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1994 84: 390-396
CD44 can mediate the adhesion of platelets to hyaluronan
I Koshiishi, M Shizari and CB Underhill
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RAPID COMMUNICATION
CD44 Can Mediate the Adhesion of Platelets to Hyaluronan
By lchiro Koshiishi, Mehran Shizari, and Charles B. Underhill
CD44 represents a family of glycoproteins that are present
on the surfacesof some types of lymphocytes,macrophages, and epithelial cells. In the present study, we have
found that CD44 is also present on murine megakaryocytes
and peripheral blood platelets as judged by immunohistochemical staining. Western blotting of platelet proteins indicated that this CD44 was predominantly of the 85-kD form.
This form of CD44 also had the capacity to bind hyaluronan,
because detergent extracts of platelets as well as intact
platelets could bind soluble [3H1hyaluronan, and this property was blocked by antibodies directed against CD44. More
importantly, isolated platelets could attach to the hyaluronan-containing extracellular matrix produced by cultured
rat fibrosarcomacells.This attachment took place in the
absence of divalent cations and could be blocked by pretreating the ratfibrosarcoma cells with hyaluronidase or by
the addition of an antibody to CD44. Theseresults suggested
that CD44 was responsible for the attachment of platelets
to hyaluronan. Histochemical staining also showed that hyaluronan was present immediately beneath the endothelial
cells of many blood vessels of various tissues, such as the
dermis, lamina propria of the intestinal tract, the lungs, and
the pericardium. Thus, it is possible that CD44 plays an important role in theattachment of platelets to thesurface of
exposed connective tissue after injury to endothelial cells.
0 1994 by The American Society of Hematology.
P
the extracellular domain of CD44 specifically recognizes a
six sugar sequence of hyaluronan.8This property is responsible for the hyaluronan-dependent adhesion seen in several
types of cells. For example, when hyaluronan is added to
suspensions of pulmonary macrophages, they are induced to
aggregate, and this process can be blocked by antibodies
specifically directed against CD44.9.'" Similar phenomena
occur in a number of other cell types as well."-" Thus, the
fact that murine platelets express CD44 suggests that they
too may be able to bind to hyaluronan. The CD44-mediated
adhesion to hyaluronan may be an important aspect of platelet function, because hyaluronan is a prominent component
of the subendothelial extracellular matrix in some tissues.
LATELETS PERFORM the critical first step in hemostasis, namely the adhesionto exposed extracellular
matrix underlying endothelial cells. To perform this step,
platelets use different mechanisms depending on the circumstances.' Under conditions of high flow rates, such as that
found in arterial circulation, platelets adhere to von Willebrand factor attached to subendothelial surfaces. This interaction involves the GPIb-IX glycoprotein on the surfaces of
platelets and occurs onlyunder conditions of high shear
rate.' In contrast, under conditions of low flow rates associated with the venous circulation, receptors of the integrin
family mediate the attachment of platelets to proteins in
the extracellular matrix such as fibronectin, collagen, and
laminin.' In addition, nonintegrin receptors for collagen have
also been identified.' Some of these receptors bind ligands
constitutively, whereas others require the activation of the
platelets to be effective. Clearly, platelet adhesion is a complexphenomenonthat
involves a number of cell surface
receptors.
In the present study, we present evidence that platelets
from mice also express CD44, the receptor for hyaluronan.'.'
CD44 represents a family of cell surface glycoproteins ranging in size from 80 to greater than 200 kD that are present
on a variety of different cell types, including both leukocytes
and epithelial cells.5 Some, butnot all, isoforms of CD44
have the capacity to bind hyaluronan; however, the structural
For these isoforms,
basis for this difference is not
From the Department of CellBiology,GeorgetownUniversiw
Medical Center, Washington, DC.
Submitted March 5, 1994; accepted April 19, 1994.
Supported by U S HealthServiceGrants
No. HLA1565 und
H026758 to C.U .
Address reprint requests to Charles Underhill, PhD, Department
of Cell Biology, Georgetown Medical Center,
3900 Reservoir Rd,
Washington, DC 20007.
The publication costs of this article were defrayedin part by page
chargepayment. This article must therefore he herebymarked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8402-0039$3.00/0
390
MATERIALS AND METHODS
Preparationofbiochemicals. KM-201 is a rat monoclonalantibody (MoAb) against mouse CD44, which was originally isolated
by Miyakeetandblockstheinteractionbetweenhyaluronan
andCD44.Theantibody
was purified fromascites fluid of nude
micebychromatographyondiethylaminoethyl(DEAE)
Affi-gel
' ~was coupled to biotin (bblue columns as described p r e v i ~ u s l y .It
KM-201) with sulfosuccinimidyl6-(biotinamido)hexanoate (Pierce,
Rockford, IL) using the methods of Updyke and Nicolson."The rat
MO)
IgG used as a control was purchased from Sigma (St Louis,
and biotinylated in the same fashion (b-rat IgG).
The binding probe for hyaluronan (b-PG) was prepared from cartilageextractsasdescribedpreviously."Thisprobeconsists
of a
mixture of the link protein and a fragment of the proteoglycan core
protein. Previous studies have shown that the b-PG probe binds to
hyaluronan with high affinity and specificity.".Ix
The ['Hlhyaluronan waspreparedusinga
protocol previously
in 90%
described." For this, rat fibrosarcomacellsweregrown
Dulbecco's modified Eagle's medium (DMEM),
1 0 8 fetal calf serum supplemented with ['Hlacetate for 1 day. The conditioned medium was collected, digestedwith pronase, and dialyzed extensively
against distilled water. The ['Hlhyaluronan
was purified by precipitation with cetylpyridinium chloride and then redissolved in a saline
solution. The amount of hyaluronan was determined
by auronic
acid assay.*" The preparation of [3H]hyaluronan used in this study
had a specific activity of 8.9 X l o 4 c p d y g Na hyaluronan.
Isolation of platelets. Whole blood was collected from anesthetized mice, and mixed with 1/10 vol of 3.8% Na citrate. After lowspeedcentrifugation,thesupernatant
of platelet-rich plasma was
carefully removed and mixed with an equal volume of ACD buffer
Blood, V01 84, NO 2 (July 15). 1994: pp 390-396
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391
PLATELET ADHESION TOHYALURONAN
(0.13 mol/L NaC1, 20 mmol/L citrate, 1.0 mg/mL glucose, pH 6.0).
This was subjected to high-speed centrifugation and the precipitated
platelets were resuspended in ACD buffer. The platelet preparation
was washed two additional times by centrifugation followed by resuspension in ACD buffer. In some experiments, the platelets were
suspended in CMF-Tyrode's solution containing the following: 137
m o l / L NaCI, 2.7 mmol/L KC], 11.9 mmol/L NaHC03, 0.42 mmoV
L NaHZPO4,5.6 mmol/L glucose, 10 mmoVL HEPES, and 0.4 mg/
mL bovine serum albumin, pH 7.4.
Histochemistry. The liver and other tissues of a newborn mouse
were fixed overnight in a 3.7% formaldehyde solution in saline, then
dehydrated in a graded series of ethanol and water solutions, and
then embedded in 90% polyester wax, 10% ethanol as described by
Kusakabe et al." Sections 7-pm thick were cut on a cryostat at OT,
layered on a water bath, and then taken up on slides subbed with
egg albumin. The slides were dried overnight at 4°C and stored at
this temperature.
For staining, the sections were rehydrated in a graded series of
ethanol and water solutions and then incubated for 5 to 10 minutes
in 10% H,Oz to inactivate endogenous peroxidase. The slides were
incubated for 1 hour with the primary reagent that was dissolved
in 10% calf serum, 90% calcium- and magnesium-free phosphatebuffered saline (CMF-PBS). For the detection of CD44, the primary
reagent consisted of 8 pg1mLof b-K"201, and the background
was determined by substituting an equal concentration of control brat IgG. For the detection of hyaluronan, the primary reagent consisted of 10 pg1mL b-PG and the background was determined by
premixing theb-PG with 100 pg/mL hyaluronan. After thorough
washing, the sections were incubated for 20 minutes with peroxidase-labeled streptavidin (Kirkegaard and Perry, Gaithersburg, MD)
diluted 1 to 250 in 10% serum, 90% CMF-PBS. Finally, the sections
were incubated for 20 to 30 minutes in a peroxidase substrate consisting of 0.03% HzOl and 0.2 m g h L 3-amino-9-ethyl
The sections were counterstained for 1 minute in Mayer's hematoxylin followed by 0.5 mom Tris, pH 8.6, for 5 minutes, andthen
preserved in CrystaVMount (Biomeda, Foster City, CA). Coverslips
(1.5 weight) were attached using Histomount (National Diagnostics,
Highland Park, NJ).
Western blotting. The concentration ofproteinin
the extracts
was determined using the bicinchoninic acid method (Pierce). Samples containing 50 pg of protein were precipitated by the addition
of 70% ethanol and then redissolved in Laemmli sample buffer in
the absence of reducing agents.23After placing in boiling water for
5 minutes, the samples, along with prestained molecular weight
standards (high molecular weight; Bio-Rad, Richmond, CA), were
electrophoresed on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The proteins were then electrophoretically transferred to
a sheet of nitrocellulose (Immobilon; Millipore, New Bedford, MA)
at 0.9 A for 30 minutes using a transblot apparatus (Idea, Corvallis,
OR). The sheet of nitrocellulose was incubated in 5% nonfat milk
overnight to block residual protein binding sites. The sheet was then
immunostained by incubation in the following solutions: (1) 4 pg/
mL of b-KM-201 in 10% calf serum, 90% CMF-PBS containing
0.05% Tween 20 (blotting buffer) for 1 hour; (2) a 1 to 500 dilution
of peroxidase-labeled streptavidin in blotting buffer; and (3) a peroxidase substrate consisting of 0.03% HzOl and 0.2 mg/mL 3-amino9-ethyl carbazole in 0.05 mol1L Na acetate, pH 5.0.**
Binding of ['Hlhyaluronan. Hyaluronan binding activity was determined on both detergent-extracted and intact platelets. In the case
of the detergent-extracted platelets, the binding assay was performed
as previously de~cribed.'~,'~
Purified platelets were dissolved in DOC
buffer (0.1% Na deoxycholate, 0.5 m o m NaC1, and 0.02 mol/L
Tris, pH 8.0), and 460-pL aliquots of this extract were mixed with
2 pg of ['Hlhyaluronan and the volumes were adjusted to 500 pL.
After shaking for 20 minutes, 500 pL of saturated (NH4),S04 was
added to each tube followed by 50 pL of nonfat milk, andthe
samples were centrifuged at 9,OOOg for 5 minutes. The tubes were
rinsed twice with 50% saturated (NHJ2S04, and the pellets were
dissolved in water and processed for scintillation counting. The background level of binding was determined by including an excess
of nonlabeled hyaluronan (200 pg) in the assay mixture. In some
experiments, antibodies (KM-201 and nonspecific rat IgG) were included in the assay mixture.
In the case of intact platelets, the binding assay was performed
by a modification of a previously described technique.24The purified
platelets were suspended in CMF-Tyrode's solution and aliquots
were distributed into microfuge tubes (1 to 2 X 10' platelets in 500
pL), to which was added 2 pg of [3H]hyaluronan.After 30 minutes
of incubation, the samples were centrifuged (9,OOOg for 2 minutes)
and washed twice with the appropriate buffer. The cell pellets were
finally solubilized in a detergent solution and processed for scintillation counting. The background level of binding was determined by
including a large excess of nonlabeled hyaluronan in the assay mixture. The protein content was determined from representative samples extracted in detergent.
Attachment of platelets to rat fibrosarcoma cells. Rat fibrosarcoma cells that produce a large extracellular coat of hyaluronan were
cultured in 90% DMEM, 10% fetal calf serum in an atmosphere of
5% COz, 95% air.zs The cells were transferred to chambered slides
(3 to 5 X IO' cells/cmz, 4 cm'khamber; Nunc, Naperville, IL) and
grown for 2 days before fixation with 3.7% formaldehyde in PBS
for 10 minutes. In some cases, the cell surface hyaluronan was
digested by incubating the cells with Streptomyces hyaluronidase
(1 17 U1200 pL/chamber; Sigma) at 37°C for 1 hour. Mouse platelets
were fluorescently tagged by 5 minutes of incubation in CMF-Tyrode's solution containing 10 pmol/L 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate succinimidyl ester (Molecular Probes,
Eugene, OR).26 The platelets were then centrifuged, washedwith
ACD solution, and resuspended in CMF-Tyrode's solution. The
platelet suspension was transferred to the chambers (500 pL/chamber) containing the fixed rat fibrosarcoma cells and incubated for 1
hour. In some cases, 100 pg of the KM-201 MoAb was included in
the assay. The chambers were gently washed and then fixed with
3.7% formaldehyde in PBS for 10 minutes. The attachment of platelets to the hyaluronan-containing matrix surrounding the rat fibrosarcoma cells was evaluated by fluorescent microscopy.
RESULTS
Histochemical localization of CD44. In the course of an
immunohistochemical survey for CD44, we observed that
this protein was associated with a group of large cells with
lobulated nuclei present in the livers of new-born mice (Fig
l a and b show specific and control staining, respectively).
Similar cells were also found in bone marrow smears of
adult mice (data not shown). Based on their location and
morphology, these cells were identified as megakaryocytes.
It was interesting to note that, in these megakaryocytes, the
CD44 was not restricted to the cell surface, but was present
throughout the cytoplasm. This finding suggests that CD44
may be associated with the demarcation system, an internal
network of membranes continuous with the plasma membrane.
The fact that megakaryocytes expressed large amounts of
CD44 suggested that platelets derived from these cells may
also possess large amounts of CD44. To test this possibility,
platelets were isolated from theblood of adult mice and
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KOSHIISHI,SHIZARI, AND UNDERHILL
392
q:;w*
e,
,;
Fig 1. lmmunostaining of
CD44 on megakaryocytes and
platelets. The tissues were incubated with b-KM201 for CD44
immunostaining (a and c) or brat lgG for background staining
(b and d), followed in each case
bycounterstaining with hematoxylin. (a) Newborn mouse liver
stained with b-KM-201 shows
the presence of hepatic megakaryocytes that express CD44.
Note thelarge lobulated nucleus
and the presence of immunoreactivitythroughoutthecytoplasm of the cell. (b) Liver
stained with a nonspecific antibodyshows a lack of specific
staining (control fora). (c) Platelets
from
peripheral
blood
stained with b-KM-201 show the
presence of CD44. (dl Platelets
stained with a nonspecific antibodyshow a lack of staining.
Bars = 25 pm.
histochemically stained for CD44. As shown in Fig IC, the
isolated platelets were uniformly positive for CD44 (Fig I C
and d show both specific and control staining of platelets,
respectively).
Phvsical characteristics of platelet CD44. CD44 comes
in a wide variety of isoforms that differ in their molecular
weight and other physical characteristics. The isoform of
CD44 expressed by platelets was identified byWestern blotting. As shown in Fig 2, the majority of CD44 immunoreactivity of platelets was associated with a discrete band of 85
kD. Higher molecular weight isoforms were not observed.
To determine whether this isoform of CD44 could bind
hyaluronan, binding studies were conducted on detergent
extracts of platelets. Platelets isolated from mouse blood
were solubilized in a detergent solution of high ionic strength
and then examined for ['HH]hyaluronan binding by (NH&S04
precipitation as described in the Materials and Methods. As
shown in Fig 3, the extract of platelets did bind the isotopically labeled hyaluronan and this binding was blocked by
the addition of either an excess of nonlabeled hyaluronan or
by the KM-201MoAb directed against CD44. However,
nonspecific rat IgG had no effect on the binding. Thus, the
CD44 present in the platelets was capable of binding hyaluronan.
Binding of platelets hvaluronan.
to
To determine
whether platelets could bind hyaluronan under physiologic
conditions, intact platelets were suspended in CMF-Tyrode's
solution and mixed with ['Hlhyaluronan. After centrifugation, the amount of label presentin the pellet was determined.
Figure 4 shows that the intact platelets could bind the ['HIhyaluronan under physiologic conditions, and this binding
was again blocked by both nonlabeled hyaluronan and the
KM-201 MoAb. It should be noted that the amount of binding was significantly lower than that observed after detergent
205
115
45
-
Fig 2. Western blotofplatelet
CD44. Platelets isolatedfrom
mouse blood were electrophoresed on a 10% SDS-polyacrylamide
gel in the absence of reducing agents. The gel was electrophoretically
transferred t o a sheet of nitrocellulose that was then
stained for CD44
with b-KM-201. Only one immunopositive bandof approximately 85
kD wasapparent (indicated by arrow on the right).
The positions of
the molecular weight standards are indicated on the left.
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393
PLATELETADHESION TO HYALURONAN
solubilization because the assay was performed under conditions of low ionic strength. Previous studies have indicated
that binding of hyaluronan is a direct function of the ionic
strength of the medium.%
We then examined the binding of the platelets to insoluble
hyaluronan. For this examination, we used the rat fibrosarcoma cell line that produces a large pericellular coat of hya l ~ r o n a n ?Previous
~
studies have indicated that hyaluronan
is covalently linked to the surface of these cells and is retained after fixation with f~rmaldehyde.”~~~
Figure 5a shows
hyaluronan on the surfaces of fixed rat fibrosarcoma cells
after staining with the b-PG probe derived from cartilage.
Most of this staining is lost after treatment with Streptomyces
hyaluronidase (Fig 5b). Interestingly, much of the hyaluronan on the surface of the rat fibrosarcoma cells was associated with microvilli-like structures that projected from the
surface of the cells (see arrows inFigSa and c). These
structures may account for the large extent of pericellular
coat associated with these cells.
To measure the binding of platelets, the rat fibrosarcoma
10
8
T
T
6
4
T
2
T
0
None
HA KM-201 IgG
Component Added
3 t
T
2 -
1 -
0-
None
HA KM-201 IgG
Component Added
Fig 3. Binding of I’Hlhyaluronan by detergent-solubilizedplatelets. Isolated platelets were extracted in DOC buffer and samples
(331 p g protein) were mixed with [’Hlhyaluronan (2.0 pglsamplel.
Nonlabeied hyaluronan (200 pghamplel or KM-201 MoAb (100 &g/
sample) was added to some of the samples. Thefinal volume of each
sample was adjusted to 500 &L. After incubating at room temperature for 20 minutes, the samples were mixed with an equalvolume
of saturated (NHJzSO, along with 50 pL of nonfat milk as carrier.
After centrifugation, the amounts of label presentin the precipitates
were determined. The error bars represent the range of triplicate
determinations.
Fig 4. Bindingof {’Hlhyaluronan by intact platelets. Platelets were
isolatedfrom mouse blood and suspendedin CMF-Tyrode‘s solution,
and aliquots were mixed with [3Hlhyaluronan (2.0 p g l in a final volume of 500 CL. Nonlabeled hyaluronan(200pglsample) or antibodies
[KM-201or rat IgG, 100 pglsample) wera included in some samples.
After shaking at room temperaturefor 30 minutes, the samples were
centrifuged and the amounts of label presant in the pellets were
determined. The protein content was determined from representative samples after extraction in detergent. The error barn represent
the range of triplicate determinations.
cells were grown on slides and then fixed withformaldehyde.
Next, platelets that had been labeled with fluorescent dye
were added to the fixed rat fibrosarcoma cells and allowed
to settle. After 1 hour, the cultures were gently washed and
the attached platelets were examined by fluorescent microscopy. Figure 6a shows that a large number of platelets
attached to the fixed cultures of rat fibrosarcoma cells, and
most of this binding occurred on the microvilli that projected
from the surfaces of these cells. However, if the rat fibrosarcoma cells were pretreated with Streptomyces hyaluronidase,
then the attachment of platelets was diminished (Fig 6b),
suggesting that hyaluronan was required for the attachment.
Furthermore, the addition of KM-201 greatly diminished the
extent of binding (Fig 6c), whereas the addition of control
rat IgG had no apparent effect on this binding (Fig 6d).
These results indicate that CD44 mediates the attachment of
platelets to hyaluronan on the rat fibrosarcoma cells.
Distribution of hyaluronan in subendothelial connective
tissue. One important consideration is whether platelets
have the opportunity to come in contact with hyaluronan
after injury to endothelial cells. To address this issue, we
examined the distribution of hyaluronan in a variety of vas-
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
394
KOSHIISHI,SHIZARI, AND UNDERHILL
7
trast, in other tissues, large amounts of hyaluronan were
associated with blood vessels. Figure 7 shows the distribution of hyaluronan in the dermis, the lamina propria of the
intestinal track, the stroma of the lungs, and the pericardium
of the heart. In these tissues, hyaluronan was generally associated with the intima of veins and venules, immediately
beneath the endothelial cell lining. In contrast, in arteries, it
was generally reduced or absent from the intima, but was
present in the adventitia. Thus, there are a number of locations in which disruption of the endothelial cell lining could
result in the exposure of hyaluronan to the platelets.
DISCUSSION
b
'
Fig 5. Hyaluronan containing extracellular matrix surrounding
cultured rat fibrosarcoma cells. In each case, the cultured cells were
stained with theb-PG probe followed by counterstainingwith hematoxylin. (a) Cultured rat fibrosarcoma cells were stained for hyaluronan with the b-PG probe. The arrows point to microvilli-like structures that project from the cell surface. (b) Rat fibrosarcoma cells
treated with Streptomyces hyaluronidase show a decreased staining
for hyaluronan.IC)A higher magnificationview of a cell in (a) showing
the microvilli-like structures projecting from the surface of the rat
fibrosarcoma cells, which stain positively for hyaluronan (indicated
by arrows). Bar = 25 pm.
cular tissues. The results of this survey showed that the
distribution of hyaluronan varied depending on the tissue
and the type of the blood vessel. In some tissues, such as
the liver and spleen, only small amounts of hyaluronan were
associated with the blood vessels (data not shown). In con-
The present study presents several lines of evidence that
CD44 could, at least in mice, play a role in the attachment
of platelets to subendothelial connective tissue. First, CD44
ispresenton
platelets as demonstrated by immunohistochemistry and Western blotting, which also showed that it
consisted of the most common isoform with a mass of 85
kD. Secondly, this isoform of CD44 could interact with hyaluronan, because antibodies directed against this protein
blocked the binding of [3H]hyaluronan by both intact and
detergent solubilized platelets. Thirdly, CD44 mediated the
attachment of platelets to a substratum of hyaluronan consisting of fixed ratfibrosarcomacells. This adhesion occurred
under physiologic conditions of ionic strength and pH, and
was blocked by enzymatically removing the hyaluronan or
by blocking the CD44 with antibodies. And finally,the distribution of hyaluronan as assessed by histochemical staining
was compatible with a functional role in hemostasis. As
reported here and in other studies, hyaluronan is a prominent
component of the intima of some blood vessel^."^^^ Indeed,
biochemical and electron microscopic studies haveshown
that it is often closely associated with some types of collagen.3oThus, if the endothelial cells were damaged in some
fashion, then in many cases hyaluronan may be one of the
major components of the extracellular matrix encountered
by platelets.
We also observed that hyaluronan was not uniformly distributed in the walls ofallbloodvessels.
In general, the
amount of hyaluronan associated with the blood vessels was
a reflection of the amount of hyaluronan in the tissue as a
whole. For example, hyaluronan is abundant in the blood
vessels of the skin but not of the spleen, tissues that have
large and smallamounts of hyaluronan, respectively. In addition, we have observed that more hyaluronan was associated
with the intima of veins and venules than with arteries and
arterioles. In the case of the aorta, hyaluronan appeared to be
absent from the intima but was prominent in the ad~entitia.~'
These findings are consistent with earlier studies suggesting
that hyaluronan is synthesized by vascular smooth muscle
cells and adventitial fibroblast$ but not by endothelial
However, Amanuma and M i t d 3 have noted that transformation of endothelial cells induces the synthesis of hyaluronan. Thus, it is possible that, under certain situations, endothelial cells maybe able to produce hyaluronan to which
platelets can adhere.
Previous studies have noted that, under normal conditions,
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
395
PLATELET ADHESION TO HYALURONAN
Fig 6. The bindingofplatelets t o hyaluronan. Fixed rat
fibrosarcoma cells wereincubatedfor 1 hour with fluorescently labeled mouse platelets,
rinsed, and thenexamined under
a fluorescent microscope. (a) In
the absence of additional treatments, the fluorescently tagged
platelets attached t o hyaluronan
containingmatrixsurrounding
the fixed rat fibrosarcoma cells.
(bl Pretreating the rat fibrosarcoma cells with Streptomyces
hyaluronidase (117 Ulchamber)
reduced the attachment of platelets. (cl The addition of the KM201 MoAb (100 pglchamber)
also reduced the number of
attached platelets. (dl The addition of control rat IgG (100 p g l
chamber) did not prevent the attachment of platelets t o rat fibrosarcoma cells. Bar = 50 pm.
the level of hyaluronan circulating in the blood is maintained
at low levels.2"~z4 Although hyaluronanin the body is turned
over at a rapid rate, most of it is removed from the lymph
by thelymphnodesbeforereachingtheblood,and
that
which does enter theblood is rapidly removed by endothelial
cells lining the sinusoidsof the liver.z4 It is possible that this
system has evolved to remove hyaluronan, so that it does
not interfere with the interaction of platelets withhyaluronan
in the subendothelial connective tissue.
Along theselines,Bracey ethavedescribed
a patient
with Wilm's tumor who had abnormally high levels of hyaluronan circulating in the blood (8.5 mg/mL). The platelets
of this patientdid not function normally, resulting in a severe
clotting disorder. This disorder wasresolved when the tumor
was removed and the level ofcirculating hyaluronan returned
to normal.A similar type of disorder was obtainedwhen
rabbitswereinjectedwith
hyaluronan.'5 Although part of
the platelet dysfunction was attributed to the viscosity of the
hyaluronan, it is possible that an inhibition
of the platelet
adhesion system could also have been involved.
c
F*>,&
"
I
-
_."V
...
.,.*e-
'
.
.
.
Fig 7. The distribution
of
hyaluronan in subendothelial connective tissue. Sections of the(a)
skin, (b) intestine, (cl lung, and
(d) heart were stained for hyaluronan with theb-PG probe. The
arrows in each panel point t o the
hyaluronan foundimmediately
beneath endothelial cells of
veins and venules. In (bl, the arteriole of the righthas a layer of
smooth muscle that is deficient
in hyaluronan. Please note that
the open spaces surrounding the
blood vessels in (a) and (b) are
artifacts caused by tissue shrinkage during processing. Bar = 25
Pm.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
KOSHIISHI, SHIZARI, AND UNDERHILL
396
In conclusion, the CD44-mediated adhesion mechanism
should be considered as part of a multifunctional system that
platelets useto initiate hemostasis. Platelets use different
mechanisms of adhesion depending on the flow conditions
and the structure of the blood vessel. Given the distribution
of hyaluronan, the CD44-mediated adhesion of platelets is
most likely to occur in the venous circulation of the skin,
intestines, heart, and other organs containing a hyaluronanrich extracellular matrix.
ACKNOWLEDGMENT
The authors thank Dr Martine Culty and Prasit Pavasant for their
help in performing this study.
REFERENCES
1. Roth GJ: Platelets and blood vessels: The adhesion event. Immunol Today 13:100, 1992
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