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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Mount Sinai School of Medicine, Department of
Medicine, New York, NY.
The adhesive mechanisms leading to the mobilization of
hematopoietic progenitor cells (HPCs) from the bone marrow into the blood are poorly understood. We report on a role for selectins and
fucoidan in progenitor mobilization. Baseline levels of circulating HPCs are increased in endothelial selectin-deficient (P/E Circulating hematopoietic progenitor cells
(HPCs) were first observed several decades ago,1,2 and it
was subsequently shown that the number of circulating HPCs could be
augmented by chemotherapy, endotoxin administration, or
stress.3-5 It is now recognized that multiple agents from
various families of cytokines, including hematopoietic growth factors,
inflammatory cytokines, and chemokines, are capable of increasing
circulating progenitor counts in vivo.6,7 These mobilized
HPCs are routinely used in the clinic as a source of hematopoietic stem
cells for bone marrow transplantation. Mobilization is achieved with
variable kinetics; for example, granulocyte colony-stimulating factor
(G-CSF) requires days to achieve peak circulating HPC
numbers,8 whereas interleukin 8 (IL-8) acts within minutes
and its effect is short-lived.9
The adhesive mechanisms leading to the extravasation of mature
leukocytes to areas of inflammation have been well characterized in the
past decade. The initial tethering and rolling steps are largely
mediated by selectins and their ligands, whereas the Distinct among the families of molecules mediating adhesion events
within the vasculature, the selectin family consists of 3 members that
contain a calcium-binding lectin domain.12-14 E-selectin expression by endothelial cells is induced by inflammatory cytokines, whereas P-selectin is stored in granules of endothelial cells and
platelets, and can be rapidly transported to the cell surface after stimulation. L-selectin is constitutively present on the microvilli of mature leukocytes as well as on hematopoietic
progenitors.15,16
Specific carbohydrate or polypeptide modifications have been defined as
critical for selectin binding.17 These include
sialylation, fucosylation, and sulfation. Certain sulfated glycans have
been shown to interact with P- and L-selectin but not
E-selectin.18,19 A prototypic example is fucoidan, a
sulfated polysaccharide extracted from the brown seaweed Fucus
vesiculosus, which can inhibit leukocyte rolling and inflammatory
responses in vivo.20-22
Insight into the function of adhesion molecules in vivo was provided by
gene-targeted null mutations.23,24 Mice lacking both
endothelial selectins, for example, display impaired leukocyte extravasation to inflammatory sites leading to spontaneous skin infections.25,26 Although all 3 selectins appear to
participate in leukocyte recruitment to inflammatory sites, analyses of
mice harboring all possible combinations of selectin null mutations suggest distinct roles for each selectin member in leukocyte
homeostasis and trafficking.27,28 In addition to
abnormalities in leukocyte rolling and extravasation, endothelial
selectin-deficient mice also exhibit alterations in hematopoiesis,
including severe leukocytosis, elevated levels of hematopoietic
cytokines, splenomegaly, and increased splenic hematopoietic
progenitors.25 These findings suggested that endothelial
selectins expressed in the bone marrow play a role in hematopoiesis.
It is interesting that the bone marrow constitutively expresses
E-selectin and VCAM-1.25,29,30 We recently showed that endothelial selectins and VCAM-1 were both required for progenitor-bone marrow (BM) endothelium interactions31 and for optimal
recruitment of progenitors to the bone marrow after
transplantation.32 Recent studies also indicate that
immature (CD34+ CD38 Here, we evaluated the role of selectins and sulfated glycans in
hematopoietic progenitor mobilization. We show that endothelial selectin deficiency or blockade increases circulating HPCs. Sulfated selectin inhibitors Animals and reagents for in vivo studies
Rat antimouse P-selectin MAb RB40.34 (IgG1), rat IgG1, and rat
IgG2a control antibodies were obtained from Pharmingen (San Diego, CA). Commercially obtained antibodies were endotoxin-tested (endotoxin level less than or equal to 0.01 ng/µg of protein by Limulus amebocyte lysate assay [LAL]). Rat anti-L-selectin
(IgG2a), purified from supernatant of a MEL-14-producing
hybridoma cell line (American Type Culture Collection, Rockville, MD),
was a kind gift from Dr J. Frenette (Laval University, Quebec, Canada). Fucoidan (Fluka Lot No 385468/1) was resuspended in endotoxin-free phosphate-buffered saline (PBS). Endotoxin levels in antibody and
fucoidan preparations were tested by LAL assay (sensitivity 0.06 EU/mL;
BioWhittaker, Walkersville, MD), and if detectable, contaminating
endotoxin was removed using a polymixin B column (Detoxi-Gel, Pierce,
Rockford, IL).
Desulfation, carbohydrate, and sulfate analyses
Isolation of cells and colony-forming units in culture assays Blood was harvested by retro-orbital sampling of mice anesthetized with tribromoethanol and collected in polypropylene tubes containing EDTA. Blood counts were obtained using an automated cell counter (Serono-Baker Diagnostics, Allentown, PA) and differential counts were determined from Wright-stained smears. Mononuclear cells were isolated by underlaying 400 µL of blood diluted in 3 volumes of PBS with lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) and by centrifugation at room temperature at 280g for 30 minutes. Contaminating erythrocytes (RBCs) were lysed in 0.8% NH4Cl and the remaining nucleated cells were washed thrice in RPMI. Bone marrow cells were harvested by aseptically flushing both femora of each animal in RPMI using a 21-gauge needle. A single cell suspension was obtained by gently aspirating several times with the same needle and syringe. Cells of both femora were pooled and the volume of each cell suspension was determined with a graduated pipette.For CFU-C assays, one volume of hematopoietic cells was added to 9 volumes of methocult M3434 media (Stemcell Technologies, Vancouver, BC,
Canada). Cells were plated in triplicate assays. Burst-forming
units-erythroid (BFU-E) and granulocyte-macrophage colony-forming units
(GM-CFUs) were scored on days 7-8. GM-CFUs and BFU-E showed similar
changes in mobilization studies, therefore only the total numbers of
CFU-C are reported. For mobilization experiments in chimeric mice (see
below), CFU-Cs were grown in 0.8% methylcellulose (methocult M3100;
Stemcell Technologies) containing 30% fetal bovine serum (FBS)
(Intergen, Westchester), 1% bovine serum albumin (BSA),
10 Progenitor mobilization Progenitor mobilization using antiadhesion antibody was accomplished by administering to wild-type and E / mice
anti-P-selectin antibody or isotype-matched IgG control at a dose of 1 mg/kg, via the tail vein, daily for 3 days. Blood and bone marrow cells were collected 24 hours after the last dose and assayed for progenitor content as above. Fucoidan-induced progenitor mobilization was induced
in wild-type or selectin-deficient mice by administering fucoidan at 25 mg/kg intraperitoneally (ip). Our preliminary studies showed that
fucoidan is systemically absorbed via this route because this dose can
inhibit leukocyte rolling in the cremaster muscle (L.W. and P.S.F.,
unpublished observations). In the first protocol, fucoidan or vehicle
(PBS) were administered at 25 mg/kg intraperitoneally for 6 doses. The
first 2 doses were given on day 0 (approximately12:00 and 20:00), 3 doses on day 1 (approximately 8:00, 12:00, and 20:00) and the final
dose on day 2 ( approximately 8:00). In the second protocol, only 2 doses (25 mg/kg ip) were given over 6 hours. In both protocols, blood
and femoral bone marrow cells were harvested 2 hours after the
last dose.
Mobilization in chimeric mice We developed an assay to compare directly the ability of L/
and L+/+ progenitors to be mobilized after fucoidan treatment. Mice
chimeric for L-selectin and EGFP expression were generated by adoptive
bone marrow transfer. P/E/ animals were used as recipients and were
lethally irradiated (12.0 Gy) in 2 split doses, 3 hours apart, from a
cesium source (Model I-68A, J.L. Shepherd, San Fernando, CA).
Irradiated P/E/ mice were then injected via the lateral tail vein
with a mixture of nucleated bone marrow cells obtained from L/ or
wild-type control mice combined with bone marrow cells from EGFP
trangenic mice (2 × 106 EGFP bone marrow nucleated cells
mixed with 2 × 106 nonfluorescent bone marrow cells
[L+/+ or L/]). After transplantation, mice were kept in a
microisolator unit and fed ad libitum with sterile chow food and water.
To avoid any contribution to circulating progenitors from the spleen,
mice were splenectomized 2 weeks after BM transplantation as described
by Frenette et al32 and progenitor mobilization was induced
as described above after an additional recovery period of at least
1 month.
Long-term competitive reconstitution To assess the ability of mobilized progenitors to competitively repopulate the bone marrow of a lethally irradiated host, progenitors were mobilized by treating E/ mice (whose leukocytes bear the
CD45.2 antigen) using 2 doses of fucoidan or vehicle as above (see
scheme in Figure 4A). Blood from 5 to 6 animals per group was harvested
and pooled 2 hours after the last dose. Erythrocytes were removed by 2 rounds of 0.8% NH4Cl lysis (1 part blood: 9 parts buffer)
and washed thrice in RPMI. Fresh bone marrow competitor cells were
obtained from femora of Ly 5.2 congenic mice (whose leukocytes harbor
the CD45.1 antigen). CD45.1 bone marrow nucleated cells
(1 × 105 per recipient mouse) were mixed with blood
nucleated cells (1 mL per recipient mouse) from fucoidan or
vehicle-treated E/ mice (CD45.2) and injected into the tail vein of
lethally irradiated CD45.1 recipient mice. Mice were housed in a
microisolator and fed sterile food and sterile water containing
antibiotics (during the first week only; trimethoprim, 24 mg/dL and
sulfamethoxazole, 120 mg/dL). The proportion of leukocytes bearing
CD45.1 and CD45.2 antigens was determined monthly after transplantation
by flow cytometry. Competitive repopulating units (CRUs) were
calculated as follows: CRU = % (C) / (100-%) where % is the
measured percentage of donor cells and C is the number of fresh
competitor marrow cells per 105.42
Flow cytometry To stain the leukocyte CD45 antigen, whole blood (150 µL) was incubated, after blockade of Fc receptors with rat antimouse CD16/CD32 monoclonal antibody 1:50, with fluorescein (FITC)-conjugated mouse anti-CD45.2 and biotinylated mouse anti-CD45.1 antibodies 1:50 (all from Pharmingen). Blood cells were washed once in PBS/BSA 0.06% and stained with phycoerythrin (PE)-conjugated steptavidin (Jackson Immunoresearch, West Grove, PA). Erythrocytes were lysed in NH4Cl and the remaining leukocytes were washed thrice in PBS/BSA. To assess L-selectin expression in transplanted P/E/ mice,
50 µL of whole blood was incubated with MEL-14 antibody (1:50 of 2 mg/mL), washed with PBS/BSA and stained with FITC-conjugated goat
antirat IgG 1:250 (Cappel, Durham, NC). RBCs were then lysed and
leukocytes were washed. In some experiments, day 8 CFU-Cs were
individually picked under microscopy in PBS/BSA containing 5 mmol/L
EDTA. Cells were dissociated, washed once, resuspended in PBS
containing 10% mouse serum, and stained for L-selectin expression as
above. Analysis of 10 000 events was performed on a FACSCalibur flow
cytometer (Becton Dickinson, Mountain View, CA).
Statistical analysis All values are reported as mean ± standard error of the mean. Statistical significance for 2 unpaired groups was assessed by the Student t test. Multiple comparisons were analyzed using 1-way analysis of variance (ANOVA), Bonferroni's test.
Absence or inhibition of endothelial selectins increases circulating hematopoietic progenitors We previously observed an increase in the number of residual circulating progenitors in P/E/ mice after bone marrow
transplantation.32 To evaluate the influence of
endothelial selectins on circulating progenitors during steady-state
conditions, we assayed HPCs in the blood of wild-type and P/E/
mice. As shown in Figure 1A, circulating
HPCs were increased approximately 7-fold in P/E/ mice compared with
wild-type controls. Higher numbers of circulating progenitors were also
observed in splenectomized P/E/ animals (not shown), suggesting
that HPCs did not originate from their enlarged spleens.25
Given the multiple abnormalities present in endothelial selectin
knockouts, it was possible that deficiency from birth may have
indirectly affected the numbers of circulating HPCs.
To address this issue, we used mice lacking only E-selectin that have
no significant phenotype.25,26,43 To induce a transient inhibition of one or both endothelial selectins, we injected into the
tail vein of wild-type and E Fucoidan rapidly mobilizes hematopoietic progenitors As mentioned previously, fucoidan can inhibit the function of P-selectin and L-selectin but not E-selectin.18,19 To assess whether this selectin inhibitor would also augment circulating progenitor counts, we treated E/ mice with fucoidan or with vehicle
(PBS) as control for 6 doses over 48 hours. This way, fucoidan
administration to E/ mice transiently inhibits all 3 selectins.
Blood and bone marrow were collected 2 hours after the last dose and
progenitor content was assessed. As shown in Figure
2A, fucoidan treatment increased the
number of circulating progenitors in E/ animals, compared with
vehicle alone. Together, the data using antibody or fucoidan inhibition
indicate that short-term blockade of more than one selectin increases
the numbers of circulating progenitors. Unlike antibody treatment,
fucoidan also reduced the numbers of bone marrow nucleated cells (not
shown), suggesting that fucoidan might act through mechanism(s) other
than blocking P-selectin function.
Despite its relatively short half-life, fucoidan appeared equally
effective in increasing the numbers of circulating HPCs as antiselectin
antibody administration (Figures 1B and 2A). To investigate whether
fewer doses of fucoidan given at a shorter interval could achieve
similar mobilization, E
To evaluate the role of both endothelial selectins in this activity,
mice deficient in P- and E-selectins were treated with 2 doses of
fucoidan. As shown in Figure 2B, P/E L-selectin may also participate in fucoidan-induced mobilization The leukocyte selectin (L-selectin) is expressed on immature progenitor cells.15,16 Because it binds fucoidan, L-selectin is a potential candidate receptor to participate in progenitor mobilization. To test the role of L-selectin in fucoidan-induced HPC mobilization, we treated L-selectin-deficient (L/) and wild-type control mice using the 2-dose fucoidan
administration protocol. As shown in Table
2, fucoidan mobilized progenitors equally
well in the absence of L-selectin. Because the spleen of L/ animals is slightly enlarged,45 we also evaluated progenitor
numbers in the spleen of mice treated with vehicle and fucoidan.
Although fucoidan administration to L/ animals tended to increase
splenic progenitor numbers (P = .08; Table 2), there was
no significant difference in baseline (PBS-treated) splenic progenitor
counts between L/ and wild-type mice. Fucoidan treatment of L/
mice also produced leukocytosis, reduced platelet counts, and lowered bone marrow nucleated cell numbers to an extent similar to their wild-type counterparts (Table 2). Although the above results suggest no
role for L-selectin in fucoidan-induced HPC mobilization, it is
possible that other adhesion molecules have compensated in L/
animals and masked a potential difference.
To further evaluate the function of L-selectin in HPC mobilization, we
developed a competitive assay to compare within the same animal the
ability of L Progenitor mobilization in these chimeric mice was induced by
administering 2 doses of fucoidan (25 mg/kg) or vehicle alone. Blood
and bone marrow nucleated cells were plated to assay their progenitor
content. After 8 days of culture, the numbers of nonfluorescent and
green fluorescent colonies were determined (Figure
3A,B). To verify L-selectin expression in
the chimeras, CFU-Cs were sampled, stained for L-selectin, and analyzed
by FACS. As predicted, both "green" and nonfluorescent colonies
derived from L+/+/EGFP chimeras expressed L-selectin at similar levels,
whereas in L
Long-term bone marrow repopulating cells are mobilized after fucoidan treatment To evaluate the ability of fucoidan-mobilized progenitors to reconstitute the bone marrow of a lethally irradiated recipient, we used congenic mice that harbor a leukocyte antigen (CD45.1) distinguishable from that of selectin knockout mice backcrossed in the C57BL/6 background (CD45.2) by FACS analysis. Mice lacking E-selectin were treated with either fucoidan or vehicle for 2 doses over 6 hours (Figure 4A). After the treatment period, blood was harvested from the retroorbital venous plexus, pooled (5 mice per group), and erythrocytes were lysed. Concomitantly, fresh bone marrow cells were obtained from the femora of CD45.1 congenic mice. These fresh bone marrow cells were used as competitor cells and were mixed with mobilized blood nucleated cells (1 × 105 bone marrow competitor cells [CD45.1] mixed with nucleated cells from 1 mL of blood [CD45.2]). The 1 × 105 bone marrow cells contain sufficient numbers of HPCs to allow recipient mice to survive if no long-term reconstituting stem cells were present in the tested sample. The cell mixture was injected into the tail vein of lethally irradiated CD45.1 mice. Transplanted mice were allowed to recover for 1 month, at which point CD45 expression on leukocytes was assessed by flow cytometry. In 2 independent experiments pooled in Figure 4B, fucoidan-mobilized progenitors contributed greatly to the blood leukocyte composition at all timepoints tested. At 6 months, the competitive repopulating ability of fucoidan-mobilized blood cells was approximately 700-fold greater than that of the PBS group. In addition, CD45.2-positive cells were represented among the various subsets of leukocytes defined by light scatter characteristics (Figure 4C). These data indicate that administration of fucoidan can indeed mobilize long-term repopulating stem cells.
Critical role of sulfation To investigate the role of sulfate groups in fucoidan-induced HPC mobilization, fucoidan was desulfated by solvolysis. This treatment led to approximately 99% desulfation as measured by barium sulfate precipitation. E/ mice were treated with 2 doses (25 mg/kg) of
desulfated or native fucoidan. As shown in Figure 5A, desulfated fucoidan was ineffective
in inducing progenitor mobilization, compared with native fucoidan.
Importantly, desulfated fucoidan did not produce leukocytosis and
thrombocytopenia suggesting that sulfate groups are critical for the
various hematologic effects of fucoidan in mice.
Mammalian cells also harbor sulfated glycans that have been shown to
bind selectins. For example, cell membrane-associated sulfated
glycosphingolipid (3'-sulphogalactosylceramide, or sulfatide) is found
on myeloid cells46 and heparin is characteristically stored
by mast cells.47 Although not fucosylated, both sulfatide and heparin, like fucoidan, have been shown to interact with P- and
L-selectins.18,19 To evaluate the function of these
sulfated molecules in HPC mobilization, we treated E
In this study, we evaluated the role of selectins and sulfated
inhibitors of selectin function in the mobilization of hematopoietic progenitor cells from the bone marrow to the blood compartment. We
found that antibody blockade and/or absence of endothelial selectins
led to peripheralization of HPCs. The combination of anti-P-selectin
antibody administration in E Endothelial selectins may influence progenitor mobilization in different ways. It is conceivable that P- and E-selectins expressed on the bone marrow endothelium might retain progenitors in the bone marrow. This is suggested by the varying degrees of mobilization achieved, depending on the type and the extent of selectin blockade. However, their restricted expression in the bone marrow stroma (eg, endothelial only) argues against this possibility. As we previously described, endothelial selectins play a role in progenitor homing to bone marrow.32 It is likely that under steady state conditions or during induction of mobilization, traffic between the bone marrow and the blood compartments is bidirectional. Therefore, blocking or absence of endothelial selectins may prevent progenitors from reentering the bone marrow and tilt this equilibrium toward the circulating HPCs. Our studies indeed suggest that blocking adhesion receptors acting on progenitor homing increases circulating progenitor numbers and that it may represent a very useful addition to other agents currently used to mobilize HPCs for clinical bone marrow transplantation. Although the lack of one or both endothelial selectins enhanced the
effect of fucoidan, expression of the leukocyte selectin appeared to be
an advantage in a competitive setting as progenitors expressing
L-selectin were mobilized in greater numbers than those lacking it
(Figure 3C). L-selectin expression was not required because
fucoidan-induced HPC mobilization was similar between wild-type and
L Several lines of evidence suggest that fucoidan acts through
mechanism(s) other than interacting with selectins alone. First, the
effect of fucoidan appears more powerful than the influence of
antiselectin antibody administration or selectin gene knockouts (compare Figure 2B and Figure 1). Second, fucoidan treatment produces a
reduction in the total nucleated cell numbers in the bone marrow, whereas these changes were not observed in nontreated P/E Arguably, the most striking observation of our studies is the profound
effect of fucoidan on progenitor mobilization. Fucoidan was first shown
by Stoolman and Rosen52 to inhibit lymphocyte binding to
high endothelial venules in a Stamper-Wooddruff assay. Subsequent work
confirmed this finding,53 and it was later demonstrated that this sulfated polysaccharide could influence leukocyte-endothelial interactions and inflammatory responses through inhibition of P-selectin and L-selectin function.20-22 Although our
study clearly suggests a target other than selectins for
fucoidan-induced mobilization, the targeted bone marrow cell or
compartment and the nature of this "ligand" are unclear at present.
It is noteworthy that fucoidan can bind thrombospondin and
laminin,54 extracellular matrix (ECM) proteins expressed
in the bone marrow, and that low concentrations of fucoidan may produce
endothelial cell retraction in vitro.55 Thus, fucoidan
might compete with HPCs for binding to ECM proteins or proteoglycans
that could contribute to HPC release. It is also conceivable that the
sulfated polysaccharide might directly interact with progenitor or
mature hematopoietic cells via nonselectin ligands. Evidence exists for
such ligands on lymphocytes.56-58 Surprisingly,
administration of multiple doses of fucoidan to E Of note is the 2-fold reduction in platelet counts after fucoidan treatment. Although, to our knowledge, a reduction in platelet numbers in fucoidan-treated animals has not been previously described, this effect was highly reproducible in our studies and was independent of selectin expression (Tables 1 and 2). Interestingly, Durig and colleagues59 showed that a low concentration (10 µg/mL) of fucoidan could induce platelet activation in vitro, and this effect was more prominent in high molecular weight fractions. In addition, fucoidan exhibits an anticoagulant activity similar to that of heparin.60-62 During our studies, however, mice appeared to tolerate fucoidan therapy very well and no spontaneous bleeding was observed. This may result from the fact that the reduction in platelet counts is relatively small and the anticoagulant potency of fucoidan is much lower than that of heparin.62 Our data also indicate that sulfation, but not fucosylation, of the glycan appears to be critical for its effect because the desulfated fucose polymer is inactive and sulfated glycosphingolipids display activity in HPC mobilization (Figure 5). Anionic charges provided by sulfated groups are not likely to be solely responsible for the effect of fucoidan because the charge density per saccharide residue is greater in heparin than in fucoidan,47,63 and heparin was not capable of significant mobilization (Figure 5). The fact that sulfated galactosylceramide (sulfatide) also induced HPC mobilization, albeit to a lesser extent than fucoidan, suggests that the proper spacial arrangement of the anionic groups rather than the type of saccharide is important. Despite their structural disparities, fucoidan and sulfatides share
many characteristics such as binding P- and
L-selectins,18,19 inducing tyrosine
phosphorylation,49,50 and interacting with extracellular
matrix proteins.54 In contrast to fucoidan, which is not
found in mammals, sulfatides are produced by human myeloid cells.46 It has been estimated that 108
granulocytes may excrete as much as 5 µg of sulfatides in 12 hours.64 Interestingly, recent data indicate that mature
leukocytes have a critical role in chemokine-induced HPC mobilization.
For example, IL-8-induced mobilization is inhibited by anti-
We thank Drs Richard Hynes and Masaru Okabe for their gifts of breeding pairs of L-selectin knockout and EGFP transgenic mice, respectively. We are also grateful to Sylviu Tamasdan for his help in carbohydrate determination and desulfation, Drs Schickwann Tsai and Rona Weinberg for providing cell lines and reagents for CFU-C assays, Dr Juan Badimon for sharing his automated cell counter, and Drs George Atweh and Denisa Wagner for their helpful comments.
Submitted February 2, 2000; accepted May 24, 2000.
Supported in part by a Junior Faculty Scholarship from the American Society of Hematology and a scholarship from the Comprehensive Manhattan Sickle Cell Center (P60 HL28381).
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.
Reprints: Paul S. Frenette, Mount Sinai School of Medicine, Department of Medicine, 1 Gustave L. Levy Pl, Box 1079, New York, NY 10029; e-mail: paul.frenette{at}mssm.edu.
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© 2000 by The American Society of Hematology.
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Top
Abstract
Introduction
2 integrins and
immunoglobulin superfamily members mediate the subsequent firm adhesion
and diapedesis.
) human progenitor cells
express a functional P-selectin ligand.
4
integrin or VCAM-1 increased circulating blood progenitors and that
this process required an intact c-kit ligand.
fucoidan in particular
1).
