Surface membrane clearing of receptor-ligand complexes in human
blood platelets
O. BEHNKE
Institute of Anatomy C, University of Copenhagen, The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen X
Summary
Human blood platelets were challenged sequentially in vitro with polyclonal anti-platelet antibodies and cationized ferritin. Both ligands
bound to the surface membrane and were sequestered by internalization into a surface-connected
membrane system (SCS) with a cleared surface
membrane as the eventual result. Patching and
capping of bound antibody preceded internalization, and platelets cooperated in the clearing
process by adhering to each other at capped areas
and by mutual covering up, a process dubbed
platelet hugging. The internalization process was
repeated upon challenge 'with the second ligand,
the two ligands being sequestered as separate
deposits in the SCS, mirroring the two cycles of
internalization. Platelets were activated during
the internalization process and tended to aggregate. In aggregates, surfaces exposed to the medium were cleared of ligand, which accumulated
in the intercellular spaces and within the SCS of
the aggregated platelets. Aggregation but not internalization and hugging was prevented by adenosine and adenosine monophosphate.
Introduction
granule contents during the platelet release reaction
(White, 1972a), and various particulate substances are
taken into the SCS (White, 19726; Zucker-Franklin,
1981; Suzuki et al. 1985; Kawakami & Hirano, 1986).
It remains unsolved, however, whether small particulates just percolate through and become trapped in the
system or whether binding to the surface membrane
proper is a prerequisite for uptake. Furthermore it is
not clear whether material is taken into the 'preexisting
SCS', or whether new imaginations of the surface
membrane arise and thus by definition become SCS.
Mammalian blood platelets have three main functions:
they play a key role in haemostasis and thrombosis
(Frojmovic & Milton, 1982; Silver, 1981), they supply
a growth factor (Rosenfeld et al. 1984; Heldin et al.
1985), and they participate in sequestering foreign
matter in plasma (e.g. see review by Gordon & Milner,
1976). While the former activities have received much
attention, the implications of the platelet as a sequestering unit seem less appreciated, despite the fact that the
mass of circulating platelets in adult man represents a
substantial, reactive surface membrane area of about
25 m 2 , to which should be added the area of the
labyrinthine surface-connected system (SCS) (Behnke,
1967). In addition, the activated platelet can increase
its surface area considerably by transforming to a spiny
sphere or through adhesion to and spreading on a
wettable surface (Frojmovic & Milton, 1982); in the
latter case the surface area may increase up to four
times (Behnke & Tranum-Jensen, 1978).
The SCS is a dynamic membrane system, reflecting
the platelet's ability to invaginate its surface membrane. The system serves as a secretory pathway for
Journal of Cell Science 87, 465-472 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
Key words: blood platelets, membrane dynamics,
antibodies, sequestration, spreading.
Here structural observations are reported on the
response of platelets to a challenge by heterologous
anti-platelet antibodies and to a non-specific challenge
of anionic surface groups by cationized ferritin.
Platelets showed the same response to both challenges:
individual platelets cleared the ligands from their
surface by internalization, and sequential challenges
elicited repeated cycles of internalization. Platelets
cooperated in covering up ligand-blocked surfaces by
forming small aggregates in which only cleared surfaces
were externally exposed.
465
Materials and methods
Platelet-rich plasma (PRP)
This was produced by differential centrifugation of citrated
human venous blood at 37 °C in a thermostatically controlled
centrifuge. The PRP was kept at 37°C until used for
experiments (usually within 30min).
Washed platelets
The method of Walsh (1972) was used, with a minor
modification, to separate platelets from plasma. Briefly, 10 ml
of PRP was layered on top of 2 ml of 10% bovine serum
albumin (BSA, Sigma, fraction V) dissolved in phosphatebuffered saline (PBS) or Ca2+-free Tyrode solution. Below
the 10% BSA was then layered 40% BSA in Ca2+-free
Tyrode (1 ml to 10 ml of PRP) and the tube centrifuged for
15min at 2800revsmin~' (=1000 £). Platelets sedimented
onto the 40% BSA. The platelet-poor plasma was removed
together with the 10% BSA layer, 9 ml of Ca2+-free Tyrode
was then added and the platelets resuspended. This procedure was repeated once, and the final suspension was
named a Walsh suspension.
Antibodies
The polyclonal rabbit anti-human antibody towards platelet
membranes (APA) was a generous gift from Dr O. Bjerrum,
Copenhagen (Hagen et al. 1979). The antibody was not
adsorbed. The horseradish peroxidase(HRP)-conjugated
swine anti-rabbit anti-immunoglobulin used as second antibody was from Dakopatts, Copenhagen.
times (2-4-8-15, 30, 60min) by adding 3 vol. 2 % glutaraldehyde in 02M-sodium cacodylate buffer at pH7. Second
antibody-labelled platelets were fixed for 15min by adding
1 vol. of glutaraldehyde, then sedimented and rinsed for
30min in 02M-cacodylate buffer. Peroxidase activity was
visualized in a diaminobenzidine medium (Graham &
Karnovsky, 1966).
To prevent aggregation due to ADP release during rewarming, some experiments were done with adenosine or
adenosine monophosphate ( 1 0 ~ 4 M ) added to all media.
To observe the effects of APA alone, it was added directly
to 37°C Walsh suspensions for 15 or 30min, or samples of
platelets labelled with APA in the cold were fixed during
rewarming at the times given above.
Specimens were postfixed in 1 % osmium tetroxide in
0-2M-cacodylate buffer, and embedded in Epon.
Repeated
challenge incubations. APA-PAG-labelled
platelets were washed through BSA after 15 or 30min of
rewarming, resuspended and cationic ferritin (CF, MilesYeda Ltd, lot CF/9) was added at a concentration of
20^gml~' platelet suspension. The mixtures were incubated
at 37°C for 5, 15 or 30min. Platelets were then sedimented,
fixed in glutaraldehyde and processed for electron microscopy.
'Inert tracers'. Walsh suspensions were incubated at 37°C
or 0-4 c C for 1 h with BSA-stabilized or haemoglobinstabilized colloidal gold (100/tl stock suspension ml" 1 platelet
suspension). The 37°C suspensions were then fixed directly;
the cooled suspensions were rewarmed to 37CC for 1 h in the
continued presence of the colloidal gold, and then fixed.
Whole-mount preparations
Staphylococcal protein A-gold conjugates (PAG)
Colloidal gold suspensions (=12nm (Frens, 1973) and
=5 nm (Faulk & Taylor, 1971)) were stabilized with staphylococcal protein A (SpA, Pharmacia) following established
procedures (Geoghegan & Ackerman, 1977; Horisberger &
Roset, 1977; Horisberger & Vauthey, 1984). Stabilized
particles were sedimented and resuspended in PBS with
Carbowax 20000M r ( 0 - 2 m g m r ' ) (Horisberger, 1979) or
gelatin was added ( 0 ' l % , Merck, Bloom number 60—100)
(Behnke et al. 1986). The sedimented particles from 100ml
of suspension were resuspended in 2ml PBS ('stock solution'), stored at 0-4°C, and used within 3-4 weeks.
Gold suspensions (=12nm) stabilized with BSA (Sigma,
fraction V)) or human haemoglobin (twice crystallized,
Sigma) were used in control incubations.
Incubation procedures
Anti-platelet antibodies. Walsh suspensions were incubated with APA at 0-4°C for 1 h. The concentration of
antibody was adjusted to the number of platelets in each
experiment (30/ig protein/100000 platelets).
After incubation, platelets were washed twice at 0-4°C
through 10 % BSA onto 40 % BSA as described, incubated at
0-4°C for 1 h with either PAG (5 or 12 nm, using 50-100 /<ml
stock solution ml~' platelet suspension) or with the HRPconjugated second antibody (60 ^g protein ml" 1 suspension),
and finally rinsed through BSA and resuspended. Samples of
the suspension were fixed while still at 0 c C, the remainder
was rewarmed to 37°C and samples were fixed at various
466
O. Behnke
Suspensions of APA-PAG-labelled platelets were applied to
Formvar-carbon-coated, plasma-glowed electron microscope
grids while still at 0—4°C or after 15 or 30min of rewarming,
the grids were kept in a moist chamber for 15 min, then
rinsed in PBS, and negatively stained with 2 % sodium
silicotungstate at pH 7'2.
Light microscopy
Drops of APA-PAG-labelled platelets were applied to glass
microscope slides while still at 0— 4CC and studied with phaseinterference optics during rewarming to room temperature.
APA-PAG-labelled platelets were also studied after rewarming to 37°C, and double-challenged platelets after the second
challenge was completed.
Electron microscopy
Sections were contrasted with uranyl acetate followed by lead
citrate.
Results
Internalization of APA
Platelets exposed to APA in the cold and fixed before
rewarming were labelled exclusively at the surface
membrane proper. Upon rewarming to 37°C the PAG
marker patched, particularly at areas of the surface
where openings to the SDS were present (Figs 1,2),
but openings were also observed at non-patched areas.
In some platelets a single cap was observed. Platelets
did not regain their discoid shape during rewarming,
and they appeared activated as expressed by the
disappearance of granules. Internalized PAG accumulated in what appeared as empty granule cavities in
continuity with the SCS. Most platelets had internalized bound PAG within 30min of rewarming (Fig. 3)
and possessed only a few profiles of SCS devoid of gold
I-'
.•
Fig. 1. Platelet incubated in the cold with APA followed
by PAG, then rewarmed to 37°C and fixed after 8min.
PAG label has capped (arrow) at a segment of the surface.
The arrowhead indicates an entrance to the SCS in which
PAG label has accumulated. X29 000.
Fig. 2. Procedure as for Fig. 1, but fixed after 30min of
rewarming. PAG label (5 nm) has patched at several
regions (arrows). Multiple entrances to the SCS are seen
(arrowheads). The platelet is degranulated. X30000.
label. Openings of the SCS towards the exterior were
almost absent in single, non-aggregated platelets.
Internalization of the second surface challenger
Cationic ferritin (CF) binds to cell surfaces, primarily
to anionic, sialic acid-rich glycoproteins and gangliosides. Platelets that had cleared their surface membrane
of APA-PAG complexes readily accepted a second
challenge by CF, which in turn became internalized
and cleared from the surface proper (Fig. 4). The two
labels were largely confined to separate compartments
of the SCS that were in continuity with one another.
The compartment containing PAG did not contain
ferritin, while the compartment containing ferritin
showed some variable contamination with PAG.
Platelet cooperation in surface clearing
Some aggregation occurred during rewarming of
platelets tagged with APA in the cold. A characteristic
pattern of aggregation, described in the following as
'platelet hugging', was constantly observed both among
platelets labelled with PAG (Figs 5-6) or with a second
antibody (Fig. 7) and among platelets that had been
incubated with APA alone (not illustrated).
Platelets adhered to each other at patched and
capped membrane areas, and internalization of label
seemed to occur preferentially where two caps were
apposed (Fig. 5) because openings to the SCS were
located here exclusively. Frequently one platelet appeared to spread over another (Fig. 5), or the pair of
platelets hugged each other, forming a Yin-Yang
figure (Fig. 6). In both configurations the capped
membranes and the entrances to the SCS were thus
shielded from the medium.
Fig. 3. Procedure as for Fig. 2. The surface membrane of
this platelet is cleared of PAG label that has acccumulated
in the SCS. The platelet is degranulated. A few profiles of
SCS without label are seen (arrow). X26000.
Receptor-ligand complexes in platelets
467
The SCS cavities of cooperating platelets formed a
continuum. This was readily appreciated when bound
APA was visualized by the HRP-conjugated second
antibody. In Fig. 7 a triplet has cleared the surface
membranes, and the HRP/DAB reaction product is
•
*
_
Fig. 4. Platelet incubated in the cold with APA followed
by PAG and rewarmed to 37°C for 30min, then exposed to
CF for 5 min. The surface is cleared of both labels, which
are present in separate compartments of the SCS. The
compartment that contains PAG (arrow) contains no CF,
while there is some admixture of PAG to the compartment
containing CF (arrowhead). X30000.
•
confined to a tortuous channel system, representing the
SCSs of the three platelets in open continuity with each
other via the intercellular space.
In larger aggregates (Fig. 8) of doubly challenged
platelets the surface of the aggregate was in general free
of ligand, while apposed membranes in the interior of
the aggregates exhibited varying amounts of ligand.
The SCS of aggregated platelets opened onto apposed
Fig. 6. Two platelets hugging each other producing a
Yin-Yang figure. The SCS of one platelet is seen in
continuity with capped, adhering membranes (arrowhead).
Same procedure as for Fig. 5. X29 000.
•
••?• - - , . / •
Fig. 5. Platelet cooperation in surface clearing (twin
formation). Two platelets adhere to each other at capped
segments of their surface membranes. The inter-platelet
space is in continuity with the PAG-filled SCS of the
platelet to the left. The platelet to the right appears to
spread over the other. Both platelets are activated.
Procedures as for Fig. 3; 15 min rewarming. X32000.
468
O. Behnke
Fig. 7. Platelets incubated in the cold with APA followed
by HRP-conjugated second antibody, rewarmed for 30 min,
fixed and DAB-reacted. Section contrasted briefly with lead
citrate. The SCSs of a triplet are filled with DAB reaction
product and are in continuity with apposed platelet
membranes, forming one continuous cavity. X 19 000.
surfaces only, thus creating a large continuous labyrinth shielded from the surrounding medium.
Because PAG- and HRP-conjugated second antibody are multivalent probes, the possibility exists that
hugging and aggregation might be induced by crosslinking of platelets by PAG or second antibody bound
to APA on neighbouring platelets. However, parallel
incubations employing only APA revealed the same
morphological reaction as did platelets in which bound
APA had been visualized by a second marker.
Control incubations
Platelets incubated in Walsh medium at 0—4°C with
PAG, BSA-stabilized and haemoglobin-stabilized colloidal gold did not bind gold particles. When rewarmed
in the continued presence of the gold probes the
Effect of adenosine and adenosine monophosphate
Adenosine and adenosine monophosphate inhibit
ADP-induced platelet aggregation. Both compounds
prevented aggregation of APA-labelled platelets during
rewarming, but did not have any appreciable effect on
internalization, twin formation or platelet hugging.
Internalization as seen hi whole mounts
Platelets that had internalized bound antibody,
retained their ability to spread. In the light microscope
the bright red gold label was particularly well seen with
interference contrast optics, and spread, single
platelets showed label accumulated at their centres
(Fig. 9, inset). Small aggregates also spread, and gold
label accumulated in the centre of the aggregates. After
a second challenge, platelets spread poorly.
In platelets spread on grids, internalized PAG was
seen in the SCS in the central area. The surfaces of
spread platelets were essentially free of label. Platelets
that had formed small aggregates spread as an entity
(Fig. 10) with the PAG-containing SCS continuum in
the centre of the aggregate.
Fig. 8. Aggregate from double-challenge experiment. The
exposed platelet surfaces are almost devoid of the markers
that have accumulated in the SCSs and intercellular spaces
of the aggregate. X 15 000.
Fig. 9. Whole mount of platelet incubated in the cold with
APA followed by PAG. The platelet was then allowed to
rewarm during spreading on a grid for 30min at room
temperature, and negatively stained with sodium
silicotungstate at pH 7. PAG has accumulated in the SCS
at the centre of the platelet and the surface of the platelet is
essentially free of label. The arrow indicates an entrance to
the SCS. X 13 500. Inset: Platelets treated as for Fig. 9 but
spread on glass and photographed with Nomarski optics.
Both the spread single platelets (arrows) and the small
aggregates have accumulated PAG at their centres. X800.
Fig. 10. Same procedure as for Fig. 9. In this small
aggregate (consisting of five platelets) PAG has
accumulated at the centre of the aggregate. X7500.
Receptor-ligand complexes in platelets
469
platelets regained their disc shape and showed no signs
of activation or aggregation. Random sections showed
that 2-4 % of platelets contained a few gold particles in
the SCS.
Preincubation of APA-treated platelets with SpA
completely prevented subsequent binding of PAG,
provided SpA was continuously present during PAG
treatment (Behnke et al. 1986).
Discussion
Heterologous, polyclonal APAs induce thrombocytopenia in experimental animals (Ebbe & Stohlman,
1970) and provoke aggregation and release in human
platelets (Colman et al. 1977). Monoclonal antibodies
against epitopes on the platelet membrane glycoproteins GP Ib, l i b and Ilia have been shown to exert
different effects (Santoso et al. 1986); only antibodies
against epitopes of GP l i b and GP Ilia caused
patching and capping. Monoclonal antibodies to the
lib—Ilia complex have also been shown to have
different effects on various platelet functions (Heinrich
et al. 1985). We used a heterologous, polyclonal APA
(Hagen et al. 1979) and the observed effects are thus
the net result of binding of APA to a number of
epitopes.
The present study documents differences in platelet
behaviour when exposed to supposedly inert tracers
such as BSA- and haemoglobin-conjugated gold particles, and to compounds that interact specifically with
receptors in the surface membrane. A small number of
platelets exhibited a few particles of inert tracers in the
SCS; these platelets regained their disc shape when
rewarmed after cooling and they showed no sign of
activation. In contrast, platelets responded vigorously
to the formation of membrane ligand-receptor complexes and the ensuing events illustrate the emphasis
the platelet places on clearing its surface of the complexes. Polyvalent, non-immunological ligands such as
concanavalin A have also been described, in lightmicroscopical studies, to cause patching and capping of
platelet membranes (Bourgignon, 1984; Alexandrova
& Vasiliev, 1982).
Internalization of ligand-receptor complexes in
platelets may conceivably occur by one of two mechanisms: (1) complexes may patch/cap by lateral flow in
the plane of the membrane and the flow may continue
into a preexisting SCS; alternatively (2) the entire
membrane fabric at patched/capped areas may flow
into the platelet interior, perhaps at the preexisting
entrances to the SCS, or new invaginations may form.
Because the second-challenge internalization of CF
occurred into platelets that showed few or no openings
to the SCS (Kawakami & Hirano, 1986) and little or no
'vacant SCS' (Fig. 3), and because the ligands remained separated after the two internalizations had
470
0. Behnke
been completed (Fig. 4), the second mechanism seems
more likely. If so, the internalization calls for the
mobilization of a membrane reserve to compensate for
the internalized area.
A considerable reserve of membrane components
must be available as a precondition for the platelet's
impressive and rapid capacity for spreading (Behnke &
Tranum-Jensen, 1978; Frojmovic & Milton, 1982) but
its precise localization in the platelet is unknown. The
fact that platelets were able to spread after the first
internalization indicates that a membrane reserve was
still available; the poor spreading after the second
internalization suggests reserve exhaustion.
CF binds to anionic sites on cell membranes (Danon
et al. 1972), and is internalized by human (ZuckerFranklin, 1981; Yamazaki et al. 1984; Kawakami &
Hirano, 1986), rabbit (Suzuki et al. 1985) and rat
platelets (Behnke, unpublished); it is a potent platelet
activator causing aggregation and release, in contrast to
native ferritin that does not have these effects (Patscheke & Dierichs, 1986). Platelets that had been
activated by APA readily bound CF and repeated a
wave of internalization; thus activated platelets possess
anionic sites at the APA-cleared surface.
Platelets take up large particles such as opsonized
latex particles (Movat et al. 1965; White, 19726;
Zucker-Franklin, 1981; White & Clawson, 1982). The
particles are present in membrane-bound cavities continuous with the surface membrane, i.e. by definition
the SCS. However, during uptake latex particles may
be found in shallow invaginations of the surface independent of SCS openings (Movat et al. 1965; ZuckerFranklin, 1981), suggesting that large particles may be
incorporated by a mechanism different from that
operating during incorporation of small particles
(Zucker-Franklin, 1981).
We suggest that internalization of an APA-blocked
surface membrane, spreading on a wettable surface and
incorporation of large particles may all reflect the
platelets' reaction to a foreign surface. In the case of
APA binding the presence of receptor—ligand complexes all over the surface may be registered as 'overall
contact' with foreign matter; since the platelet is in
suspension and cannot spread, it internalizes its membrane instead. Spreading on a surface would be tantamount to internalizing an object of infinite size. The
hugging phenomenon might indicate that platelets that
have internalized APA-epitope complexes have surfaces that are mutually recognized as 'foreign' and
spreading/mutual incorporation is elicited.
The very common hugging phenomenon was not
inhibited by adenosine or AMP. The studies of Colman
et al. (1977) suggested that APA-induced platelet
aggregation was in part mediated by mechanisms
independent of ADP, because removal of ADP by
apyrase inhibited aggregation by only 50%. The
platelet cooperation phenomenon may explain the
observation of Colman et al.
HAGEN, I., BJERRUM, O. I. & SOLUM, N. O. (1979).
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