ether 550, PEG 35,000, and PEG dimethyl ether 2,000, respectively. Crystals were cryoprotected in the same buffer with a final glycerol concentration of 30% and 50 μM inhibitor. The H143A variant complexed with an acetylated tetrapeptide substrate (N-acetyl-arginine-histidine-acetyllysine-acetyllysine-coumarin) was crystallized using a precipitant buffer of 50 mM Tris-HCl (pH 8.0), 50 mM MgCl₂, 150 mM KCl, 13% PEG 6000, 2 mM TCEP, and 3.2 mM substrate. Crystals were subsequently transferred to a cryoprotectant buffer of 50 mM Tris-HCl (pH 8.0), 25 mM MgCl₂, 75 mM KCl, 20% PEG 6000, 1 mM TCEP, 20% glycerol, and 50 μM substrate as described previously for Y306F HDAC8 (25 (link)).
Diffraction data were measured on beamline F1 at the Cornell High Energy Synchrotron Source (CHESS, Ithaca, NY) for crystals of the HDAC8-TSA complex, and beamlines XL12-B and X29 at the Brookhaven National Synchrotron Light Source (NSLS, Brookhaven, NY) for crystals of the HDAC8-APHA and D101E HDAC8 variant-M344 complexes. Diffraction data for the remaining D101 HDAC8 variant-M344 complexes and the H143A HDAC8-substrate complex were collected at beamlines ID-24C/E at the Advanced Photon Source, Northeastern Collaborative Access Team (APS, NECAT, Argonne, Il). Crystal and data collection statistics are recorded in Table 1.
Data were indexed and merged using HKL2000 (28 ) and MOSFLM (29 ). Molecular replacement calculations were performed with AMoRe (30 ) using the atomic coordinates of an inhibited form of HDAC8 less inhibitor and solvent atoms (PDB accession code 1W22) (18 (link)) as a search probe for rotation and translation functions for the HDAC8-TSA complex. This refined solution was used for molecular replacement with the HDAC8-APHA complex. Similarly, molecular replacement calculations for the D101 HDAC8 variant-M344 complexes and the H143A HDAC8-substrate complex were performed with AMoRe (30 ) or PHASER (31 ) using the structure of wild-type HDAC8 minus ligand and solvent atoms as a search probe (PDB accession codes 1T67 and 2V5W, respectively) (17 (link), 25 (link)). Iterative cycles of refinement and model building were performed using CNS (32 (link)) and O (33 (link)), respectively, in order to improve each structure as guided by R_free values. Strict noncrystallographic symmetry (NCS) restraints were initially used during the first few cycles of refinement of each complex and relaxed into appropriately-weighted restraints early in refinement. In the HDAC8-TSA and HDAC8-APHA complexes, residues M1–Q12 at the N-termini of all monomers, residues A32-K33 of monomer A, and residues G86-E95 of monomer C of the HDAC8-TSA complex appeared to be disordered and were excluded from the final model. For the HDAC8-APHA complex, residues Q84 to E106 of monomer C appeared to be disordered and were excluded from the final model (APHA was not observed to bind to monomer C). For the HDAC8-D101 variants complexed with M344, disordered regions correspond to those previously identified for the wild-type enzyme (17 (link)). Finally, residues M1-S13 in all monomers of the H143A HDAC8-substrate complex are disordered and excluded from the final model. All refinement statistics are recorded in Table 1.