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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Vascular Medicine and
Pharmacology, "G.Bizzozero" Laboratory of Blood and Vascular Cell
Interactions, Istituto di Ricerche Farmacologiche Mario Negri,
Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy; and
Università Cattolica del Sacro Cuore, Centro di Ricerca e
Formazione ad Alta Tecnologia nelle Scienze Biomediche, Campobasso,
Italy.
Adhesion of polymorphonuclear leukocytes (PMNLs) to
activated platelets requires a P-selectin-triggered, tyrosine
kinase-dependent adhesiveness of Mac-1 and is accompanied by tyrosine
phosphorylation of a 110-kd protein (P-110) in PMNLs.
Inhibitors of SRC tyrosine kinases were found to inhibit PMNL adhesion
to activated platelets or to P-selectin expressing Chinese hamster
ovary (CHO-P) cells and the tyrosine phosphorylation of P-110. Adhesion
of PMNLs to activated platelets or to CHO-P cells stimulated activity
of LYN and HCK. Monoclonal antibody blockade of P-selectin or
Polymorphonuclear leukocyte (PMNL)-platelet
interactions occur at sites of vascular injury, and inflammatory and
thrombotic states are associated with circulating PMNL-platelet
aggregates. Recent studies in patients and in experimental animal
models suggest that PMNL-platelet aggregates may play a role in the
vascular response to injury that occurs after erosion or rupture of an atherosclerotic plaque or during coronary angioplasty or stent placement.1 The interaction of PMNLs with activated
platelets is coordinated by an adhesion cascade in which P-selectin
binds to P-selectin glycoprotein ligand-1 (PSGL-1)2,4 on
leukocytes to promote the initial tethering of the cells. Subsequent
firm adhesion is mediated by integrin The ability of the Tyrosine kinases belonging to the SRC family have been implicated in
the outside-in signaling that follows In the present study, we demonstrate a crucial role of the SRC family,
most probably LYN and HCK, in the regulation of Mac-1 adhesiveness
triggered by P-selectin. Moreover, our results support the concept that
the SRC kinase-dependent signaling initiates from the integrin itself
and regulates Mac-1-cytoskeleton interaction and Mac-1 clustering at
the site of cell-cell contact. This study highlights a new role of SRC
kinases in a regulatory loop by which Mac-1 promotes its own function.
Chemicals
Antibodies
Preparation of polymorphonuclear leukocytes, platelets, and culture of CHO-P cells Blood was collected from healthy volunteers who had not received any medication for at least 2 weeks. PMNLs and platelets were isolated as previously reported.8 After they were washed in HEPES-Tyrode buffer containing 2 µM PGE1 and 5 mM EGTA, platelets were stimulated with 0.5 U/mL thrombin for 2 minutes at room temperature and fixed with 1% PFA at room temperature for 1 hour. CHO cells stably transfected with the cDNA encoding for human P-selectin were kindly provided by Genetics Institute (Cambridge, MA) and cultured as previously reported.29 Immediately before the experiments, CHO-P cells were detached by incubating the monolayer with 5 mM EGTA and 5 mM EDTA for 10 minutes, washed twice in HEPES-Tyrode, and resuspended in the same buffer at a concentration of 107/mL.Experimental conditions All the experiments were performed under the following standard conditions: cells were incubated in a final volume of 500 µL in siliconized glass tubes (internal diameter, 6 mm; ChronoLog; Mascia Brunelli, Milan, Italy), and the tubes were placed in an aggregometer (Platelet Ionized Calcium Aggregometer; Mascia Brunelli) at 37°C with stirring (1000 rpm) obtained by an iron bar (4 mm long) rotated under a magnetic field. Although the shear rate produced by this stirring speed cannot be precisely quantified, it should approximate 250 seconds 1.30 PMNLs were incubated in standard
conditions alone or with platelets (1:5 or 1:10 ratio) or CHO-P cells
(5:1 ratio) or 10 µg/mL P-selectin-IgG chimera. Given that
P-selectin-IgG chimera contains the Fc portion of the human IgG, PMNLs
were preincubated for 10 minutes with 50 µg/mL nonimmune human IgG,
before stimulation with P-selectin-IgG chimera, to exclude a possible
effect of this portion mediated by the Fc receptor on PMNLs. For PSGL-1
cross-linking experiments, PMNLs were preincubated at 4°C for 15 minutes with 20 µg/mL anti-PSGL-1 antibody PL2, rapidly centrifuged,
and incubated in standard conditions with 10 µg/mL rabbit
anti-mouse IgG F(ab')2 fragment.
For blocking studies, antibodies were preincubated at saturating concentration (20 µg/mL) with the desired cell fraction for 15 minutes at 4°C. PMNLs were preincubated with tyrosine kinase inhibitors for 1 minute at room temperature or with cytochalasin D for 2 minutes at room temperature. For adhesion experiments, PMNLs were stained with the vital red fluorescent dye HE (20 µg/mL per 5 × 107 PMNL/mL) for 30 minutes at 4°C. Platelets and CHO-P cells were loaded with the green fluorescent dye BCECF by incubating PRP or CHO-P cell suspensions with 2 µg/mL acetoxymethyl ester (BCECF-AM) for 30 minutes at 37°C. The formation of PMNLs platelets and PMNLs-CHO-P cells mixed conjugates was evaluated by double-color flow cytometry, as previously described.8 Percentages of PMNLs binding platelets (PMNL (+) %) and of CHO-P cells binding PMNLs (CHO-P (+) %) were reported. In vitro kinase assay PMNLs were pretreated with 2 mM di-isopropyl fluorophosphate for 30 minutes at 4°C and washed with HEPES-Tyrode buffer before incubation with platelets (1:5 ratio) or CHO-P cells (5:1 ratio) in standard conditions. Samples were immediately centrifuged at 200g for 10 minutes at 4°C. Cell lysis was performed by adding to the cell pellet RIPA (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate) or Triton buffer (1% Triton X-100, 25 mM Tris-HCl, pH 7.4, 37.5 mM NaCl) containing protease and phosphatase inhibitors (1 mM EDTA, 5 µg/mL aprotinin, leupeptin and pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 10 µM PAO, 200 µM sodium orthovanadate, and 1 mM dithiothreitol). After 1 hour at 4°C, lysates were clarified by centrifugation, adjusted to the same protein concentration (300 µg for FGR and LYN and 600 µg for HCK) with lysis buffer, and precleared with anti-mouse rabbit IgG bound to immobilized Protein A on Trysacryl (Pierce).Immunoprecipitations were performed by incubating the samples for 3 hours at 4°C with 10 µL protein A, previously preadsorbed with
specific anti-LYN, anti-HCK, or anti-FGR antibodies. Immune complexes
were washed and divided in half. One part was resuspended in kinase
buffer (20 mM HEPES, pH 7.5, 10 mM MnCl2, 1 mM DTT) containing 10 µCi [ Isolation of polymorphonuclear leukocyte cytoskeleton Samples were lysed for 10 minutes at 4°C with 500µL of 2× cytoskeleton stabilization buffer (CSK)31 as follows: 50 mM PIPES pH 6.9, 8 M glycerol, 2 mM MgCl2, 0.4% Triton X-100, 4 mM EGTA, 4 mM EDTA, 4 mM sodium orthovanadate, 2 mM PMSF, 20 µg/mL aprotinin, 20 µg/mL leupeptin, 10 µg/mL pepstatin A, and 20 µM PAO. Then cell lysates were centrifuged for 10 minutes at 14 000 rpm in Eppendorf microfuge, and pellets (corresponding to Triton X-100-insoluble fractions) were washed once with 1× CSK, resuspended with RIPA buffer (30 minutes at 4°C) and added with the same volumes of 2× Laemmli boiling buffer containing protease and phosphatase inhibitors. Amounts of Triton X-100-insoluble fraction corresponding to 0.5 × 106 PMNLs were subjected to 6% SDS-PAGE and transferred to nitrocellulose sheets, and CD18 was analyzed by the anti-CD18 monoclonal antibody KIM 127 (0.1 µg/mL; 1 hour at room temperature) followed by goat anti-mouse IgG peroxidase conjugate.Tyrosine phosphorylation experiments PMNLs were incubated alone or with platelets or CHO-P cells, as described for adhesion experiments. The reaction was stopped by adding 1 volume of 2× reducing Laemmli buffer. Samples were boiled for 10 minutes and centrifuged for 10 minutes at 7000g. Then 100 µL aliquots, corresponding to 1.25 × 106 total PMNL lysate, were loaded into 7.5% to 12.5% gradient SDS polyacrylamide gels and transferred onto nitrocellulose sheets, and the tyrosine-phosphorylated proteins were revealed with the recombinant horseradish peroxidase-conjugated antiphosphotyrosine antibody RC20 (0.1 µg/mL, 30 minutes at 37°C). Detection was performed by chemiluminescence using the ECL kit, and bands were visualized by autoradiography. Western blot analysis of lysates of platelets and CHO-P cells alone indicated that tyrosine-phosphorylated proteins, including P-110, found in mixed PMNLs-platelets and PMNLs-CHO-P cells lysates were derived from PMNLs. This was further supported by the evidence that the pattern of protein tyrosine phosphorylation in PMNLs-platelets, PMNLs-CHO-P cells, and PMNLs challenged with soluble P-selectin was overimposable (not shown).Confocal microscopy Samples were incubated with 5 µg/mL anti-CD11b antibody OKM10 for 30 minutes at 4°C and fixed with 2% PFA. After they were washed, samples were treated for 15 minutes at 4°C with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 50 mM NH4Cl, to quench autofluorescence and to block nonspecific interactions, and they were incubated for 30 minutes at 4°C with 20 µg/mL goat anti-mouse Alexa Fluor 488 IgG antibody. In samples in which PMNL aggregation was induced by antibody-mediated PSGL-1 engagement, Mac-1 was stained by FITC-conjugated mAb 44 before fixation. After Mac-1 staining, PMNLs were then permeabilized with PBS containing 0.05% saponin and 0.5% BSA for 30 minutes at 4°C, and filamentous actin (F-actin) was stained with 1 µg/mL rhodamine-phalloidin in PBS containing 0.01% saponin and 0.5% BSA for 30 minutes at 4°C. Samples were resuspended in Mowiol, plated on a glass coverslip, and observed with a LSM 510 laser scanning microscope equipped with Axiovert 100 M-BP (Zeiss, Milano, Italy). Optical Z-sections from each sample were taken with 0.3 µm Z-step from the top to the bottom of the cell.
Activity of tyrosine kinases belonging to the SRC family is required for polymorphonuclear leukocyte adhesion to P-selectin-expressing cells and for P-110 phosphorylation We previously reported that the adhesion of PMNLs to activated platelets requires a P-selectin-triggered, tyrosine kinase-dependent adhesiveness of Mac-1 and is accompanied by tyrosine phosphorylation of P-110 in PMNL.2 To identify the tyrosine kinases involved in the regulation of Mac-1 adhesiveness stimulated by P-selectin, specific inhibitors of different classes of tyrosine kinases, which have already been implicated in the regulation of integrin function in leukocytes,31-34 were tested for their ability to prevent PMNL adhesion to activated platelets. Inhibitors of SRC tyrosine kinases PP1 and PP235 inhibited the formation of mixed PMNL-platelet conjugates in a concentration-dependent manner with the IC50 of 17.2 and 3.8 µM, respectively, whereas the inactive analog PP3 was ineffective (Figure 1A). In contrast, the Jak-2 tyrosine kinase inhibitor tyrphostin AG 490,36 the Syk inhibitor piceatannol,37 and the MEK inhibitor PD9805938 did not significantly modify PMNL-platelet adhesion (Figure 1B). Likewise, only SRC kinase inhibitors prevented the tyrosine phosphorylation of the P-110 protein (Figure 1C). This indicates that SRC tyrosine kinase activity may be required for Mac-1 adhesiveness triggered by P-selectin.
To confirm this hypothesis, CHO-P cells or a soluble recombinant
P-selectin-IgG chimera was used to stimulate
Polymorphonuclear leukocyte adhesion to platelets or CHO-P cells induces a CD18-dependent activation of LYN and HCK PMNLs express the SRC tyrosine kinases LYN, HCK, and FGR.15 To determine the activity of these enzymes, we immunoprecipitated the kinases and measured their autophosphorylation using an in vitro kinase assay. A rapid stimulation of the activity of LYN and HCK occurred in PMNLs adherent to activated platelets or CHO-P cells (Figure 3A,B). Western blot analysis with biotinylated anti-LYN or anti-HCK antibodies showed that equal amounts of both kinases were immunoprecipitated in the samples. Lysates of fixed platelets or CHO-P cells alone did not show any detectable LYN and HCK kinase activity (data not shown). The activity of FGR was not consistently stimulated after PMNL coincubation with platelets or CHO-P cells (data not shown). mAbs to either P-selectin or 2-integrins reduced the activation of both LYN and HCK in PMNLs
mixed with activated platelets or CHO-P cells (Figure 3C,D),
indicating that P-selectin and 2-integrin-mediated events are
necessary for the activation of SRC tyrosine kinases.
P-selectin triggers CD18 redistribution to Triton X-100-insoluble cytoskeletal fraction and colocalization of Mac-1 clusters with F-actin patches at the site of cell-cell contact: role of SRC tyrosine kinases SRC tyrosine kinases are essential for actin cytoskeletal re-organization39 and may modulate integrin-cytoskeleton interactions,18 which, in turn, regulate2-integrin
adhesiveness.20,40 We investigated whether SRC tyrosine
kinases promote the adhesive function of Mac-1 triggered by P-selectin
through the regulation of integrin-cytoskeleton interaction and
integrin distribution on the PMNL membrane. The association of
2-integrins with the Triton X-100-insoluble cytoskeletal fraction
was examined in PMNLs incubated alone, in mixed PMNL-platelet
aggregates, and in PMNL aggregates induced by soluble P-selectin-IgG
chimera. A rapid increase in the amount of CD18 in the Triton
X-100-insoluble fraction occurred when platelets were added to PMNLs
(Figure 4A). The redistribution of CD18
to the Triton X-100-insoluble fraction was maximal at 30 seconds and
declined thereafter, reflecting the kinetics of PMNL-platelet conjugate
formation (data not shown and reference 2). Exposure of PMNLs to
P-selectin-IgG chimera also resulted in a rapid association of CD18
with the cytoskeleton (Figure 4B). Western blot analysis using the
specific anti-CD11b mAb OKM10 confirmed that Mac-1 was redistributed to
the Triton X-100-insoluble cytoskeleton in PMNLs aggregated by
P-selectin (data not shown).
To determine whether association with the Triton X-100-insoluble cytoskeleton correlated with surface redistribution, PMNLs were dual labeled with mAb OKM10 to CD11b and rhodamine phalloidin to detect F-actin and were examined by confocal fluorescence microscopy. In resting PMNLs (Figure 4C), CD11b was almost evenly distributed over the PMNL surface (i), and F-actin was homogeneously distributed inside the cell (ii). In PMNL aggregates induced by P-selectin-IgG chimera, large clusters of CD11b were localized at the site of cell-cell contact (v) and were superimposed on or surrounded by F-actin patches (iv and vi). The role of SRC tyrosine kinases in Mac-1 association with the
cytoskeleton and clustering triggered by P-selectin was investigated using inhibitors of their activity. At concentrations needed to inhibit
P-selectin-induced PMNL adhesion, PP1 impaired CD18 redistribution to
the Triton X-100-insoluble fraction in PMNLs stimulated by P-selectin-IgG chimera (Figure 5A) and
in PMNL-platelet mixed aggregates (data not shown). Similar results
were obtained with the SRC tyrosine kinase inhibitor PP2 (data not
shown). Confocal microscopy (Figure 5B) showed that in PMNLs pretreated
with PP1 before stimulation with P-selectin, CD11b was uniformly
distributed as a peripheral ring (v) and F-actin was homogeneously
distributed (iv). These data demonstrate that SRC tyrosine kinase
activity is required for Mac-1 association to the cytoskeleton and
clustering at the sites of cell-cell contact in PMNL aggregates induced
by P-selectin.
Engagement of PSGL-1 by a monoclonal antibody triggers formation of Mac-1 clusters and F-actin patches in polymorphonuclear leukocytes Because PSGL-1 is the major leukocyte ligand for P-selectin, we examined the effect of PSGL-1 cross-linking on the cellular localization of Mac-1. PSGL-1 was engaged by mAb PL2 and was cross-linked by anti-mouse IgG F(ab')2 fragment. Cross-linking of PSGL-1 induced PMNL aggregation, organization of F-actin (Figure 6A), clustering of CD11b (Figure 6B), and colocalization of CD11b and F-actin at the sites of PMNL-PMNL adhesion (Figure 6C). These results support the conclusion that P-selectin induces Mac-1 activation through the binding to PSGL-1.
Integrity of the cytoskeleton is required for P-selectin-induced polymorphonuclear leukocyte adhesion and Mac-1 clustering The finding that Mac-1 associates with the cytoskeleton and that Mac-1 clusters colocalize with F-actin patches at the site of cell-cell contact suggested the involvement of actin polymerization in Mac-1 adhesiveness promoted by P-selectin. Therefore, we evaluated the effect of cytochalasin D, an agent that impairs actin filament polymerization, on P-selectin-induced PMNL adhesion and Mac-1 clustering. Treatment with cytochalasin D inhibited PMNL homologous aggregation induced by soluble P-selectin and PMNL adhesion to activated platelets (Figure 7A) in a concentration-dependent manner. PMNLs pretreated with 20 µM cytochalasin D and stimulated with P-selectin-IgG chimera showed areas completely devoid of F-actin and areas containing dense clumps or even compact foci of F-actin forming a discontinuous ring beneath the cell membrane (Figure 7Biv). The formation of large clusters of Mac-1, normally localized at the site of cell-cell boundaries (Figure 7Bii), was almost completely prevented by cytochalasin D treatment. Small patches of Mac-1 appear distributed along the cell membrane (Figure 7Bv). This result indicates that the assembly of F-actin is required for the formation and maintenance of the Mac-1 clusters at the site of cell-cell contact and that it plays a key role in Mac-1 adhesiveness triggered by P-selectin.
Ligand binding to Mac-1 is required for the formation of Mac-1 clusters and F-actin patches in polymorphonuclear leukocytes challenged with P-selectin Because mAbs to2-integrins inhibited SRC tyrosine kinase
activation in PMNLs adherent to P-selectin-expressing cells (Figure 3), we investigated whether ligand binding to Mac-1 was required for
clustering and colocalization with F-actin at cell-cell boundaries. Blockade of either P-selectin by WAPS 12.2 (Figure
8D-F) or 2-integrin by IB4 resulted in
a complete inhibition of Mac-1 clustering (Figure 8H) and F-actin
accumulation (Figure 8G), providing further support for the
interpretation that Mac-1 binding to its ligand is an essential
preliminary step to switch on the intracellular machinery that mediates
the formation of Mac-1 clusters and F-actin accumulation at the
adhesion sites.
Adhesion of PMNLs to activated platelets proceeds through the coordinated action of P-selectin and Mac-1.2,5-8 In the present study, we report that the activity of tyrosine kinases belonging to the SRC family is required for Mac-1 adhesive function triggered by P-selectin. Blockade of SRC kinase activity by specific inhibitors prevented PMNL-platelet adhesion and tyrosine phosphorylation of P-110, the major protein undergoing phosphorylation in PMNLs adherent to activated platelets.2 SRC tyrosine kinases can be activated in PMNLs as a consequence of
stimulation by soluble and particulate agonists41 and after Engagement of Integrin-cytoskeleton linkage plays a crucial role in Although it cannot be excluded that P-selectin binding to its
receptor(s) could stimulate changes in integrin affinity, the evidence
of Mac-1 clustering indicates that increased avidity is involved in
Mac-1 adhesiveness triggered by P-selectin. In fact, clustering on the
plasma membrane results in high-avidity of integrins that strengthens
the interaction with the ligand.22,49,50 Moreover,
clustering of integrins on the cell surface colocalizes at the
cytoplasmic side signaling molecules, including kinases and their
substrates.51 The formation of integrin clusters requires an early phase of integrin release from the cytoskeletal constraints that allows its mobility in the plasma membrane.52-54 Once
the integrin has bound ligand, establishment of new connections between integrin and the cytoskeleton may be required for firm adhesion. In our
study, treatment of PMNLs with cytochalasin D prevented PMNL-platelet
adhesion and PMNL-PMNL aggregation induced by soluble P-selectin and
the formation of Mac-1 clusters usually observed at the boundaries of
PMNL-PMNL aggregates; only small clusters of Mac-1 randomly distributed
along plasma membrane could be observed. These data support the
hypothesis that the cytoskeleton plays a key role in Mac-1 adhesiveness
triggered by P-selectin. As in P-selectin stimulation, the
PSGL-1 is the predominant P-selectin ligand in PMNLs.57 In
the present study, we showed clustering of Mac-1 and accumulation of
F-actin in PMNL aggregates triggered by the engagement of PSGL-1 by a
mAb. These data confirm and extend our previous observations showing
that cross-linking of PSGL-1 by a mAb was able to trigger tyrosine
kinase-dependent Mac-1-mediated homotypic aggregation and protein
tyrosine phosphorylation in PMNLs.2 The role of PSGL-1 as
a signaling molecule was originally suggested by Hidari et
al,58 who showed that the ligation of PSGL-1 by P-selectin and by mAbs results in the activation of the 42-44 kd MAP kinase and in
the tyrosine kinase-dependent production of IL-8 in PMNLs. However, the
evidence that PSGL-1 can signal activation of human PMNLs is
contradictory.14,59 It is important to note that in our
experimental conditions, signaling due to P-selectin binding to PSGL-1
and signaling induced by engagement of Mac-1 by its ligand could not be
differentiated, in agreement with the hypothesis that the 2 adhesive
systems are tightly coordinated with each other. In fact, in our model,
the activation of LYN and HCK in PMNLs interacting with
P-selectin-expressing cells was prevented by blocking anti-CD18
antibody, indicating that not only P-selectin stimulation but also the
binding of Mac-1 to its ligand is essential for kinase activation. In
agreement, the blockade of CD18 prevents the formation of Mac-1
clusters and their colocalization with F-actin patches. Thus
P-selectin, interacting with its receptor on PMNLs, directly triggers
an initial Mac-1 interaction with its ligand responsible for SRC kinase
activation; these, in turn, mediate the remodeling of
cytoskeleton-integrin linkages, integrin diffusion, and clustering that
finally strengthen cell-cell adhesion. A multistep model, in which
initial ligand binding stimulates post-receptor events leading to
integrin clustering and stabilization of integrin-ligand interaction,
has already been proposed for In summary, the mechanism proposed for Mac-1 activation triggered by
P-selectin highlights a new role of SRC tyrosine kinases in a
regulatory loop by which the
We thank Dr S. Smyth (Division of Hematology, Health Science Center, Stony Brook, NY) for helpful discussion and suggestions. We also acknowledge Dr R. Polishchuk for expert help in using confocal laser scanning microscopy and Dr L. Fumagalli (Institute of General Pathology, University of Verona, Italy) for help in setting up the in vitro kinase assay. We thank Drs R. P. McEver, K. L. Moore, M. K. Robinson, and G. Berton and the Ortho Diagnostic System for valuable monoclonal antibodies and the Genetics Institute for the P-selectin-IgG fusion proteins and CHO-P cells.
Submitted November 28, 2000; accepted February 12, 2001.
Supported in part by Fondation Segré, Geneva, Switzerland; Ministero della Sanità, Convenzione n. ICS060.2/RF99.74, IRCCS Aviano; and the Italian National Research Council (Convenzione CNR-Consorzio Mario Negri Sud). R.S. was the recipient of a fellowship by Fondazione Italiana Ricerca sul Cancro.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Presented in part at the 42nd Annual Meeting of the American Society of Hematology, December 1-5, 2000, San Francisco, CA. Reprints: Paola Piccardoni, Department of Vascular Medicine and Pharmacology, "G.Bizzozero" Laboratory of Blood and Vascular Cell Interactions, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy; e-mail: piccardo{at}cmns.mnegri.it.
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A. Massaguer, S. Perez-del-Pulgar, P. Engel, J. Serratosa, J. Bosch, and P. Pizcueta Concanavalin-A-induced liver injury is severely impaired in mice deficient in P-selectin J. Leukoc. Biol., August 1, 2002; 72(2): 262 - 270. [Abstract] [Full Text] [PDF]
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M. P. Scott, F. Zappacosta, E. Y. Kim, R. S. Annan, and W. T. Miller Identification of Novel SH3 Domain Ligands for the Src Family Kinase Hck. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP), WASP-INTERACTING PROTEIN (WIP), AND ELMO1 J. Biol. Chem., July 26, 2002; 277(31): 28238 - 28246. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
M
-32P]ATP and enhanced chemiluminescence Western
blotting system (ECL kit) and the ECL protein biotinylation module were
from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire,
United Kingdom). fMLP was dissolved in dimethyl sulfoxide (DMSO) at
concentrations of 100 µM, stored at 
20°C, and diluted in isotonic
saline just before use. Thrombin was dissolved in saline at
concentrations of 50 U/mL and was stored at 
), PP2 (
), PP3 (
); (B) tyrphostin
AG 490 (
), PD98059 (
). Mixed-cell
suspensions were coincubated in standard conditions, the interaction
was stopped at 2 minutes, and samples were processed for FACS analysis.
Alternatively, cytochalasin D-pretreated PMNLs were challenged with
P-selectin-IgG chimera (10 µg/mL) (
T cells.
J Immunol.
1994;153:3917-3928