© 1997 American Heart Association, Inc.
Articles |
Native Macromolecular Heparin Proteoglycans Exocytosed From Stimulated Rat Serosal Mast Cells Strongly Inhibit Platelet-Collagen Interactions
From the Wihuri Research Institute, Helsinki, Finland.
Correspondence to Riitta Lassila, Wihuri Research Institute, Kalliolinnantie 4, SF-00140 Helsinki, Finland. E-mail riitta.lassila{at}wri.fimnet.fi.
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Abstract |
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Abstract Mast cells, the major source of tissue heparin, line the vascular system. On stimulation, rat serosal mast cells release soluble heparin proteoglycans (HEP-PGs) of very high molecular weight (750 000). We compared the effects of HEP-PGs and standard heparins (average molecular weights, 15 000 and 5 000) on platelet-collagen interactions in vitro. In contrast with the standard heparins, HEP-PGs completely inhibited collagen-induced platelet aggregation and serotonin release in platelet-rich plasma. The inhibition caused by HEP-PGs depended on its macromolecular structure. In flowing blood, HEP-PGs also inhibited platelet deposition on a collagen-coated surface both at low and high shear rates. Although HEP-PGs did not block glycoprotein (GP) Ia/IIa-mediated platelet adhesion, they attenuated subsequent platelet activation and aggregation, as well as fibrinogen binding to platelets after collagen stimulation. HEP-PGs did not bind to platelets but bound tightly to von Willebrand factor (vWf) and enhanced its binding to collagen. Although platelet adhesion at high shear rate and vWf binding to GP Ib after ristocetin stimulation were not markedly affected, HEP-PGs reduced thrombin-induced aggregation and vWf binding to GP IIb/IIIa. These findings imply that activation of vascular mast cells with ensuing secretion of HEP-PGs may locally attenuate the thrombogenicity of matrix collagen by inhibiting its platelet-activating capacity.
Key Words: collagen • heparin • mast cells • platelet • thrombosis
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Introduction |
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Mast cells are prevalent in the adventitial layer of vessel walls and in the perivascular areas of venules.1 2 They are also present in the arterial intima, the site of atherogenesis,3 and activated mast cells have been found to infiltrate into the inflammatory shoulder regions of coronary atheromas, the most common site of rupture.4 5 On activation, such as that occurring during inflammation, mast cells degranulate and exocytose an array of potent vasoactive mediators, of which the short-lived leukotrienes and prostaglandins, platelet-activating factor, and histamine are known to stimulate platelets. Histamine releases endothelial vWf and P-selectin, two factors that are important adhesive signals to platelets and leukocytes.6 7 Furthermore, mast cells secrete glycosaminoglycans from which the clinically used heparins are derived,8 whereas activated platelets secrete platelet factor 4, a heparin-neutralizing factor, and heparitinase, a heparin-cleaving endoglycosidase.9 10 Thus, there appears to be an interplay between these two cell types after their activation, which has been, however, poorly characterized at present.
Whether mast cell activation is involved in hemostasis can be evaluated during anaphylaxis and in mastocytosis, two clinical conditions in which mast cells become excessively activated.11 Yet in these conditions, thrombosis is not prevalent despite the release of platelet agonists and potent inflammatory mediators, which cause changes in vascular endothelium, ie, downregulation of its nonthrombogenic properties, induction of adhesive molecules, increased permeability, and even exposure of subendothelial structures.6 7 11 It therefore seems likely that activated mast cells are also able to counteract their own thrombogenicity. Previously, in addition to their anticoagulant potential, the clinically useful HMWH glycosaminoglycans (average MW, 15 000) have been shown to inhibit the platelet aggregation induced by low-dose collagen.12 13 Interestingly, this inhibitory effect of heparin was found to be directly related to the MW of the heparins used.
The aim of the present study was to assess the effects of mast cell-derived HEP-PGs on platelet-collagen interactions. We used rat serosal mast cells as a model. These are filled with cytoplasmic secretory granules composed of HEP-PGs with a MW of 750 000 (range, 750 000 to 1 000 000), each monomer containing, on average, 10 heparin glycosaminoglycan chains with a MW of 75 000 (range, 50 000 to 100 000).14 15 On activation, mast cells expel some of their granules into the extracellular fluid where a fraction of the granule HEP-PGs becomes solubilized.15 We found that these soluble HEP-PGs strongly inhibited collagen-induced platelet aggregation and platelet interaction with immobilized collagen. The findings imply that mast cell HEP-PGs of very high molecular weight (macromolecular heparin) may attenuate the reactivity of platelets to the vascular extracellular matrix, thereby counteracting the other, potentially thrombogenic, effects of mast cells.
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Methods |
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HEP-PGs Exocytosed by Stimulated Mast Cells
Mast cells were isolated from rat peritoneal and pleural cavities as described.14 In a standard assay, 10 to 13x106 mast cells were incubated in 1 mL of PBS buffer containing 0.1 mg/ml of HSA (Red Cross Transfusion Service) and 5.6 mmol/L glucose. After preincubation (15 minutes, 37°C) the cells were incubated for 15 minutes with compound 48/80 (Sigma Chemical Co) (5 µg/ml), a specific mast cell agonist, to induce mast cell degranulation. Control experiments showed that compound 48/80 does not affect platelet aggregation. The degranulated mast cells were then sedimented by centrifugation at 150g for 10 minutes, the supernatant was centrifuged for a further 15 minutes at 12 000g to sediment the exocytosed granules, and the granule-free supernatant was analyzed for its content of Alcian blue-reactive (Fluka) material.15 In the experiments performed in the absence of plasma, soybean trypsin inhibitor (Sigma Chemical Co) was included to inactivate mast cell-derived neutral serine proteases. Throughout the experiments, HEP-PGs were compared with up to 300-fold concentrations (measured as Alcian blue-reactive material) of commercial porcine HMWH (Leiras) (average MW, 15 000; 1% <7500; 1 mg=100 IU USP) and LMWH (Fragmin, Kabi Pharmacia) (average MW, 5000; 25% >7500; 1 mg=152 anti Xa units). HEP-PGs did not alter the content of ionized calcium or magnesium in the buffers or plasma (Microlyte 6, Kone Instruments).16 HEP-PGs were radiolabeled by incubating mast cells with sodium [35 S]sulfate (Amersham International), as described.15 In some experiments HEP-PGs were treated with chondroitinase ABC and heparinase (both from Seikagaku Kogyo Co).
Inhibition of Thrombin
The relative potencies of HEP-PGs, HMWH, and LMWH were measured
with thrombin time in pooled citrated plasma and with a
chromogenic assay using a thrombin substrate (S-2238,
Chromogenix, Kabi Pharmacia).17 In the latter
assay, 1 U/ml (110 U/mg) of thrombin (Dade, Baxter Healthcare Co) was
the selected dose after titrating the effects of the
glycosaminoglycan concentrations used. Exogenous
thrombin activity was assessed in the presence of antithrombin III
(Kabi Pharmacia) alone and at two plasma dilutions (1:5 and 1:40 in
Tris-NaCl-HSA, pH 8.2) as a control for the competitive binding of the
glycosaminoglycans to plasma proteins. In the
absence and presence of plasma (1:40 dilution), exogenous antithrombin
III was used at two concentrations, 7.5 and 10 mU/ml. The reagents were
applied to 96-well microtiter plates (Falcon 3072, Becton Dickinson) on
ice and incubated for 10 minutes at 37°C. S-2238 was added, the
reaction was stopped with 20% acetic acid, and residual thrombin
activity was assessed spectrophotometrically (405 nm) (Labsystems
Multiscan MCC, Labsystems).
Platelet Preparation
The study was approved by an institutional review board. Blood
for the studies was donated by healthy volunteers not using any
medication. Nine volumes of free-flowing blood were collected via a
polytetrafluoroethylene cannula (Viggo)
into 1 volume of PPACK (Calbiochem) (200 to 400 µmol/L)
or acidic citrate dextrose anticoagulant (pH 4.9 for aggregation [pH
7.3 in PRP] and pH 4.5 for gel filtration). PRP was separated by
centrifugation (180g, 12 minutes, 22°C)
and used for platelet aggregation studies and adhesion assays. For
detecting deposition of serotonin-positive platelets
and release reaction, the platelets in PRP were labeled with
[14 C]serotonin (specific activity 8
µCi/ml, final concentration of serotonin 40
nmol/L) (Amersham) for 15 minutes at 37°C. In blood perfusion
studies, the labeled PRP was added to the remaining blood. The method
of platelet detection by serotonin labeling has been
previously controlled with the determination of deposited protein and
with electron microscopy.18
Gel-filtered platelets were prepared from PRP after a single washing step in the presence of prostaglandin E1 (25 ng/ml) and apyrase (1 U/ml) (both from Sigma Chemical Co), and the platelet suspension was then passed through a Sepharose CL-2B column (Pharmacia LKB). After gel filtration, ristocetin (1.0 mg/ml) (Sigma Chemical Co) did not induce a platelet response, indicating that vWf was lacking, and the cell suspension was also devoid of antithrombin III activity, as shown by crossed immunoelectrophoresis.19 Gel-filtered platelets were used for aggregation studies, for studying Mg2+-dependent platelet adhesion to collagen, and for binding the ligands (fibrinogen and vWf) that mediate platelet-to-platelet interaction. The elution buffer was HEPES with 1 mmol/L Mg2+.20 Usually, 2 mmol/L Ca2+ was added to the suspension of gel-filtered platelets, but when assaying Mg2+-dependent (2 mmol/L) adhesion, Ca2+ was omitted.21 HSA (4%) solution with 2 mmol/L Ca2+ and 1 mmol/L Mg2+ was used when the platelet-collagen interaction was studied in flowing reconstituted blood without plasma factors.22 After centrifugations and reconstitutions, the final platelet suspension was allowed to stabilize for 30 minutes before the assays.
Platelet Aggregation
Aggregation in PRP and in gel-filtered platelet suspension
was studied with a Payton aggregometer (Payton Associates Ltd).
Pepsin-extracted collagen (Sigma Chemical Co, platelet aggregation
kit) and fibrillar type I bovine collagen from the Achilles'
tendon,23 thrombin, ristocetin, ADP (Sigma
Chemical Co), and epinephrine (Bioanalytical Systems Inc) were
used as agonists, each added in a volume of 30 µL/270 µL of
platelet suspension. The effects of HEP-PGs, HMWH, and LMWH were
studied by adding them either during the 1-minute preincubation or
simultaneously with the agonist (collagen). In some
instances, HEP-PGs were added 10 and 20 seconds after the collagen. The
response was assessed as the slope of primary aggregation (rate, 1/min)
and as maximal aggregation (percentage).
Immobilization of Isolated Fibrillar Collagen
Fibrillar collagen had been extracted from bovine Achilles
tendon by acetic acid extraction and salt precipitation without
pepsin.23 Collagen (at a concentration of 0.36
mg/ml) was kept in 0.5 mol/L acetic acid, and fibril
formation was induced by neutralizing with 60 mmol/L TES
buffer (1:1) and incubating at 35°C for 90 minutes in a humid
atmosphere.24 25 For adhesion studies, this
fibrillar collagen solution was sprayed five times on ethanol-washed
round (diameter 1.5 mm) Thermanox coverslips (Nunc). The
successive sprayings of collagen suspension were made just before the
droplets dried. Collagen settled as a homogeneous layer of
fibril-containing droplets ranging from 50 to 200 µm with both
diameters and interspaces, as assessed by scanning electron microscope
(JEOL JSEM 820). The coverslips were kept in a humid atmosphere before
use on the same day. For perfusion studies, collagen was
immobilized, and native-type fibrils were allowed to be
formed in situ in polytetrafluoroethylene
tubing (Optinova) by adding TES and incubating the stoppered tubing at
35°C for 90 minutes. After incubation, the tubing was rinsed with
PBS.
Platelet Interaction With Collagen in PRP or in
Mg2+ Buffer
Platelet adhesion to immobilized collagen was
studied both in PRP (PPACK) and in gel-filtered platelets in HEPES
with 2 mmol/L
Mg2+.21 Collagen-coated
Thermanox coverslips were placed on the bottom of the 24-well plates
(NUNC) (precoated with 2% HSA) and 1 mL of
[14 C]serotonin-labeled PRP or
gel-filtered platelets with platelet counts adjusted to 100 or
300x106/ml (Thrombocounter C, Coulter
Electronics) was added. Before the assay, the
14 C-scintillation activity in the platelet
suspension and the release of serotonin into plasma were
measured in tubes with imipramine-formaldehyde on ice
(centrifuged at 9500g for 2
minutes).26 After incubation for 30 minutes
either at 22°C without rotation (to study adhesion of
100x106/ml platelets) or at 37°C during
rotation at 100 rpm (to study aggregation on adhesion of
300x106/ml platelets), the coverslips were
removed, rinsed three times in buffer, and subjected to scintillation
counting. The number of platelets deposited on the collagen-coated
coverslip was calculated from the number of platelets added and
from their specific activity. The release of serotonin from
the platelets to plasma was also measured as described, and it was
constantly <5%. To assess the role of GP IIbIIIa under these
conditions, PRP was preincubated (15 minutes, 37°C) with a mAb
against GP IIbIIIa (m7E3, kind gift from Dr Barry Coller) before the
adhesion assay.27
Platelet Interaction With Collagen in Flowing Whole Blood or in
Reconstituted Blood
To study platelet interaction with collagen in
PPACK-anticoagulated blood (30 mL) containing preincubated
[14C]serotonin-labeled
platelets, blood was recirculated for 5 minutes through the
collagen-coated tubing, which was connected to a perfusion pump (Cole
Parmer). To induce different shear rates (200, 700, and 1700
s-1), at a flow rate of 10 mL/min, tubings of
different diameters (1.1, 1.5, and 1.9 mm) were used. The collagen
surface was stabilized by perfusing it with PBS (at 37°C for 15
seconds) before the blood perfusion. After the perfusion, the
unattached platelets were rinsed off by perfusing with PBS for 30
seconds. The adherent platelets were detached by incubating them in
2% SDS twice for 30 minutes, and the lysates were subjected to
scintillation counting. In some instances, scanning electron
micrographs were obtained from the surface after perfusion.
Platelet counts, background radioactivity of the blood, and
serotonin release were measured, as described for the
adhesion assay. To study platelet-collagen interaction in the
absence of plasma proteins, reconstituted blood with washed red cells,
buffy coat, and gel-filtered
[14C]serotonin-labeled
platelets in HSA solution was used.22
Interaction Between Platelets and HEP-PGs
Binding of HEP-PGs to resting platelets was assessed by
incubating 35 S-labeled HEP-PGs with PRP or with
gel-filtered platelets at 37°C for 15 minutes. The
35 S-scintillation activity was then recovered in
plasma fractions and in platelets using sedimentation or gel
filtration. HEP-PGs were also immobilized on Thermanox
coverslips by incubation for 30 minutes. The quantity of bound HEP-PGs
was determined from the 35 S-binding, and it was
56±6 ng/cm2 (n=4). Interaction of
platelets with HEP-PGs was then determined using the platelet
adhesion assay, as described above.
Binding of vWf and Fibrinogen to Activated Platelets
vWf (specific activity 200 U/mg protein)
(CRTS)28 and fibrinogen were
radioiodinated with 125I (Amersham)
by the method of Bolton and Hunter.29 The
structural stability of vWf and fibrinogen was confirmed by
analysis with gradient (4% to 21%) SDS gel electrophoresis.
The function of vWf was confirmed by ristocetin-induced aggregation of
gel-filtered platelets and that of fibrinogen by thrombin-induced
coagulation. Gel-filtered platelets were stimulated with ristocetin
(1 mg/ml) or thrombin (0.1 U/ml) for 3 minutes. In some tubes
thrombin, 60 s after its addition, was inhibited with 3 U/ml of
hirudin. Then 125I-vWf (15 µg/ml) was
added, and the platelets (1x108) were
incubated at 37°C without stirring. To separate the platelet-free
and platelet-bound activities, the platelet suspension was
layered on top of 0.3 mol/L sucrose with 1.35% HSA and
centrifuged at 950g for 5 minutes to sediment the
platelets. The supernatant was collected, the tip was cut off, and
both fractions were counted for their radioactivity. The binding of
125I-fibrinogen (100 µg/ml) to
ADP-stimulated and collagen-stimulated (stirred) platelets was
studied similarly. The data were subjected to Scatchard
analysis.
vWf Binding to Collagen and HEP-PG
The effects of HEP-PGs, HMWH, and LMWH on vWf binding to
collagen were assessed according to Lawrie et
al.30 For this purpose 96-well microtiter wells
(Maxisorb, Nunc) were coated for 2 hours at 37°C with pepsinized type
I collagen (dialyzed against 67 mmol/L phosphate, pH 7.2)
(at 50 µg/ml), then washed, and blocked with 3% bovine serum
albumin. vWf (0.1 µg/ml) was then added to the plates
and incubated for 2 hours in the presence of different concentrations
of HEP-PGs, HMWH, and LMWH. Subsequently, bound vWf was quantified
using peroxidase-conjugated polyclonal anti-vWf antibody (Dako A/S). In
addition, vWf (1 µg) was incubated with HEP-PGs (0.5 µg) for 10
minutes at 22°C and applied to a cellulose acetate plate (Helena
Laboratory). The plate was electrophoresed for 30 minutes at 180 V in
5 mmol/L HEPES, pH 7.4, containing 2 mmol/L
Ca2+ and 2 mmol/L
Mg,2+ and stained with Alcian blue to visualize
HEP-PGs or Ponceau red to visualize vWf.
Statistical Analysis
Results are given as mean±SD. The statistical significance of
the difference between sets of values was determined by Student's
t test for paired values or factorial ANOVA as
indicated.
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Results |
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Inhibition of Thrombin
Mast cell-derived soluble HEP-PGs at concentrations exceeding 1.0 µg/ml significantly prolonged thrombin time when studied in 1:3 plasma dilution (Fig 1
). However,
HEP-PGs inhibited thrombin significantly less effectively than HMWH or
LMWH. Also, as measured by the chromogenic assay, HEP-PGs
were less effective than HMWH in inhibiting thrombin in the presence of
1:5 plasma dilution. This difference could be observed at the various
thrombin concentrations used (0.5 to 3 U/ml) (data not shown).
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To study whether plasma proteins affected the ability of HEP-PG to
potentiate exogenous antithrombin III, the effects of various
concentrations of HEP-PGs and HMWH on residual thrombin activity were
measured in the presence and absence of plasma. At a plasma dilution
(1:40), HEP-PGs did not differ from HMWH (Fig 2A). In the absence of plasma, however,
HEP-PGs were more potent than HMWH in enhancing antithrombin III
activity (Fig 2B). Thus, HEP-PGs were able to potentiate antithrombin
III, but this ability was impaired in the presence of plasma
proteins.
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, HMWH; 
,
HEP-PGs.
Platelet Aggregation and Serotonin Release in
Platelet-Rich Plasma
Mast cell-derived HEP-PGs strongly inhibited collagen-induced
platelet aggregation in both types of PRP investigated. When
studied in acidic citrate dextrose-anticoagulated PRP, HEP-PGs were
inhibitory at a concentration of as low as 1.0
µg/ml (Fig 3A). At this
concentration, HMWH and LMWH did not impair aggregation, and these
heparins were without effect even if a 300-fold excess (300
µg/ml) was used. We next treated the HEP-PGs with alkali to
dissolve their protein components and to obtain isolated
glycosaminoglycan chains. The
inhibitory action of the
glycosaminoglycan chains (average molecular weight,
75 000) was significantly weaker than that of the native HEP-PGs. In
contrast to HEP-PGs, HMWH impaired collageninduced aggregation
only in citrated PRP and at low collagen concentrations (<2.0
µg/ml).
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The dose-dependent effects of HEP-PGs on collagen-induced aggregation
in citrated and in PPACK-anticoagulated PRP is shown in Fig 3B
. The
inhibitory effect of HEP-PGs was independent of collagen
concentration up to 150 µg/ml and more pronounced in
cation-depleted plasma than in PPACK-anticoagulated PRP, in which total
inhibition was reached only at 3 µg/mL. Inhibition was total,
irrespective of whether HEP-PGs and collagen were added
simultaneously, or HEP-PGs were added 10 seconds after
collagen. HEP-PGs also reduced the release of platelet
serotonin from 50% to the background level (10%) in PRP,
even at the highest collagen concentration tested (150 µg/ml)
(Fig 3C).
In additional experiments we treated HEP-PGs with heparinase or chondroitinase ABC. We found that treatment with heparinase totally abolished the ability of HEP-PGs to inhibit collagen-induced platelet aggregation, whereas treatment of HEP-PGs with chondroitinase ABC did not lessen their inhibitory potential (not shown). The macroaggregated HEP-PGs complexes forming the granule remnants, ie, the residues left over after release of the soluble proteoglycans from the exocytosed granules,45 had no inhibitory effect on collagen-induced platelet aggregation compared with the same amount of soluble HEP-PGs. However, when the granule remnants were first disintegrated into HEP-PG monomers by treatment with 2 mol/L NaCl and then added to the platelets, the inhibitory effect equaled that observed with soluble HEP-PGs.
The concentration of HEP-PGs that completely abolished the
collagen-induced responses of platelets (3 µg/ml; Fig 3)
was selected for testing the effects of HEP-PGs on platelet
aggregation induced with agonists other than collagen. As shown in
Table 1, HEP-PGs inhibited platelet
aggregation induced with ristocetin, inhibition being total at a
ristocetin concentration of 0.60 mg/ml. Inhibition was also
considerable at the two higher ristocetin concentrations, 0.75 and 1.0
mg/ml. HMWH and LMWH did not inhibit ristocetin-induced
aggregation to the same extent as did HEP-PGs. Furthermore, HEP-PGs did
not markedly modify platelet aggregation in response to ADP or
epinephrine (1 to 10 µmol/L) (not shown).
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Aggregation of Gel-Filtered Platelets
When HEP-PGs were added to suspensions of gel-filtered
platelets, the collagen-induced platelet aggregation was only
incompletely abolished. With 25 µg/ml of collagen the
inhibitory effect of 3 µg/ml of HEP-PGs ranged
between 25% and 60%. We also studied thrombin-induced (0.1 and 0.25
IU/ml) aggregation of gel-filtered platelets. Again, platelet
aggregation was more effectively inhibited by HEP-PGs than by HMWH or
LMWH (all at 3.0 µg/ml) (not shown). HMWH, if used at a
100-fold concentration (300 µg/ml), led to full inhibition at
the two thrombin concentrations used.
Interactions Between Platelets and Collagen in
Mg2+-Containing Buffer and in PRP
In the following, we assessed the interaction of platelets
with immobilized collagen. When
100x106/ml platelets were studied at 22°C
under static, Mg2+-dependent conditions, HEP-PGs
(3 µg/ml) did not affect the formation of a monolayer of
adherent platelets, which also spread normally (Figs 4A and 5A
and 5B). In contrast, HEP-PGs significantly inhibited the subsequent
platelet-platelet interaction, when
300x106/ml platelets were rotated at 37°C
(Figs 4B and 5C and 5D). In PPACK-anticoagulated PRP, however, HEP-PGs
did not significantly decrease the interaction (Fig 4C). Under the
corresponding conditions, the mAb against GP IIbIIIa (m7E3 at 10
µg/ml) inhibited collagen-induced platelet deposition by
20%, 75%, and 80%, respectively.
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Interactions Between Platelets and Collagen in Flowing
Blood
When PPACK-anticoagulated whole blood was perfused at different
shear rates through tubing coated with collagen, HEP-PGs (3
µg/ml) significantly inhibited platelet deposition, but
not adhesion, on the collagen. Inhibition was evident both at a low
shear rate (200 s-1) and at higher shear rates
(700 and 1700 s-1) (Fig 6A). When the same experiment was
repeated (at 700 and 1700 s-1) using
reconstituted blood without plasma, the platelets adhered to the
collagen to the same extent whether HEP-PGs were present or not
(not shown). Scanning electron micrographs of the platelets
covering the collagen-coated surface after perfusion with whole blood
at 1700 s-1 demonstrated complete absence of
aggregates when HEP-PGs were present (Fig 6B versus 6C). At this
shear rate platelet adhesion was not significantly diminished in
the presence of HEP-PGs. Surface coverage was 22±4% in the absence
and 17±5% in the presence of HEP-PGs (n=3).
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Binding of Fibrinogen and vWf to Platelets
HEP-PGs tended to reduce the binding of fibrinogen to
collagen-stimulated platelets: from 2.4±1.2 to 1.5±0.7
pmol/108 platelets (n=4,
P.06), the background being 0.8±0.4
pmol/108 platelets, but did not affect
ADP-induced binding (not shown). However, HEP-PGs (3 µg/ml)
inhibited vWf binding to thrombin-stimulated platelets by 40% (234
versus 349 ng/108 platelets) (Table 2). HWMH at the same concentration was
without significant effect, but at 100-fold excess (300 µg/ml)
if completely blocked vWf binding to platelets. HEP-PGs did not
inhibit vWf binding to ristocetin-stimulated platelets. A similar
result was obtained with HMWH. Again, 100-fold excess (300
µg/ml) of HWMH significantly inhibited vWf binding to
ristocetin-stimulated platelets.
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Interaction Between Platelets and Heparin
Proteoglycans
When HEP-PGs were immobilized instead of collagen, and
platelets in PPACK-anticoagulated PRP were allowed to attach, the
level of platelet deposition was
0.36±0.17x106
platelets/cm2 (n=4), which did not differ
from the value obtained with immobilized albumin
(0.50±0.31x106
platelets/cm2) (n=4). The finding that
platelets did not bind to HEP-PGs was confirmed by experiments in
which 35 S-labeled HEP-PGs (3 to 10
µg/ml) were incubated in PRP, and after the incubation the
platelets were sedimented and counted for their
35 S-scintillation activity. No
35 Scintillation activity was present in the
sediments, indicating that HEP-PGs did not cosediment with the
platelets. Furthermore, when washed platelets were incubated
with 35 S-HEP-PGs and subsequently subjected to
gel filtration, 35 S-HEP-PGs were eluted
separately after the platelet population.
vWf Binding to Heparin Proteoglycans and to Collagen
vWf and HEP-PGs were electrophoresed either alone or together on a
cellulose acetate plate, and the plates were stained for both protein
and glycosaminoglycans to visualize the individual
components. The addition of vWf to HEP-PGs reversed their mobility from
anodic to cathodic, implying an association between vWf and HEP-PGs
(Fig 7). Since HEP-PGs had inhibited
platelet-collagen interaction at a high shear rate (Fig 6) and also
interfered with the other vWf-mediated platelet functions (Table 1), we studied whether HEP-PGs affected vWf binding to collagen, using
an enzyme-linked immunosorbent assay. As shown in Fig 8, vWf binding to collagen was not
inhibited, but, on the contrary, was markedly enhanced. This result
differed completely from those obtained with HMWH and LMWH. Even at a
10-fold excess concentration (30 µg/ml), compared with
HEP-PGs, HMWH only slightly increased the binding of vWf to collagen,
and LMWH was without any effect.
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Interaction Between HEP-PGs and Collagen
We also tested the binding of HEP-PGs to collagen under conditions
mimicking those in which HEP-PGs inhibited the platelet-collagen
interaction (ie, at similar concentrations of HEP-PGs and collagen and
a similar incubation time). When collagen (whether pepsinized or
fibrillar) was immobilized, it did not interact with
HEP-PGs. These results were obtained using
35 S-HEP-PGs or detecting
glycosaminoglycans with Alcian blue. Furthermore,
after incubation of collagen with HEP-PGs, the pellet obtained by
centrifugation through a sucrose cushion failed to show
Alcian blue-reactivity.
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Discussion |
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We first determined the ability of mast cell-derived HEP-PGs to inhibit thrombin activity. Particularly, we were interested in comparing the inhibitory effect of this very HMWH species with the well-known anticoagulative function of standard heparins, which essentially relies on the potentiation of antithrombin III activity.8 The assays focusing on the differential capacities of HEP-PGs, HMWH, and LMWH to inhibit thrombin (Figs 1
and 2) showed that, in the absence of plasma proteins, HEP-PGs
had a better functional ability than HMWH to potentiate antithrombin
III directly, whereas in the presence of plasma, HEP-PGs were less
potent than HMWH. These data can be understood if HEP-PGs, more
effectively than HMWH, bind to plasma proteins, such as vWf (Fig 7),
which may compete with antithrombin III for binding to heparin. It has
been shown that the greater the molecular weight of the heparin, the
higher is its affinity for plasma proteins.31 32
In all, the inhibitory potential of HEP-PGs on the observed
platelet-collagen interaction (see below), compared with HMWH and
LMWH, cannot be due to antithrombin III-dependent thrombin
inhibition.
The major findings of the present study were the total inhibition
by HEP-PGs of (1) collagen-induced platelet aggregation (Fig 3A and 3B), (2) subsequent dense granule release (serotonin) (Fig 3C), and (3) platelet deposition on immobilized
collagen in flowing blood under both low and high shear rate conditions
(Fig 6A). Previously, HMWH (at concentrations >6 µg/ml) had
been shown to impair platelet aggregation induced with a low-dose
collagen in cation-depleted PRP.12 13 In our
study HEP-PGs, in contrast with HMWH, totally inhibited
collagen-induced platelet aggregation and serotonin
release, irrespective of collagen concentration. Curiously, HEP-PGs
were able to prevent the action of collagen on platelets even when
added to the PRP 10 seconds after collagen. HEP-PGs inhibited
platelet aggregation by collagen more effectively in
citrate-anticoagulated PRP than in PPACK-anticoagulated PRP, showing
that HEP-PGs were most potent in blocking the cation-dependent
platelet functions on collagen stimulation (Figs 3B and 4B and 4C).
The heterogeneity of platelet GP IaIIa results in
variability of platelet responses among
individuals,33 and this may have been reflected
in the somewhat variable inhibitory effect of HEP-PGs
on the aggregation of the gel-filtered platelets.
Since, under the conditions used, HEP-PGs appeared not to bind directly
to collagen or to resting platelets, HEP-PGs disturbed
platelet-collagen interaction through other mechanisms. The finding
that although HEP-PGs did not inhibit
Mg2+-dependent platelet adhesion, but did
impair the subsequent platelet aggregation (Figs 4 and 5) implies
attenuated transmission of the activation signal from GP IaIIa to GP
IIbIIIa. Thus, the detected decrease in fibrinogen binding could have
been secondary to the impairment of collagen-induced platelet
activation. On the other hand, HEP-PGs did not significantly affect the
platelet deposition on collagen in PRP (PPACK), which seems GP
IIbIIIa-dependent (Fig 4C). GP IIbIIIa plays a role in platelet
activation with fibrillar collagen, as shown by the significant
inhibition obtained with m7E3 (present study), a finding that
agrees with previous observations.34
Additionally, GP IIbIIIa participates in collagen-induced adhesion and
subsequent platelet activation indirectly via plasma
proteins.34 35 The suggestion that HEP-PGs do not
directly interfere with GP IIbIIIa is supported further by normal ADP-
and epinephrine-induced aggregation and fibrinogen binding in
the presence of HEP-PGs.
The present findings imply an impairment of activation that follows
adhesion of the platelets on collagen (Figs 4 to 6), leading to
inhibited platelet recruitment in flowing blood at both low and
high shear rates. Because HEP-PGs did not attenuate
Mg2+-dependent platelet adhesion, direct
inhibition of GP IaIIa as the underlying mechanism could be
excluded.36 HEP-PGs, being macromolecules with a
strong negative charge that inhibited not only collagen-induced
aggregation but also thrombin-induced aggregation, could have disrupted
the outward movement of negatively charged platelet membrane
phospholipids during activation with these two
agonists.37 After GP IaIIa-mediated adhesion to
collagen, the subsequent decrease in platelet function could have
been mediated by the reduced ligand binding to GP IIbIIIa. Indeed,
HEP-PGs decreased fibrinogen binding to collagen-stimulated
platelets. Under flow conditions, platelet recruitment to
collagen depends crucially on vWf and its binding to platelet GP Ib
and GP IIbIIIa as well as to collagen.38 39 GP
IIbIIIa can be triggered by thrombin to bind vWf, especially when
hirudin is used to freeze its proteolytic
actions,40 and under these conditions HEP-PGs
also reduced the binding of vWf to platelets (Table 2). In summary,
after platelet adhesion to collagen, HEP-PGs blocked platelet
activation and also, by binding tightly to vWf, reduced its
availability to GP IIbIIIa.
HEP-PG, by binding to vWf (Fig 7), could also result in decreased
interaction of vWf not only with GP IIbIIIa, but also with GP Ib. Thus,
ristocetin-induced platelet aggregation was markedly reduced by
HEP-PGs (Table 1). At the concentration of HEP-PGs used, the vWf
binding to platelets stimulated with ristocetin (static conditions)
was not affected, although a 100-fold excess of HMWH did inhibit the
vWf binding. In blood flowing at high shear rates, in which HEP-PGs
inhibited platelet-collagen interaction but not surface coverage,
these macromolecules could certainly have a more potent effect on
vWf-dependent platelet activation than in the static binding
assays. Indeed, HMWH has previously been reported to severely impair
vWf-dependent platelet functions both in vitro and in
vivo.41 However, HEP-PG enhanced, rather than
inhibited, the binding of vWf to collagen (Fig 8), which is mediated
also by other domains of the vWf molecule than the A1 domain, where the
GP Ib- and heparin-binding areas are located.42
The enhanced binding of vWf to collagen may have sealed the
platelet-activating domains of collagen.
In the above studies, the HEP-PGs were released from mast cell granules
after their exocytosis. The residual proteoglycans that form the
insoluble matrix of the granules after release of the soluble HEP-PGs
(the granule remnants; diameter 0.5 to 1.0 µm) are composed
solely of heparin glycosaminoglycan
chains.14 15 On the other hand, the soluble
proteoglycans released from the granules into the extracellular fluid
contain heparin and to a small extent also chondroitin sulfate
glycosaminoglycan chains.15
The differential effects of heparinase and chondroitinase treatment on
the soluble HEP-PGs, the former decreasing their inhibitory
activity and the latter not, reveal that the inhibitory
effects on platelet-collagen interaction are due to the heparin
glycosaminoglycan component of the HEP-PGs. In
structural analysis of the soluble HEP-PGs, the composition of
disaccharide units is typical of heparin (J–p. Li, P. Kovanen,
and U. Lindahl, unpublished results). Therefore, the observed
functional differences between HEP-PGs and commercial heparins must
depend on factors other than the composition of the
glycosaminoglycan chains. Thus, the ability of
intact HEP-PG (MW, 750 000) to inhibit platelet function was
greater than that of the heparin glycosaminoglycan
chains (MW, 75 000) released from HEP-PGs, which, again, was greater
than that of HMWH (MW, 15 000) or LMWH (MW, 5 000) (Fig 3A). Taken
together, the above findings indicate that the large size of the
heparin chains and their attachment to a core protein (to create
"macromolecular" heparin) are the most important factors
contributing to the observed inhibition.
Platelets interacting with collagen exposed by vascular injury are thought to play a crucial role in both hemostasis and atherothrombosis. The importance of vascular collagen in platelet-vessel wall interactions is evident in patients with bleeding disorders due to defective collagen synthesis and platelet glycoprotein receptors for collagen. The same is also evidenced experimentally by mAbs against GP IaIIa, and by the enhanced thrombogenicity of smooth muscle cell matrix when collagen synthesis is optimized.43 44 45 46 HEP-PGs can be secreted locally into the subendothelium and adventitia where mast cells are present and where they can be activated by various stimuli.1 47 The significant inhibitory capacity of HEP-PGs in platelet reactivity toward collagen implies a novel mast cell-dependent physiologic mechanism regulating hemostasis in the vascular wall.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The constant encouragement of Dr Vesa Manninen, Wihuri Research Institute, is highly appreciated. The skillful technical assistance of Tuula Järvenpää, Marja Lemponen, and Päivi Hiironen is gratefully acknowledged, as are the generous gifts of Fragmin and human serum albumin by Kabi Pharmacia, Helsinki, Finland, and human von Willebrand factor concentrate from CRTS, Lille, France (Dr Burnouf). Many thanks to Dr Barry S. Coller for m7E3, platelet GP IIbIIIa antibody, to Dr Matti Vauhkonen for providing fibrillar collagen type I and to Pia Siljander for the WF ELISA. This study was supported financially by the Finnish Heart Research Foundation and the Paavo Nurmi Foundation.
Received February 26, 1997; accepted August 22, 1997.
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