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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1318-1324
Platelet-Endothelial Interactions in Inflamed Mesenteric Venules
By
Paul S. Frenette,
Caitlin Moyna,
Daqing W. Hartwell,
John B. Lowe,
Richard O. Hynes, and
Denisa D. Wagner
From the Center for Blood Research, Departments of Pathology and
Medicine, Harvard Medical School, Boston; Howard Hughes Medical
Institute, Center for Cancer Research, Department of Biology,
Massachusetts Institute of Technology, Cambridge, MA; and Howard Hughes
Medical Institute, University of Michigan Medical School, Ann
Arbor, MI.
 |
ABSTRACT |
The selectins are membrane glycoproteins promoting adhesive events
between leukocytes, platelets, and endothelial cells. We have
previously demonstrated that platelets roll on P-selectin expressed on
stimulated endothelium. In this study, we wished to examine the
function of both the platelet and endothelial selectins, P- and
E-selectins, in mediating platelet-endothelial interactions during
inflammation. We demonstrate, using intravital microscopic examination
of venules inflamed with tumor necrosis factor- (TNF-), that
resting platelets interact with both P- and E-selectins and that the
leukocyte (1,3)fucosyltransferases FucT IV and FucT VII do not
provide platelets with selectin ligand activity. We also show that
after thrombin activation of wild-type (+/+) platelets, platelet
P-selectin can mediate interactions on a TNF--inducible endothelial
ligand. To evaluate the potential role of platelet P-selectin in the
recruitment of leukocytes to inflammatory sites, we reconstituted the
bone marrow of mice deficient in both P- and E-selectins (P/E /)
with wild-type (+/+) or P-selectin-deficient (P/) bone
marrow containing megakaryocytic precursors. Providing +/+
platelets to P/E/ mice by bone marrow transplantation did not
rescue the immunodeficient phenotype, suggesting that platelet P-selectin does not have an active function in the recruitment of
leukocytes into inflammatory sites. To participate in inflammatory or
hemostatic responses, platelets may use the endothelial selectins.
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INTRODUCTION |
MAINTENANCE OF VASCULAR integrity requires
the normal function of both the vascular endothelium and circulating
platelets. Although endothelial cells normally act as a nonthrombogenic
surface, they may become procoagulant in certain
circumstances.1 For example, after stimulation with
endotoxin, interleukin-1 (IL-1), or tumor necrosis factor-
(TNF-), endothelial cells synthesize tissue factor and other
procoagulant proteins, which may promote acute thrombosis in patients
with chronic inflammatory diseases. In contrast, inherited defects in
platelet adhesion molecules are associated with bleeding diathesis.
Stimulated endothelial cells also express a variety of adhesion
molecules. Among these are two selectins, members of a family of
receptors involved in adhesive interactions among leukocytes, platelets, and the endothelium. P-selectin is stored in Weibel-Palade bodies in the endothelial cells and in the -granules of platelets. It is rapidly translocated to the surface of both cell types after stimulation with various secretagogues.2-4 E-selectin
expression is induced by endotoxin or inflammatory cytokines such as
IL-1 and TNF-.5 Endothelial selectins bind carbohydrate
ligands expressed on the surface of leukocytes. Sialylated and
fucosylated oligosaccharides, such as sialyl-Lewis,x appear
crucial for binding activity.6 Most selectin-binding carbohydrate structures are synthesized by (1,3)fucosyltransferases (FucTs), of which FucT IV and FucT VII are expressed in leukocytes and
thought to be crucial for the biosynthesis of selectin ligands. FucT
VII, in particular, appears to direct the expression of
sialyl-Lewisx.7,8
The generation via gene targeting of animals deficient for these
receptors has provided valuable insight into their functions in
vivo.9 Whereas P-selectin knockout mice display severe
defects in leukocyte rolling and extravasation in the early phases of inflammation,10 E-selectin-deficient mice are remarkably
free of such defects.11 However, the ablation of both
endothelial selectins produces profound abnormalities in leukocyte
dynamics, indicating that P- and E-selectins cooperate in vivo to mount an appropriate inflammatory response and that they are crucial for
leukocyte homeostasis.12,13 Similarly, mice lacking FucT VII exhibit severe defects in leukocyte rolling and extravasation and
also lymphocyte homing,14 which are further exaggerated in
animals doubly deficient in leukocyte FucT IV and FucT VII (Thall et
al, manuscript submitted).
Previously, we reported that, in a manner similar to leukocytes,
platelets roll on the endothelium of venules stimulated with the
secretagogue calcium ionophore A23187 and that this interaction depended on endothelial P-selectin, with little contribution from platelet P-selectin.15 Consistent with a role for
P-selectin in hemostasis, we subsequently detected a 40% prolongation
of the tail bleeding time and greater hemorrhage after a local
Shwartzman reaction in P-selectin-deficient mice compared with
wild-type controls.16 The local Shwartzman reaction, a
model of intravascular coagulation induced by successive doses of
endotoxin and TNF-, illustrates the intricate relations between
inflammatory and hemostatic systems. TNF- administered alone does
not produce intravascular coagulation but induces leukocyte rolling in
the mouse that is dependent on both P- and
E-selectins.12,13 Here, we report on selectin-mediated
platelet-endothelial interactions during inflammation induced by
TNF- as observed by intravital microscopy of mesenteric venules. We
also examine whether wild-type platelet P-selectin can ameliorate the
immunodeficient characteristics of P- and E-selectin doubly null mice.
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MATERIALS AND METHODS |
Animals.
Male C57BL/6J/129Sv mice, wild-type (+/+) or deficient in P-selectin
(P/),10 E-selectin (E/),12 or both
P- and E-selectins (P/E/),12 weighing 18 to 22 g
(aged 5 to 6 weeks) were used for intravital microscopy. Animals were
housed at the Center for Blood Research of Harvard Medical School or
the Center for Cancer Research of Massachusetts Institute of
Technology. Blood for platelet preparation was harvested from +/+,
P/, or FucT IV/VII / mice (Thall et al,
manuscript submitted) of any sex or age. The experimental procedures
were approved by the Animal Care and Use Committee of the Center for
Blood Research.
Blood sampling and platelet preparation.
Mouse blood was obtained as previously described.15
Briefly, blood was collected from the retro-orbital venous plexus in acid-citrate-dextrose ([ACD] 38 mmol/L citric acid, 75 mmol/L trisodium citrate, and 100 mmol/L dextrose, 1/10 vol), and
platelet-rich plasma (PRP) was obtained by two sequential
centrifugations (280g for 8 minutes and 280g for 3 minutes). Platelets were isolated by filtering the resulting PRP
through a Sepharose 2B (Sigma, St Louis, MO) column
equilibrated with PIPES buffer (25 mmol/L PIPES, 137 mmol/L NaCl, 4 mmol/L KCl, and 0.1% wt/vol dextrose), pH 7.0 for experiments using
resting platelets, and pH 7.4 to optimize activation for experiments
with activated platelets.
Gel-filtered platelets were fluorescently labeled with calcein AM 0.25 µg/mL (Molecular Probes, Eugene, OR) as previously described.15 In experiments using activated platelets, the
preparation was incubated with human thrombin (Sigma) 0.2 U/mL for 15 minutes at 37°C. Thrombin was then inhibited with hirulog BG8967 1 µg/mL (generous gift from Dr John Maraganore, Biogen, Cambridge, MA). Recipient mice were injected via the lateral tail vein with 5 × 109 platelets/kg in a volume of 200 to 400 µL PIPES
buffer.
Intravital microscopy.
Recipient mice were treated with murine recombinant TNF- (Genzyme
Corp, Boston, MA) 0.5 µg in 500 µL phosphate-buffered saline (PBS)
intraperitoneally. Such treatment induces P- and E-selectin-dependent leukocyte rolling in the mesentery.12 At 3.5 hours after
TNF- injection, animals were transfused with calcein AM-labeled
platelets and immediately anesthetized with 2.5% tribromoethanol 0.15 mL/10 g. Preparation of the mesentery was as previously
described.15 A suitable venule for filming (28 to 40 µm
in diameter) was found within about 15 minutes following injection of
labeled platelets. Venules were visualized using a Zeiss
Axiovert 135 inverted microscope (objective 32X, 0.4NA, Oberkochen,
Germany) equipped with a 100 W HBO fluorescent lamp source (Opti Quip,
Highland Mills, NY) with a narrow-band FITC filter set (Chroma
Technology, Brattleboro, VT), and a silicon-intensified tube camera
C2400 (Hamamatsu, Tokyo, Japan) connected to a SVHS video
recorder AG-6730 (Panasonic, Tokyo, Japan). Recording of
one venule per animal was made for 20 minutes for resting and 10 minutes for activated platelets, respectively. Centerline erythrocyte
velocity (Vrbc) was measured using an optical Doppler
velocimeter (Microcirculation Research Institute, Texas A&M College of
Medicine, College Station, TX). The venular shear rate ( ) was
calculated based on Poiseuille's Law for a newtonian fluid, = 8(Vmean/Dv), where Dv is the
diameter of the venule and Vmean is estimated from the
measured Vrbc using the empirical correlation
Vmean = Vrbc/1.6.17
Image analysis.
The critical velocity (Vcrit) for each venule was
calculated using the equation, Vcrit = Dp/Dv × (2 D
p/Dv), where Dp is the diameter
of a platelet (2 µm). Quantitation of platelet-endothelial interactions was made by an investigator blind to the genotype of the
platelets or venules. Platelets traveling a distance of at least 30 µm at a velocity less than Vcrit were scored as
"rolling." Any platelet interacting with the endothelium at a
velocity less than Vcrit but not fulfilling the criteria
for rolling was labeled as "captured." The average number of
rolling or captured platelets per minute over a venular segment of 250 µm was determined by taking 10 counts of 1 minute (five counts in
each half of filming for resting platelets and the whole recording for
activated platelets).
Bone marrow transplantation.
P- and E-selectin doubly deficient mice and wild-type female mice aged
7 to 8 weeks received whole-body irradiation (9.5 to 11.0 cGy) in two
split doses 3 hours apart, from a cesium source (model 143; J.L.
Shepherd, San Fernando, CA). These irradiated recipient
mice were then injected with 5 × 106 fresh bone marrow
cells (in 300 µL minimal essential medium) from +/+ or P/ male
donors. Transplanted animals were transferred to a sterile
microisolator unit and fed with sterile chow food and acidified (pH
2.5) sterile water for 4 weeks. After this recovery period, blood cell
counts and the expression of platelet P-selectin were assessed and the
mice were transferred to a conventional (nonsterile) environment.
Flow cytometry.
To examine P-selectin expression, 3 to 4 drops of blood, obtained by a
retro-orbital bleed from P/E/ transplanted mice and +/+ and
P/ controls were transferred into 1 mL solution containing 9 parts Hanks balanced salt solution (without calcium and magnesium) and
1 part ACD. PRP was isolated by centrifugation (280g for 5 minutes at room temperature), and the platelets were washed five times
(centrifugation 2,000g for 6 minutes) in PIPES buffer (pH 6.1)
and resuspended in 500 µL PIPES buffer at physiologic pH (7.4) for
activation with thrombin (0.2 U/mL) at 37°C for 15 minutes. Thrombin
was removed by a single wash with PIPES buffer, and the platelets were
resuspended in PBS-0.01% bovine serum albumin (BSA). Activated
platelets were stained for P-selectin using a rabbit polyclonal
antibody 1:100 (PharMingen, San Diego, CA) and
fluorescein-conjugated sheep anti-rabbit antibody 1:250 (Cappel,
Durham, NC). Analysis of 10,000 events was performed on a
FACSCaliber flow cytometer (Becton Dickinson, Mountain
View, CA).
Thioglycollate-induced peritonitis.
Mice were injected intraperitoneally with 1 mL 2.95% thioglycollate
(Sigma). Blood was obtained by retro-orbital sampling 6 hours after
thioglycollate injection. The animals were killed, and peritoneal
lavage was performed using 10 mL PBS containing 0.5 mmol/L EDTA, 1 U/mL
heparin, and 0.1% BSA. Total cell numbers were determined with a
Coulter (Hialeah, FL) counter. The absolute number of
neutrophils, eosinophils, monocytes, and lymphocytes were calculated
from differential counts of Wright-stained cytospin preparations.
Statistical analysis.
All values are reported as the mean ± SEM. Statistical significance
for continuous variables was assessed by Student's t-test. The
log-rank test was used to determine statistical significance between
probability curves (prevalence of dermatitis).
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RESULTS |
Platelets interact with both endothelial selectins in inflamed venules.
To examine whether selectins are important in platelet-endothelial
interactions in venules during inflammation, we transfused fluorescently labeled resting +/+ platelets into TNF--treated +/+
or P/E/ recipient mice and rapidly prepared the animals for
intravital microscopy. We observed rolling interactions with the
endothelium (Fig 1A) similar to what we
described in calcium ionophore (A23187)-stimulated +/+
venules.15 Significantly lower numbers of rolling platelets
were seen in TNF--treated P/E/ recipient mice, suggesting
that platelets roll on at least one of the endothelial selectins during
inflammation. To investigate which endothelial selectin mediates
platelet rolling, we transfused resting +/+ platelets into P- or
E-selectin singly deficient mice. Venules of P/ mice treated with
TNF- displayed a reduced flux of rolling platelets compared with
that of +/+ recipient mice (Fig 1A). In TNF--treated E/
venules, the rolling activity was reduced further in comparison to +/+
venules, suggesting that under these experimental conditions platelets
interact preferentially with E-selectin (Fig 1A). To evaluate whether
platelet P-selectin contributed to this interaction, we transfused
P/ platelets into TNF--treated P/ and P/E/
recipients. The frequency of P/ platelet rolling was similar to
that of +/+ platelets, confirming that platelets roll on endothelial
selectins in inflammation.
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| Fig 1.
Resting platelets interact with both endothelial P- and
E-selectins in TNF--stimulated venules. Gel-filtered fluorescent wild-type, P/, or FucT IV/VII/ platelets were transfused
into wild-type, P/, E/, or P/E/ recipient mice. The
number of platelets (A) rolling and (B) captured over a 250-µm
venular segment was determined; n = 5 to 9. *P < .05, **P < .01: v P/E/.
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Along with the rolling activity, we noted that many platelets stuck to
the venular wall or interacted briefly without fulfilling our criteria
for rolling. Although the majority of these interactions were of a
transient-sticking type, some platelets adhered for a prolonged
duration on TNF--treated venules. The fate of sticking platelets
could not be evaluated in this system, due to the rapid bleaching (<1
minute) of the fluorescent dye. We determined the number of platelets
captured by the vessel wall but not rolling per minute over the same
250-µm venular segment (Fig 1B). Removing one endothelial selectin
does not affect the frequency of platelet capture by the vessel wall
compared with that of +/+ mice, but the absence of both selectins
diminished these interactions by about threefold. Again, similar
numbers were obtained from transfusion of resting P/ platelets
into P/ and P/E/ recipients (Fig 1B). These results suggest
an overlapping role for endothelial P- and E-selectins in platelet
interactions with inflamed venules, similar to what has been seen for
leukocyte adhesion in other systems.11-13,18,19 The fact
that hemodynamic parameters were similar among venules of various
genotypes rules out hemodynamic influences on these findings (Table
1).
FucT IV and FucT VII do not endow platelets with selectin ligand
activity.
Given the similar dependence on endothelial selectins for leukocyte and
platelet rolling, we suspected that the (1,3)fucosyltransferases responsible for leukocyte selectin ligand expression (FucT IV and FucT
VII20) might contribute to E- and P-selectin ligand
activity on platelets. We therefore isolated platelets from FucT IV/VII
double knockout mice and transfused them into TNF--treated P/
and P/E/ recipients. Surprisingly, FucT IV/VII / platelets
rolled and were captured as frequently as wild-type platelets in
P/ venules, and similarly, the interaction was greatly reduced in
P/E/ venules (Fig 1A and B). These data suggest that FucT IV and
FucT VII do not play a significant role in the synthesis of platelet
selectin ligands.
P-selectin on activated platelets can mediate their capture and
rolling on a TNF--inducible endothelial ligand.
Following the release of Weibel-Palade bodies by A23187, a few minutes
after the exteriorization of the mesentery, activated platelets
(expressing P-selectin) do not interact with P-selectin-deficient endothelium.15 To investigate whether the situation changes in inflammation, we transfused fluorescently labeled thrombin-activated +/+ or P/ platelets into TNF--treated P/E/ mice.
P/E/ animals are useful to examine interactions between activated
platelets and the endothelium during inflammation, since leukocyte
rolling is virtually absent in P/E/ mice12 (and
activated +/+ platelets tend to bind rolling
leukocytes15). Resting platelets rolled and were captured
minimally under these conditions (Fig 1). We observed numerous
interactions of +/+ activated platelets in inflamed venules of
P/E/ mice, whereas very little capture and rolling was seen when
activated P/ platelets were injected or when mice were not
treated with TNF- (Fig 2). These results
indicate that activated platelet P-selectin promotes adhesion to
inflamed endothelial cells, and that this can occur in the absence of
endothelial selectins.
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| Fig 2.
P-selectin of activated platelets can mediate
interactions with TNF--treated venules in P- and E-selectin
double-deficient mice. Number of (A) rolling and (B) captured platelets
per minute; n = 5 to 8. *P < .05 v the other 2 groups; **P = .01 v P/ platelets.
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Transplantation of wild-type bone marrow into P- and E-selectin
double-deficient mice does not rescue the immunodeficient phenotype.
It has been hypothesized that platelets might be recruited in numbers
large enough to cover segments of inflamed venules and, through their
high surface expression of P-selectin, actively participate in the
recruitment of leukocytes. Supporting this concept, recent studies have
demonstrated that leukocytes can roll and extravasate in vitro on a
monolayer of activated platelets expressing
P-selectin.21-23 To test whether platelet P-selectin could
mediate leukocyte recruitment to sites of inflammation in vivo, we
transplanted wild-type bone marrow (containing +/+ megakaryocyte precursors) into P/E/ mice. We asked three questions: Would platelets expressing P-selectin (1) reduce the severe leukocytosis of
P/E/ mice, (2) limit the incidence of bacterial dermatitis in
these mice, and (3) improve the extravasation of leukocytes in the
thioglycollate model of peritonitis?
In two independent experiments, a total of 38 P/E/ female mice
were divided into two groups (by littermate pairs) and received either
+/+ or P/ bone marrow after a lethal dose of irradiation. The
mice were allowed to recover for 4 weeks before expression of platelet
P-selectin was assessed by flow cytometry. All mice survived the
procedure. Platelets of mice transplanted with +/+ marrow displayed
mean fluorescence levels of P-selectin comparable to wild-type
nontransplanted controls, and animals reconstituted with P/
marrow remained negative for P-selectin (data not shown). At week 5 after reconstitution, blood cell counts were determined. P/E/
mice harboring +/+ circulating platelets displayed total leukocyte and
differential counts comparable to those of mice transplanted with
P/ bone marrow (Table 2). This
suggests that, unlike endothelial P-selectin, platelet P-selectin does
not play a significant role in leukocyte homeostasis. Reconstituted
animals were observed periodically for occurrence of the typical
spontaneous bacterial dermatitis seen in P/E/ mice.12
No significant difference in the incidence of skin infection was
observed over a 4-month period regardless of whether mice carried
platelet P-selectin (Fig 3).
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| Fig 3.
Probability of spontaneous occurrence of infectious
dermatitis in P- and E-selectin double-deficient mice after bone marrow transplantation. Lethally irradiated P/E/ recipient mice were reconstituted with either +/+, ( ) or / ( ) bone marrow.
Mice were periodically observed for the presence of dermatitis; n = 19. P = .31 (log-rank test).
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To investigate if platelet P-selectin would influence the recruitment
of leukocytes during experimental inflammation, we performed bone
marrow transplantation using +/+ and P/ bone marrow cells in
another cohort of +/+ and P/E/ lethally irradiated mice. Five
weeks after transplantation, P-selectin expression in platelets of mice
reconstituted with +/+ marrow was similar to that of wild-type controls, while the platelets of mice transplanted with P/
marrow, predictably, were negative (data not shown). These bone
marrow-reconstituted mice were injected intraperitoneally with
thioglycollate. After 6 hours, blood samples were collected and
peritoneal lavage was performed. P/E/ mice reconstituted with +/+
megakaryocyte precursors did not show any difference in the circulating
neutrophil count (Fig 4A) or the number of
neutrophils recruited into the peritoneum (Fig 4B) compared with
P/E/ animals transplanted with / megakaryocyte precursors.
The proportions of monocytes, eosinophils, and lymphocytes also were
not significantly different (data not shown). Similarly, neutrophil
emigration to inflamed peritoneum was not altered in +/+ mice harboring
P/ platelets compared with +/+ mice transplanted with +/+
platelets (Fig 4B). As previously demonstrated,12 the recruitment of neutrophils into the thioglycollate-inflamed peritoneum of both groups of P/E/ mice was significantly impaired compared with that of wild-type animals. Taken together, these results indicate
that despite its interactions with inflamed endothelium, platelet
P-selectin does not appear to participate in leukocyte homeostasis or
in the recruitment of leukocytes into infected or inflamed sites in P-
and E-selectin doubly deficient mice. In addition, platelet P-selectin
does not appear to potentiate neutrophil efflux to inflamed peritoneum.
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| Fig 4.
Peripheral neutrophil count and peritoneal neutrophil
influx after thioglycollate administration. +/+ or P/ bone
marrow cells were transplanted ( ) into irradiated +/+ or
P/E/ recipients. Mice were allowed to recover for 4 weeks before
being challenged with thioglycollate for 6 hours. Blood cell counts
were obtained with a Coulter counter, and (A) the absolute neutrophil
number/µL was determined from Wright-stained smears. Nucleated cell
numbers were also determined by Coulter counter on lavage fluids from the peritoneum, and (B) the neutrophil number per lavage was calculated from differential counts performed on Wright-stained cytospin preparations; n = 7 to 8. *P < .0005 versus wild type.
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DISCUSSION |
During inflammation, several adhesion molecules such as E-selectin,
P-selectin, VCAM-1, and ICAMs are induced and/or upregulated on
endothelial cells.24 This translates in vivo into an
increase in leukocyte rolling and adherence on venules.25
Our present study shows that, similar to leukocytes,
platelet-endothelial interactions are mediated by endothelial adhesion
molecules upregulated during inflammation. We analyzed both the rolling
and the capturing rates of platelets on the venular wall and found that
either P- or E-selectin could mediate these adhesion events. Although
each endothelial selectin captured free-flowing platelets equally well (Fig 1B), the platelet rolling flux was greater on E-selectin (P/
recipients) during inflammation (Fig 1A). Platelet sticking and rolling
did not require platelet P-selectin, since P/ platelets behaved
similarly to +/+ platelets (Fig 1).
Although the behavior of platelets appears to parallel that of
leukocytes in many aspects, differences are also highlighted by the
present study. Rolling is thought to be mandatory for leukocytes for
subsequent firm adhesion, but it did not appear to be a prerequisite for platelets to arrest on venular endothelium under flow in vivo. Indeed, many platelets were captured without prior rolling (Fig 1B).
Another main difference is the surprising lack of a need for leukocyte
FucT IV and FucT VII for the biosynthesis of platelet selectin
ligand(s), as demonstrated by the similar behavior of FucT IV/VII
/ platelets and platelets derived from +/+ mice (Fig 1A and B).
These two enzymes have been shown to provide most of the selectin
ligand activity expressed by leukocytes.20 However, the
fact that mice deficient in both leukocyte (1,3)fucosyltransferases have a milder phenotype (e.g., they do not develop infectious dermatitis) than P/E/ mice suggests that other selectin ligands (fucosylated or nonfucosylated) are functional in vivo. Alternatively, the relative protection from skin infections of FucT IV/VII / mice still expressing endothelial selectins might also result from
normal platelet-endothelial interactions that may be important for
normal host resistance to infections. Furthermore, our results indicate
that a ligand(s) for E- and P-selectins is constitutively expressed on
the surface of platelets. Such a ligand(s) is likely to bear specific
carbohydrate moieties carried by a protein or a glycolipid
scaffold.26 Interestingly, purified glycolipids (gangliosides) from human platelets contain sialyl-Lewisx
epitopes,27 and similar glycolipids isolated from human
myeloid cells have been shown to bind E-selectin.28,29
P/E/ venules stimulated with calcium ionophore15 or
with surgical manipulation (Fig 2) show little interaction with
transfused +/+ activated platelets. In contrast, TNF- induces in
vivo binding of activated platelets with venular endothelium that is
dependent on platelet P-selectin and does not require endothelial
selectins (Fig 2). The nature of the endothelial P-selectin ligand in
systemic venules is speculative at the moment. Notably, activated
platelets were recently described to interact in high endothelial
venules with a cognate molecule expressed constitutively that shares
characteristics with L-selectin ligands.30 In systemic
venules, an inducible endothelial L-selectin ligand has been previously
invoked to explain defects of leukocyte rolling in
L-selectin-deficient mice.31 This inducible endothelial
ligand for L-selectin may therefore be a potential candidate for
binding P-selectin in these venules. P-selectin glycoprotein ligand-1
(PSGL-1) represents another candidate counterreceptor for platelet
P-selectin. PSGL-1 is expressed constitutively on polymorphonuclear and
monocytic cells and may interact with all three selectins.2
Although immunohistochemical staining revealed PSGL-1 expression in the
venules of some pathologic tissues, it was not detected in tissues
undergoing inflammation.32
Thus, platelets may be recruited to the wall of an inflamed venule in
different ways. Resting platelets may roll on endothelial selectins or
be captured directly. Likewise, activated platelets, through platelet
P-selectin, may be captured or roll in a similar fashion on inflamed
venules. What are the biologic roles of selectin-mediated platelet-endothelial interactions? Platelets contain a wide range of
biologically active materials that may modulate certain inflammatory responses.33 For example, platelet membranes possess IgE
receptors that support the cytotoxic functions of platelets in
parasitic infections such as Schistosoma mansoni
infection34 and filariasis,35 and additionally,
platelet IgE receptors may initiate T-cell-dependent contact
sensitivity.36 Although, by itself, platelet P-selectin does not influence the immunodeficient phenotype of P/E/ mice, it
is possible that platelet and endothelial selectins together reinforce
sufficiently reciprocal interactions to allow platelets to contribute
to inflammatory responses.
As suggested by the P-selectin-dependent fibrin deposition and
leukocyte accumulation within a Dacron graft-induced
thrombus37 and the greater hemorrhagic tendency seen in
P-selectin-deficient mice,16 selectins expressed on the
endothelium or on platelets may also participate in hemostatic
mechanisms. It is interesting that platelets also roll on von
Willebrand factor (vWf) at high shear forces in
vitro.38 Complementing the dynamic adhesion of platelets
with vWf on injured vessels at high shear, selectin-mediated platelet-endothelial interactions may represent key components of a
surveillance system maintaining the integrity of inflamed venules, and
upon vascular injury, they may contribute to platelet plug formation.
Although the individual functions of selectins, vWf, fibrinogen, GpIb,
and GpIIb/IIIa in hemostasis and thrombosis have not yet been fully
defined, mice deficient in these adhesion molecules will permit further
dissection of these pathways through in vivo experimentation.
| |
FOOTNOTES |
Submitted August 15, 1997;
accepted October 2, 1997.
Supported in part by National Institute of Health Grants No. PO1
HL-56949 (D.D.W.) and PO1 HL-41484 (R.O.H.), an Investigator Award from
the Howard Hughes Medical Institute (R.O.H.), and a Fellowship from the
Medical Research Council of Canada (P.S.F.).
Address reprint requests to Denisa D. Wagner, PhD, Center for Blood
Research, 800 Huntington Ave, Boston, MA 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Mollie Ullmann-Culleré for mouse husbandry.
| |
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Abstract
Introduction
Abstract