© 1996 American Heart Association, Inc.
Articles |
Lipoprotein(a) Inhibits Collagen-Induced Aggregation of Thrombocytes
From the Institutes of Physiology (A.G., M.G., H.W., T.K.) and Medical Biochemistry (G.M.K.), Graz, Austria, and the Department of Hematology (M.I., J.J.S.), University Hospital Utrecht, Utrecht, Netherlands.
Correspondence to Dr Anna Gries, Institute of Physiology, Harrachgasse 21/V, 8010 Graz, Austria.
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Abstract |
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Abstract Lipoprotein(a) [Lp(a)] is known to interact with human platelets in vitro. In the present study the effect of physiological concentrations of Lp(a) on platelet aggregation was studied. Freshly prepared gel-filtered platelets from healthy donors were incubated for 30 minutes at 37°C with various concentrations of Lp(a); aggregation was triggered with ADP, thrombin, and collagen. Control incubations were performed with Tyrode's solution or LDL. Thrombin- and ADP-triggered aggregations were only slightly influenced by Lp(a), but aggregation of platelets stimulated with collagen (4 µg/mL) was markedly inhibited. Measurable effects occurred at low concentrations (0.05 mg/mL) of total Lp(a); at 0.5 mg/mL, maximum aggregation of platelets was inhibited by 54±20%, and the aggregation rate was attenuated by 47±19% compared with platelets incubated with Tyrode's solution. Preincubation of collagen (4 µg/mL) with Lp(a) yielded similar results. The effect of Lp(a) on platelet aggregation was accompanied by a significant reduction of serotonin release and TXA2 formation. Higher concentrations of collagen (
10 µg/mL) caused the
inhibitory effect of Lp(a) on collagen-induced
aggregation to disappear. In contrast, incubation of platelets with
5 mg/mL LDL led to a significant increase of aggregation rate, maximum
aggregation, serotonin release, and formation of
TXA2 when aggregation was induced with 4 µg/mL collagen.
In an adhesion assay using fresh whole blood, which mimics the in vivo
situation of vessel injury, Lp(a) reduced platelet adhesion at
shear rates of 300 and 1600/s by 22.6% and 11.6%, respectively. In
addition, Lp(a) reduced the size of platelet aggregates
significantly (up to 63%); this reduction was more distinct at the
higher shear rate. Unlike LDL, Lp(a) is not a proaggregatory
lipoprotein; rather, collagen-triggered aggregation in vitro is
attenuated by Lp(a).
Key Words: lipoprotein(a) • platelet aggregation • serotonin release • thromboxane A2 • platelet adhesion
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Introduction |
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The thromboatherogenic lipoprotein1 Lp(a) belongs to the class of apoB-containing cholesteryl ester–rich lipoproteins. It consists of an LDL-like particle with an additional apolipoprotein, apo(a), attached by a disulfide bridge to apoB-100.2 3 Its plasma concentration, which is genetically determined, varies from <1 to >100 mg/dL. Extensive knowledge concerning the physiological function of Lp(a) or apo(a) is lacking, but there seems to be little doubt that Lp(a) is one of the most atherogenic lipoproteins, with an apparent coronary risk threshold at a plasma concentration of 30 mg/dL, which doubles the risk of developing coronary heart disease.4 5 6 7 The postulated thrombotic nature of Lp(a) has been linked to the great similarity of the protein structure of apo(a) with that of plasminogen.8 9 Human apo(a) consists of multiple kringle repeats, closely resembling human plasminogen kringle IV, followed by one copy of kringle V and the protease domain of plasminogen.
Lp(a) is a "sticky" lipoprotein that self-aggregates, attaches to all sorts of surfaces, and precipitates not only in vitro but possibly in vivo. Moreover, Lp(a) binds to proteoglycans and glycosaminoglycans,10 11 and it has a high affinity for fibronectin,12 tetranectin,13 collagen,14 and other connective-tissue structures.15
One of the physiological roles of platelets involves binding to subendothelial tissue after vascular injury.16 The adherence of platelets to the exposed connective tissue, preferably collagen, leads to aggregation followed by the release of ADP, 5-hydroxytryptamine, and Ca2+ from their dense granules, causing passing platelets to adhere to the primary clot.17 Lipoproteins influence platelet aggregability in plasma,18 and incubation of platelets with physiological concentrations of LDL and VLDL results in enhanced platelet activation; HDL has the opposite effect.19
In the present study we investigated the influence of Lp(a) on platelet aggregation induced with various triggers, and we measured serotonin release and TXA2 formation during collagen-triggered aggregation as well as adhesion of platelets to collagen in flowing blood under the influence of Lp(a).
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Methods |
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Isolation of Lipoproteins
Lp(a) was prepared from EDTA-containing plasma samples of healthy fasting volunteers of both sexes aged 20 to 30 years. Donors were selected on the basis of their serum Lp(a) concentrations (>40 mg/dL) and their apo(a) phenotypes as determined by SDS-PAGE20 followed by Western blotting21 using the isoform standard of Immuno AG as reference. LDL was isolated from the d=1.020 to 1.050 g/mL fraction by density gradient ultracentrifugation.22 Lp(a) was prepared from the d=1.070 to 1.125 g/mL density fraction obtained by ultracentrifugation in a Ti-60 fixed-angle rotor followed by size-exclusion chromatography with Biogel A-5m.23 Lp(a) was homogenous as checked by agarose gel electrophoresis and SDS-PAGE in 3.75% gels. Special care was taken during the preparation to avoid oxidation or degradation of lipoproteins by adding EDTA (0.1 mg/mL) to all buffers. In addition, all solutions were flushed with nitrogen. Purified lipoproteins were stored under nitrogen at 4°C in the dark for not more than 5 days. Before use they were dialyzed exhaustively against Ca2+-free Tyrode's solution (pH 7.3).
Platelet Preparation and Incubation With
Lipoproteins
Platelets were isolated from apparently healthy male and
female donors after an overnight fast. The donors were free of
medication, in particular acetylsalicylic acid or
indomethacin-containing drugs, for at least 2
weeks. Platelets and lipoproteins were isolated from separate
donors. Blood was collected into a buffer containing 93 mmol/L
Na3-citrate, 7 mmol/L citric acid, and 0.14 mol/L
D-glucose, pH 6.5, at a ratio of 9:1. To obtain
platelet-rich plasma, blood was centrifuged at
200g for 10 minutes at room temperature.
GFPs were isolated by chromatography on Sepharose 2B (Pharmacia) equilibrated with Ca2+-free Tyrode's solution, pH 7.3, containing 0.2% bovine serum albumin (Sigma) and (in mmol/L) 137 NaCl, 2.68 KCl, 0.42 NaH2PO4, 1.7 MgCl2, 11.9 NaHCO3, and 5.5 glucose.24 Platelets were eluted from the column with Ca2+-free Tyrode's solution, counted in a Thrombocounter JT (Coulter Electronics), and adjusted to a concentration of 2x108/mL with the same buffer. Freshly isolated platelets were allowed to rest for 15 minutes at 37°C before incubation studies.
To study the influence of lipoproteins on the aggregability of platelets, 200 µL isolated platelets (2x108/mL) were incubated for 30 minutes at 37°C with either 50 µL Ca2+-free Tyrode's buffer, pH 7.3, or an equal volume of LDL (5 mg/mL) or Lp(a) (0.05 to 0.5 mg/mL) in Ca2+-free Tyrode's buffer. The influence of lipoproteins was studied on platelets from single donors; the aggregation parameters obtained in the absence of lipoproteins were set at 100%. Between-donor variation in aggregation behavior never exceeded 20%.
Aggregation Studies
Platelet aggregation was initiated in a 250-µL incubation
mixture by adding 10 µL of different triggers: thrombin (0.1 U/mL,
from bovine plasma grade I, Sigma), fibrillar collagen (0.1 to 1 mg/mL,
Horm collagen equine type I, Hormonchemie München), or ADP (8
µmol/L, from equine muscle grade I, Sigma). In the latter case
fibrinogen was added to yield a final concentration of 0.6
mg/mL.25 Aggregation studies were performed at 37°C
according to the method of Born26 by recording the
light transmission of the stirred platelets (900 rpm) with an Elvi
840 dual-channel aggregometer (Elvi Logos). In some cases of
collagen-induced platelet aggregation, 10 µL collagen (0.1
mg/mL) was incubated for 30 minutes at 37°C with 50 µL
Ca2+-free Tyrode's buffer or different concentrations of
Lp(a) (0.05 to 0.5 mg/mL) in Ca2+-free Tyrode's buffer.
Aggregation of 200 µL GFPs was then initiated with this pretreated
collagen.
From the aggregation curves the aggregation rate (from the slope of the curves) and the maximum aggregation (from the distance between the baseline and maximum change of light transmission) were determined. The aggregation rate is an index of the velocity of the aggregation, whereas maximum aggregation is an indicator of the completeness of the aggregation. Inhibition of both parameters by Lp(a) is expressed in percent of control observed in the absence of lipoproteins.
Dense Granule Secretion and Formation of
TXA2
For the measurement of dense granule secretion, 10 mL GFPs
(2x108/mL) were incubated for 30 minutes at 37°C
with 20 µL (1 µCi) 5-hydroxy[side
chain-2-14C]tryptamine creatinine sulfate
([14C]serotonin, specific radioactivity 1.85
to 2.20 GBq/mmol, Amersham International). Platelets were then
washed twice with Ca2+-free Tyrode's solution, and the
uptake of [14C]serotonin was determined by
measuring radioactivity in the supernatant by liquid scintillation in
10 mL of Ready-solve Hb/p (Beckmann).27
[14C]serotonin-labeled platelets were
incubated with 5 mg/mL LDL or 0.05 to 0.5 mg/mL Lp(a) and aggregated as
described above with the exception that only collagen (final
concentration, 4 µg/mL) was used for the initiation reaction. To
assay the release of [14C]serotonin, 100-µL
samples of the supernatants were collected, mixed with 25 µL freshly
prepared, ice-cold glutaraldehyde (final
concentration, 0.5% vol/vol), and centrifuged for 2 minutes at
9000g; the supernatants were counted according to standard
procedures. In some cases the aggregation of 200 µL GFPs was
initiated with collagen pretreated with various concentrations of Lp(a)
as described above. Serotonin release was expressed as
percent of total [14C]serotonin taken up by
the platelets. As control experiments using 1 mmol/L imipramine
(Sigma) to prevent reuptake of released serotonin by the
platelets did not reveal any difference, this substance was omitted
in further experiments.
TXA2 formation in platelets was determined by measuring its stable metabolite TXB2 by using a TXB2 radioimmunoassay kit (Advanced Magnetics, Inc)28 according to the instructions of the manufacturer; platelet suspensions were diluted 20-fold with assay buffer containing 10 µg/mL indomethacin before the assay was performed.
Adhesion Assay
The influence of Lp(a) on platelet adhesion was studied by
using the adhesion assay of Houdijk et al.29 Whole blood
was obtained from healthy normolipemic volunteers [plasma Lp(a)
concentration <20 mg/dL] who had not taken aspirin or other
substances that would inhibit platelet function during the
preceding week. The blood was anticoagulated with 200 U/mL
low-molecular-weight heparin (Fragmin, Kabi Pharmacia) (1/10 of
the blood volume) in 0.15 mol/L NaCl.
Surfaces
Glass coverslips (18x18 mm, Menzel) were soaked overnight in
chromosulfuric acid, rinsed thoroughly with deionized water, and air
dried. Fibrillar collagen (Horm collagen, equine type I) was sprayed
directly on the glass coverslips at a concentration of 1 mg/mL (final
density, 30 µg/cm2).29 After spraying, the
coverslips were incubated with 1% human albumin in
phosphate-buffered saline (10 mmol/L sodium phosphate and 150
mmol/L NaCl, pH 7.4) for 1 hour at room temperature.
Perfusions
Perfusions were performed in a parallel-plate rectangular
perfusion chamber.30 Duplicate coverslips were inserted in
the chamber. Whole blood was prewarmed to 37°C and circulated through
the chamber for 5 minutes at wall shear rates of 300 or 1600/s.
LDL and Lp(a) were added at different concentrations to the whole blood and incubated for 30 minutes at 37°C before perfusion. Subsequently, 15 mL prewarmed HEPES-buffered saline (10 mmol/L HEPES and 150 mmol/L NaCl, pH 7.4) was drawn through the system to wash the coverslips. Following perfusion, the coverslips were removed, rinsed with HEPES-buffered saline, fixed in 0.5% glutaraldehyde in phosphate-buffered saline, dehydrated in methanol, and stained with May-Grünwald-Giemsa.31
Image Analysis
Platelet adhesion was evaluated by using a light microscope
(magnification x1000) connected to an image analyzer (AMS
40-10). The area covered with aggregates or platelets was measured
and expressed as a percentage of the total area of the image. Aggregate
formation was analyzed by using the IBAS
image-analysis system (Zeiss/Kontron). Aggregate size was
subdivided into percentages of surface covered with aggregates with an
area between 8 and 40 µm2 and aggregates with an area
>40 µm2.
Other Methods
Agarose gel electrophoresis and immunochemical techniques were
performed according to standard procedures.32 Protein was
determined according to the method of Lowry33 by using
human serum albumin as standard. Total LDL and Lp(a) were
calculated by multiplying the protein values with a factor of 4.35 or
2.85, respectively, assuming a protein content of 23% for LDL and 35%
for Lp(a).2
Statistical analysis was performed by using a paired t test. Data are expressed as mean±SEM.
All chemicals were p.a. reagents from E. Merck unless otherwise stated.
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Results |
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Characterization of Lipoproteins
The purified lipoproteins used in this study were investigated by using agarose gel electrophoresis and SDS-PAGE. Both lipoproteins exhibited single bands in agarose gels and SDS-PAGE and were not contaminated with each other (Fig 1
). There were no
signs of fragmentation, which usually occurs if lipoproteins are
oxidized or attacked by proteases. Thiobarbituric acid–reactive
substances measured in 1% lipoprotein solutions were not higher than
blanks. The different Lp(a) preparations were
phenotyped34 and revealed all major isoforms, such
as B, S1, S2, S3, and S4 (data not shown).
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Platelet Aggregation
The effect of Lp(a) on in vitro platelet aggregation was
investigated. When unstimulated platelets were incubated with Lp(a)
for 30 minutes at concentrations up to 0.5 mg/mL total Lp(a), neither
change in light transmission nor any sign of spontaneous aggregation
could be observed. At the highest concentration of Lp(a) used (0.5
mg/mL), inhibition of maximum aggregation triggered by thrombin and ADP
was not significantly different from control (8±3% and 12±5%
respectively; results not shown). However, when aggregation was
triggered with fibrillar collagen, marked reductions of aggregation
rate and maximum aggregation were observed in the presence of Lp(a). We
therefore decided to investigate the influence of Lp(a) on
collagen-induced platelet aggregation in more detail.
When GFPs were incubated with various concentrations of Lp(a) for 30
minutes at 37°C and aggregation was initiated with a final
concentration of 4 µg/mL fibrillar collagen, the
aggregation rate and maximum aggregation diminished with increasing
amounts of Lp(a). At low concentrations of Lp(a) (0.05 mg/mL), only
slight inhibition of both parameters could be observed. At
a final concentration of 0.5 mg/mL total Lp(a), which corresponds to 50
mg/dL of Lp(a) in plasma, the aggregation rate was reduced by 47±19%
and the maximum aggregation by 54±20% compared with platelets
incubated with Tyrode's solution (Table 1). However,
the degree of inhibition by added Lp(a) was independent of the size of
apo(a) as well as of the plasma Lp(a) concentration of platelet
donors (results not shown). When LDL was added to platelets at a
final concentration of 5 mg/mL total lipoprotein, a marked increase of
both aggregation rate and maximum aggregation could be observed (Table 2). Fig 2 depicts typical aggregation
curves initiated with 4 µg/mL collagen obtained with GFPs
preincubated with Tyrode's solution, Lp(a), or LDL.
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In some experiments LDL or Lp(a) was added to platelet-rich
plasma with Lp(a) concentrations <5 mg/dL and incubated for 30 minutes
at 37°C; aggregation was initiated with 4 µg/mL collagen. Lp(a) at
0.5 mg/mL inhibited the aggregation rate by 28.4±16.9% and maximum
aggregation by 37.2±17.4% (Table 1
), whereas the influence of 5 mg/mL
added LDL was not significantly different from control (Table 2).
In subsequent experiments, collagen instead of platelets was
preincubated with different concentrations of Lp(a) for 30 minutes at
37°C, and these mixtures were added to GFPs to initiate aggregation.
Both the aggregation rate and maximum aggregation were inhibited by
Lp(a) in a concentration-dependent manner (Table 1). This effect
was even somewhat higher than in experiments in which platelets
were preincubated with Lp(a).
We next tested the ability of Lp(a) to inhibit platelet aggregation
induced with collagen concentrations >4 µg/mL. Lp(a) (final
concentration, 0.5 mg/mL) was incubated with GFPs for 30 minutes at
37°C, and aggregation was initiated with increasing amounts of
collagen. When the concentration of collagen was increased, the
inhibitory effect of Lp(a) decreased (Table 3). At concentrations of collagen >10 µg/mL the
inhibitory effect of Lp(a) disappeared, and maximum
aggregation was not significantly different from controls.
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Release of [14C]Serotonin and Formation
of TXB2
In further experiments we investigated the influence of Lp(a) on
the release of [14C]serotonin and the
production of TXB2 from stimulated platelets.
Platelets were incubated for 30 minutes at 37°C with 0.05 to 0.5
mg/mL Lp(a) or 5 mg/mL LDL followed by stimulation with 4 µg/mL
collagen (Table 4). In parallel experiments, 4 µg/mL
collagen was preincubated for 30 minutes at 37°C with the same
concentrations of Lp(a) mentioned above and used thereafter to trigger
platelet aggregation (Table 4). In both cases incubations with
Tyrode's solution served as references.
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Serotonin release in unstimulated platelet suspensions
incubated for 30 minutes at 37°C with Lp(a), LDL, or buffer and
stirred for 5 minutes in the aggregometer was <5% in all cases.
Platelets incubated in Tyrode's solution released 64% of
[14C]serotonin upon aggregation with
collagen. The addition of increasing amounts of Lp(a) led to a gradual
reduction of the release reaction, which was more pronounced when
collagen was preincubated with Lp(a). With 0.5 mg/mL Lp(a),
serotonin release was reduced to almost half (Table 4). The
diminished release of serotonin caused by Lp(a) was
accompanied by a reduction in TXB2 synthesis that was
significant at Lp(a) concentrations 0.150 mg/mL. Additionally,
preincubation of collagen with Lp(a) had a greater effect than
preincubation of platelets with Lp(a) (Table 4).
When platelets were incubated with 5 mg/mL LDL,
serotonin release was significantly increased and
thromboxane biosynthesis was almost doubled compared with
controls (Table 4). The LDL experiments confirmed earlier
results.27
Perfusions
Low-molecular-weight heparin–anticoagulated whole
blood from normolipemic donors with plasma Lp(a) concentrations <20
mg/dL was perfused for 5 minutes over Horm collagen (predominantly
equine type I) at shear rates of 300 and 1600/s with and without
addition of lipoproteins. Perfusion over the collagen surface led to
platelet aggregation at both shear rates.
Addition of 0.5 mg/mL Lp(a) to the perfusate had little
influence on the degree of platelet coverage at either shear rate.
Lp(a) (1 mg/mL) added to the whole blood, however, reduced platelet
adhesion by 22.2% at 300/s and 11.6% at 1600/s. LDL (0.5 mg/mL) did
not show any effect on platelet adhesion (Table 5).
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Table 5 also shows the influence of Lp(a) and LDL on aggregate
formation on fibrillar collagen (Horm collagen) at shear rates of 300
and 1600/s. Results are given as the ratio of the percentage of
aggregates (area >8 µm2) to the total coverage of
platelets (aggregate formation ratio). Aggregate formation was
nearly 100% at the high shear rate and was slightly lower at the low
shear rate. In neither case did Lp(a) influence the degree of aggregate
formation. LDL added at the high shear rate did not show any effect on
aggregate formation (Table 5).
Table 5 further shows the effects of lipoproteins on aggregate size
expressed as the ratio of the percentage of large (area >40
µm2) to the percentage of small (area 8 to 40
µm2) aggregates. In all cases aggregates were
significantly larger in size at the high shear rate. Addition of
Lp(a) induced a shift from large to small aggregates. At the low
shear rate significant reduction of aggregate size could be observed
only at a concentration of 1 mg/mL Lp(a), whereas at the high
shear rate aggregate size was significantly decreased at 0.5 mg/mL
Lp(a). Addition of 1 mg/mL Lp(a) to the perfusate led to a
further decrease (63±2.2%) of aggregate size (Table 5). Fig 3 compares aggregates formed on fibrillar collagen
without addition of lipoproteins (top) with aggregates formed after the
addition of 1 mg/mL Lp(a) (bottom). LDL (0.5 mg/mL) added at a shear
rate of 1600/s induced a significant increase in aggregate size
compared with controls (Table 5).
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Discussion |
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Although it is generally accepted that elevated plasma concentrations of Lp(a) are connected with premature atherosclerosis and myocardial infarction,4 5 6 7 much uncertainty remains about the influence of Lp(a) on platelet activation, a phenomenon that is believed to be involved not only in long-term processes of plaque formation but also in acute events such as stroke and myocardial infarction.35 We therefore studied the effects of Lp(a) on platelet aggregation induced with thrombin, ADP, or collagen. To avoid the interference of plasma components, platelets were isolated from plasma by gel filtration before incubation with Lp(a), which was purified from plasma for the same reason.
There is little doubt that lipoproteins interfere with platelets in vivo. This is reflected by the fact that platelets from hyperlipoproteinemic patients are hyperreactive (for reviews, see References 36 through 3936 37 38 39 ). There also seems to be agreement that atherogenic apoB-containing lipoproteins such as VLDL or LDL are proaggregatory, whereas antiatherogenic lipoproteins such as HDL exert an antiaggregatory action.36 Our study confirmed these results, as LDL caused not only an increase of collagen-induced aggregation but also an increase of serotonin release and formation of TXA2.
As Lp(a) represents an LDL-like particle, we expected an elevated platelet reactivity under the influence of this lipoprotein similar to that described for LDL.40 Surprisingly, collagen-induced platelet aggregation was inhibited by Lp(a) at trigger concentrations of <10 µg/mL in a concentration-dependent manner. This was accompanied by diminished release of [14C]serotonin and TXA2. The inhibitory effect appeared to be specific for collagen in that neither thrombin- nor ADP-induced aggregation was affected to such an extent by Lp(a). Preincubation of collagen (4 µg/mL) with Lp(a) followed by the addition of the mixture to GFPs confirmed the results, leading to an even more pronounced reduction of platelet aggregation. In both cases the attenuating influence on platelet reactivity was in a concentration range of Lp(a) that is well within physiological limits. Significant inhibition was detectable at 0.15 mg/mL (15 mg/dL) total Lp(a) in the assay (15 mg/dL is roughly the mean plasma concentration in Caucasians). This concentration is highly skewed, however; median concentration is 8 to 9 mg/dL.41 From these results we assume that Lp(a) may not be a proaggregatory lipoprotein and even exerts antiaggregatory effects when studied in vitro. Notably, we found a somewhat higher effect when collagen was preincubated with Lp(a). This points toward a direct interaction of Lp(a) with collagen.
When we compared collagen-induced aggregation using GFPs from normolipemic donors with different plasma concentrations of Lp(a), we found that the degree of inhibition by added Lp(a) was independent of whether platelet donors had high or low plasma concentrations of Lp(a) (results not shown). We have found that the plasma Lp(a) concentration of normolipemic individuals is not correlated with platelet reactivity when epinephrine, ADP, or collagen are used as triggers.42 Interestingly, there is a negative correlation between plasma concentrations of Lp(a) and ß-thromboglobulin,42 a substance that reflects the status of platelet activation in vivo.43 From all these results we conclude that Lp(a) circulating in plasma does not influence the aggregatory behavior of platelets in plasma itself, eg, by changing the membrane fluidity of the cells as described for LDL.44
To substantiate this assumption and to investigate the role of Lp(a) in platelet activation in a system that is more relevant to the situation in vivo, the influence of Lp(a) on platelet adhesion to fibrillar collagen under flow conditions using fresh blood was studied. Fibrillar collagen is one of the most thrombogenic structures in subendothelium, representing a unique ligand for platelet adhesion as it causes platelet activation and aggregate formation.45
Blood was anticoagulated with low-molecular-weight heparin to
maintain divalent cations in plasma, which are necessary for optimal
adhesion of platelets to collagen. Perfusions were performed at low
(300/s) and high (1600/s) shear rates, such as those found in small
arteries and stenotic areas, respectively.45 Our
results clearly show that Lp(a), at concentrations that reduce
collagen-induced platelet aggregation by 54%, diminished
collagen-induced platelet adhesion by 
10% at high and
20% at low shear rates. These observations make it unlikely that
the inhibitory effect of Lp(a) is due to a direct
interaction of platelet binding to collagen by the GPIa/IIa
receptor, the integrin that is the main determinant of platelet
adhesion under flow conditions.46 The partial inhibition
of platelet adhesion to collagen by relatively high amounts of
Lp(a) may reflect nonreceptor-specific independent effects. Indeed,
higher concentrations of collagen (>10 µg/mL) abolished the effect
of Lp(a) in our aggregation assay. On the other hand, platelets
encounter high local concentrations of collagen during adhesion assays
on collagen-coated surfaces. This might be why initial
microaggregate formation was not influenced by Lp(a) (Table 5). Yet we
found a significant inhibition of large aggregate formation at collagen
surfaces by Lp(a), an effect that was more pronounced at the higher
shear rate.
From these results we believe that our observations are relevant to the in vivo situation. The actual mechanism of our observations, however, deserves additional investigation. A variety of potential platelet-collagen binding sites has been described, including GPIa/IIa and GPIV.45 This underscores the complex nature of platelet-collagen interactions. Direct binding of Lp(a) to platelets has been demonstrated,47 48 which favors GPIIb as the main Lp(a)-binding protein on intact platelets. However, whether binding of Lp(a) to GPIIb has any influence on collagen-induced platelet aggregation has to be proved.
Our study indicates that Lp(a) inhibits collagen-induced platelet aggregation. It is apparent that inhibition occurs in the early phases of aggregation, as aggregation rate and release and the formation of large aggregates are significantly decreased (perhaps by interference on the level of signal transduction), particularly at low concentrations of collagen. Although extension of our data to any influence of Lp(a) on platelet function in vivo is not definitely proven, they suggest that Lp(a) does exert antiaggregatory effects under well-defined in vitro conditions.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Austrian Research Fund (grant No. SFB-702) to G.M. Kostner. The technical assistance of J.M. Kellner and H. Grillhofer is appreciated.
Received January 9, 1995; accepted February 1, 1996.
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