© 1995 American Heart Association, Inc.
Articles |
Recombinant Leech Antiplatelet Protein Specifically Blocks Platelet Deposition on Collagen Surfaces Under Flow Conditions
From the Department of Haematology (G.H. van Z., M.E.S., S. de G., J.J.S.) and Pathology (P.J.S.), University Hospital Utrecht, the Netherlands, and from the Department of Biological Chemistry (T.M.C.), Merck Sharp & Dohme Research Laboratories, West Point, Pa.
Correspondence to Dr G.H. van Zanten, Department of Haematology, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, the Netherlands. E-mail:jsixma@lab.azu.nl.
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Abstract |
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Abstract Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP). This protein was cloned and expressed in yeast and blocks collagen-mediated platelet aggregation and the adhesion of platelets to collagen-coated plates under static conditions. In the current study we investigated the effect of rLAPP on platelet deposition to collagen and collagen-rich surfaces under flow conditions. rLAPP completely inhibited platelet adhesion on collagen types I, III, and IV with IC50 values of 70, 600, and 90 nmol/L, respectively (shear rate=1600 s-1). Approximately 10-fold more rLAPP was required to obtain a similar inhibition at a low shear rate of 375 s-1. rLAPP caused a concentration-dependent inhibition of binding of 125I–von Willebrand factor (vWF) to collagen type III and was able to displace prebound vWF even after 24 hours. Since platelet adhesion at low shear rate is less dependent on vWF than at high shear rate, this property of rLAPP may explain why less rLAPP is needed at high shear rate than at low shear rate to produce the same effect. Platelet adhesion to collagen type VI was only partially inhibited by rLAPP (maximal 44% with 3 µmol/L rLAPP). rLAPP also caused a pronounced inhibition of platelet deposition to cross sections of human atherosclerotic coronary arteries but had no effect on matrices of cultured human umbilical vein endothelial cells. rLAPP is a potent platelet adhesion inhibitor at high shear rate, which binds to collagen and works by inhibiting binding of vWF to collagen.
Key Words: rLAPP • collagen • von Willebrand factor • platelet adhesion
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Introduction |
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Platelet adhesion to collagen and activation of platelets by collagen plays an important role in hemostasis1 but also in thrombosis.2
mAbs against the collagen receptor glycoprotein Ia-IIa (VLA-2) inhibit platelet adhesion to collagen under static conditions.3 4 5 One such antibody also inhibits platelet adhesion to collagen under flow conditions.6 Most other inhibitors of platelet activation by collagen are not specific for collagen.
Recently, a novel LAPP was isolated from the salivary glands of the leech Haementeria officinalis,7 cloned, and expressed in yeast.8 This recombinant protein specifically blocks collagen-mediated platelet aggregation and platelet adhesion under static conditions.8
In the current study, we investigated the effect of rLAPP on platelet deposition on collagen types I, III, IV, and VI, and on physiologically relevant collagen-rich surfaces under flow conditions. To elucidate the mechanism of the effect of rLAPP on the collagen-platelet interactions, the influence of rLAPP on both vWF binding and fibronectin binding to collagen were also investigated.
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Methods |
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Materials
rLAPP (Mr=13 642) was purified as previously described.7 Human placenta collagen types I, III, and IV were obtained from Sigma Chemical Co; human placenta collagen type VI was obtained from Heyl; glass coverslips from Menzel; the retouching airbrush from Badger Brush; human albumin from Behringwerke; RPMI-1640 medium poly-L-lysine from Sigma; unfractionated heparin and Orgaran from Organon; some blood products were obtained from the Red Cross Bloodbank Utrecht; avidin-biotin-horseradish peroxidase complex from Vector Laboratories; 3,3'-diaminobenzidine from Sigma; the platelet analyzer from Baker Instruments; the infusion-pump from Harvard Apparatus (pump 22, model 2400-004; the image analyzer from AMS; mAb CLB-RAG35 was kindly provided by Dr J.A. van Mourik (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam; Na125I was from Amersham; enzymobeads from Bio-Rad Laboratories; ELISA plates from Costar; BSA from Sigma; and Bio-gel A-15m from Bio-Rad.
Preparation of Adhesive Surfaces
Human placenta collagen types I, III, IV (1.4 mg/mL), and VI
(0.14 mg/mL) were solubilized in 50 mmol/L acetic acid, sprayed onto
clean glass coverslips with a retouching airbrush (Badger model 100) to
a surface density of 30 ng/mm2 (collagen type VI) or 300
ng/mm2 (collagen types I, III, and IV). These densities
supported optimal platelet coverages.6 9 Fibronectin
was isolated from normal human plasma by passing it over a
gelatin-Sepharose column10 11 and was sprayed onto
coverslips at a density of 50 ng/mm2. vWF was purified from
human cryoprecipitates by polyethylene glycol precipitation and gel
filtration12 and was checked for vWF antigen and vWF
ristocetin cofactor activity. Coverslips were coated by incubation with
100 µL vWF (20 µg/mL) in 0.05 mol/L TBS, pH 7.35, for 1 hour at
room temperature. All protein-sprayed or coated surfaces were
blocked by incubation with 4% HAS in 10 mmol/L HBS, pH 7.35, for 30
minutes at room temperature.
Human vascular endothelial cells derived from umbilical veins were isolated according to Jaffe et al13 with some modifications.14 The cells were cultured in RPMI-1640 containing 20% pooled human serum. Endothelial cells of passage 3 were used. After the cells had grown to confluence on glass coverslips, matrices were isolated by exposing the cells to 0.1 mol/L NH4OH.15 This step was followed by three washes with PBS, pH 7.35.
Fresh coronary arteries from explanted hearts of patients undergoing cardiac transplantation were obtained from the Department of Cardiology, University Hospital Utrecht. Frozen cross sections of the atherosclerotic coronary arteries at 6-µm thickness were cut at -20°C16 and were mounted on poly-L-lysine–coated glass coverslips. The clean glass coverslips were first coated by successive dipping into 0.05% poly-L-lysine solution (in deionized water) and into deionized water followed by air drying to prevent detachment of tissue sections in flow. Poly-L-lysine–coated coverslips, after incubation with 4% HAS, were not reactive toward platelets.
Perfusions
Fresh blood from healthy donors who denied having taken aspirin
in the preceding 10 days was anticoagulated with one-tenth volume
of 50 U/mL unfractionated heparin, with one-tenth volume of Orgaran
(a low-molecular-weight heparinoid; final
concentration 15 U/mL), or with one-tenth volume of 110 mmol/L
trisodium citrate. The choice of anticoagulant was dependent on the
experiment; Orgaran was used for studies with cross sections of
coronary arteries and trisodium citrate for studies with
fibronectin.17 For studies with collagen, blood was
anticoagulated with heparin, since we recently observed that
platelet adhesion to collagen type IV was almost absent, and
platelet adhesion to collagen type I was decreased in citrated
blood at high sr. Heparin did not cause increased platelet clumping
under the flow conditions of our experiments. Platelet deposition
under non–pulsatile-flow conditions was performed by use of a
rectangular perfusion chamber as described.18 19 The
chambers used had a slit width of 10 mm and slit heights of 0.6 mm
(sr=1600 s-1) and 1 mm (sr=375 s-1),
corresponding with flow rates of 56 and 36 mL/min, respectively. Whole
blood (15 mL) was prewarmed at 37°C for 5 minutes, duplicate
coverslips were inserted into the perfusion chamber, and the blood was
then circulated through the perfusion chamber for 5 minutes. Most
studies were performed at a wall sr of 1600 s-1 because
this sr is relevant in an arterial system.20
The system was rinsed with HBS after each perfusion. Perfusions over
collagen type III at low sr (sr=375 s-1, flow
rate=75 µL/min) were performed in a modified parallel plate perfusion
chamber with a slit height of 0.1 mm and a slit width of 2 mm. This
chamber was used in combination with a Harvard infusion pump as a
single-pass perfusion system. For collagen type III, the adhesion
pattern was the same as with the recirculating perfusion system.
rLAPP was prepared as stock solution (13.64 mg/mL in HBS, pH 7.35) and was added to whole blood 15 minutes before a perfusion was performed.
Platelet counts were measured with a Platelet Analyzer 810 with apertures set between 3.1 and 16 µm. Platelet counts before perfusion were between 150 000 and 300 000/µL. Microaggregate formation during recirculation of the blood was usually less than 30% and was not influenced by rLAPP.
Evaluation
The coverslips from the perfusion chamber were removed and
rinsed with HBS, fixed in glutardialdehyde (0.5% in
PBS) and stained with May-Grünwald-Giemsa as
described.21 Platelet deposition was quantified with a
light microscope coupled to a computerized image analyzer (AMS
40-10). Axial dependency was often observed on collagen-sprayed
coverslips. For that reason we evaluated three lines perpendicular to
the flow direction: one line in the center of the coverslip and two
lines 3 mm to the right and 3 mm to the left of the center. A clearly
decreased platelet adhesion on the downstream coverslip, caused by
the thrombogenic surface on the first coverslip, was sometimes
observed. In case platelet adhesion to the downstream coverslip was
more than 20% less than on the first coverslip, the second coverslip
was omitted. Platelet deposition was expressed as the percentage of
the surface covered with platelets and platelet aggregates.
Coverslips with cross sections of coronary arteries were fixed in paraformaldehyde (2% in PBS, pH 7.35), immersed in 0.1 mol/L glycine (in TBS, pH 7.6) for 10 minutes and rinsed in TBS. Adhered platelets and platelet thrombi were visualized by staining with a biotinylated mAb against glycoprotein Ib16 and an avidin-biotin-horseradish peroxidase complex. Cobalt chloride (0.025%) and nickel ammonium sulfate (0.020%) were added to the chromogenic substrate 3,3'-diaminobenzidine to yield a black reaction product.22 Platelet deposition was qualitatively evaluated with a light microscope.
Binding Studies
vWF and fibronectin were purified as described under
"Preparation of Adhesive Surfaces." Radiolabeling of vWF,
fibronectin, and rLAPP with Na125I was performed using
enzymobeads containing lactoperoxidase/glucose oxidase according to the
instructions of the manufacturer. Non-covalently linked
125I was removed by dialysis against TBS, pH 7.4.
Radiolabeling did not affect binding as tested by dilution with
unlabeled ligands. Results were obtained in at least two independent
experiments performed with different radiolabeled preparations
(specific activities ranged from 38 to 864 cpm/ng).
Human placenta collagen type III was solubilized in 50 mmol/L acetic acid (1 mg/mL), and subsequently dialyzed against PBS to obtain fibrillar collagen (48 hours, at 4°C). Wells of a 96-well ELISA tray were filled with 125 µL of the fibrillar collagen suspension (100 µg/mL) and were centrifuged for 15 minutes in a centrifuge with a rotor for ELISA trays (250g, room temperature) to coat the wells. Nonadsorbed collagen was then removed, and the wells were washed with deionized water. After a blocking buffer was used (50 mmol/L Tris, 100 mmol/L NaCl, 3% BSA, 0.1% Tween, pH 7.4; 1 hour at room temperature), 100 µL of the radiolabeled vWF, fibronectin, or rLAPP solution was added. Incubations in triplicate were performed for 2 hours at room temperature. After incubation, the wells were emptied and washed three times. Results were expressed as specific binding.
Washed platelets were resuspended in PBS for platelet-binding studies. Washed platelets (200x106/mL) were incubated with 125I rLAPP (41 µg/mL) or with 125I rLAPP in the presence of 20xexcess of unlabeled rLAPP (820 µg/mL) for 1 hour at room temperature. rLAPP binding to platelets was measured by placing 200 µL of the platelet suspensions (in triplicate) on top of 100 µL 20% sucrose in PBS. The tubes were spun for 2 minutes at 10 000g and the pellets were counted.
Gel Filtration
125I rLAPP (81.8 µg/mL) was incubated for 2 hours
at room temperature with vWF (30 µg/mL) in TBS+1% BSA. Of this
mixture 0.5 mL was applied to a Bio-gel A-15m (100 to 200 mesh)
column that had been previously equilibrated with TBS, 5 mmol/L sodium
citrate (pH 7.4). Fractions of 0.5 mL were collected. The amount of vWF
was measured with an ELISA.
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Results |
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Effect of rLAPP on Platelet Deposition on Collagens
Earlier studies23 indicated that rLAPP binds to collagen type I. We therefore first preincubated coverslips, sprayed with collagen types I and III, with rLAPP (30 minutes at room temperature). However, the affinity of rLAPP to bind to collagen is relatively low, and even when the coverslips were only briefly rinsed, more than 10 times higher rLAPP concentration was required to obtain the same inhibition as when rLAPP was added to the perfusate. For this reason, rLAPP was subsequently added to the perfusate for all experiments.
As shown in Fig 1A
, 1B, and 1C, rLAPP inhibited
platelet deposition to collagen types I, III, and IV at high sr
(1600 s-1) in a concentration-dependent manner.
Platelet deposition to all three types could be completely
inhibited by rLAPP. Platelet deposition on collagen types I and IV
was more sensitive to inhibition by rLAPP, with mean IC50
values of 70 nmol/L (Fig 1A) and 90 nmol/L (Fig 1C), than that to
collagen type III (Fig 1B, mean IC50=600 nmol/L). A wide
variability in response between donors was observed; both the extent of
platelet adhesion to collagen alone, as well as the efficacy of
rLAPP-mediated inhibition, varied from experiment to experiment,
resulting in distinct curves. Platelets from some donors adhered
very poorly to collagen types I and IV at high sr (1600
s-1) and were for that reason excluded. This phenomenon
was never observed for collagen type III. Aggregates on collagen type
III were in general larger and more compact than aggregates on collagen
types I and IV, in the absence of rLAPP.
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The effect of rLAPP on platelet deposition to collagen types I,
III, and IV at low sr (375 s-1) was compared with the
effect of rLAPP on platelet deposition at high sr (1600
s-1). Two concentrations of rLAPP were initially evaluated
that either partly inhibited or completely inhibited at high sr. The
lower concentration did not inhibit platelet adhesion under these
conditions at the lower sr, whereas the high concentration only
partially inhibited platelet adhesion. Subsequently, collagen type
III was selected to study the ability of rLAPP to inhibit platelet
adhesion at low sr. The comparison between low and high sr for collagen
type III was carried out with a modified perfusion chamber (slit
height=0.1 mm, slit width=2 mm) to reduce the volume of blood and thus
the amount of rLAPP required to observe inhibition. Perfusions at both
srs were performed with blood from the same donor on the same day. As
shown in Fig 2, at low sr 10 times more rLAPP was
required to inhibit platelet deposition to the same extent as at
the higher sr.
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) as
compared with the effect of rLAPP on platelet deposition at sr 1600
s-1 (
). Perfusions at both srs were performed with
blood from the same donor at the same day. Three different donors were
used. Whole blood was incubated with the amount of rLAPP as indicated.
Data are expressed as fraction of control (without rLAPP).
Effect of rLAPP on Platelet Adhesion to Collagen Type
VI
In contrast to platelet deposition to collagen types I, III,
and IV, platelets did not form aggregates on collagen type VI.
Spray-coated collagen type VI supported the adhesion of mainly
dendritic platelets. Since collagen type VI was available only in
limited amounts, we examined the effect of a high concentration of
rLAPP ([rLAPP]=3 µmol/L) that completely inhibited platelet
deposition to the collagen types described above. At this concentration
of rLAPP, platelet adhesion was only partly inhibited (43% and
27% at sr=1600 s-1 and 44% at sr=375 s-1;
Table 1). In an experiment in which coverslips were
coated with 300 ng collagen type VI/mm2 (the same as used
for types I, III, and IV), rLAPP showed the same degree of
inhibition.
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Effect of rLAPP on Platelet Deposition to Cross Sections of
Atherosclerotic Coronary Arteries
Inhibition of platelet deposition to cross sections of
atherosclerotic coronary arteries was observed with rLAPP
concentrations above 1 µmol/L. As shown in Fig 3, 6
µmol/L rLAPP produced a pronounced inhibition of platelet
deposition. The degree of inhibition was dependent not only on the
blood donor but also on the vessel wall used (not shown).
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Effect of rLAPP on Platelet Adhesion to Other Adhesive
Proteins
rLAPP had no or little inhibitory effect on
platelet adhesion to vWF or fibronectin-coated or -sprayed
glass coverslips (Table 1
). The small inhibitory effect on
platelet adhesion to fibronectin with 3 µmol/L rLAPP was not
enhanced at higher concentrations of rLAPP.
Effect of rLAPP on Platelet Adhesion to the ECM of
Endothelial Cells
rLAPP did not inhibit platelet adhesion to the ECM of
endothelial cells. Even at rLAPP concentrations up to 8
µmol/L, platelet adhesion was only slightly inhibited. In only
one of three experiments was significant inhibition of 20% observed
(P<.05, t test). At an sr of 375
s-1, rLAPP also did not inhibit platelet
adhesion to the endothelial cell matrix.
The effect of rLAPP on platelet adhesion to collagen appeared to be
specific, but it is not clear how rLAPP interacts with the collagen.
Since there is significant amount of vWF present in the ECM, in
contrast to the other ECMs in which vWF is absent, we hypothesized that
rLAPP might inhibit platelet adhesion to collagen under flow
conditions in whole blood by interacting with the binding of vWF to
collagen. In case vWF was already present, rLAPP should not be able
to interfere with this process. We tested this working hypothesis by
following two approaches: (1) Incubation of collagen-sprayed
coverslips with vWF (10 µg vWF/mL; 1 hour at room temperature). As
shown in Table 2, preincubation with vWF had no effect
on the efficacy of rLAPP to inhibit platelet adhesion. (2)
Perfusion of blood with suboptimal amounts of mAb CLB-RAG35, over ECM.
mAb CLB-RAG35 is directed against the platelet-binding domain
on vWF and almost completely inhibits platelet adhesion to ECM at
high SR (>1000 s-1).15 Inhibition by various
concentrations of mAb CLB-RAG35 resulted in a dose-response curve,
which was not affected by the addition of rLAPP to the perfusate
(results not shown).
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Effect of rLAPP on Binding of vWF to Collagen Type
III
The lack of effect of preincubating collagen type III with vWF may
have two alternative explanations: either rLAPP does not affect the
binding of vWF to collagen, or rLAPP is able to displace already bound
vWF. The second possibility was tested in binding studies with
125I vWF. As shown in Fig 4A, rLAPP competed
for the binding of 125I vWF to collagen type III when added
simultaneously with 125I vWF
([125I vWF]=0.5 µg/mL, binding of 125I vWF
was maximally 8%). When increasing concentrations of 125I
vWF (0.5 to 20 µg/mL) were added together with 0.5 µmol/L rLAPP
(suboptimal concentration) and subsequently incubated with surfaces
coated with collagen type III, we observed that the inhibition of
125I vWF binding to collagen by rLAPP was not easily
overcome by increasing the amount of 125I vWF (Fig 4B).
Binding of vWF to collagen type III becomes less reversible in time
(Fig 5). rLAPP was able to displace 70% to 80% of
125I vWF after 2 hours of preincubation of collagen with
125I vWF and 50% to 60% of 125I vWF after 24
hours. Larger amounts of rLAPP could not displace the remaining 40% to
50% of bound vWF.
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), 0.5
µmol/L rLAPP (
), 40 x excess of unlabeled vWF (+). Incubations
were performed at room temperature and for 2 hours.
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). rLAPP (2 µmol/L) (We did the following experiment to see whether the amount of vWF that was not displaced by rLAPP was able to support platelet adhesion: collagen type III–sprayed coverslips were preincubated with 100 µL vWF (20 µg/mL) during 24 hours at room temperature. Coverslips were washed 20 minutes before perfusion started and were subsequently preincubated with 5 µmol/L rLAPP (20 minutes at room temperature). Perfusions were performed with washed platelets and red blood cells in 4% HAS.18 Coverslips that were not preincubated with vWF gave no platelet adhesion; neither did the coverslips that were only preincubated with rLAPP. Coverslips that were successively preincubated with vWF (24 hours) and rLAPP (20 minutes) showed platelet coverages of 30% to 40% of control (without rLAPP). Coverslips that were simultaneously preincubated with vWF and rLAPP (24 hours at room temperature) showed no platelet adhesion at all, suggesting that the amount of vWF that was not displaced by rLAPP supported platelet adhesion.
Effect of rLAPP on Binding of Fibronectin to Collagen Type
III
Binding of 125I fibronectin (1 µg/mL) to coated
collagen fibers was maximal at 9%. As indicated in Fig 6A, rLAPP prevented this binding with a maximum
inhibition of 30%. An increase in the amount of 125I
fibronectin led to a decreased inhibition by rLAPP ([rLAPP]=3
µmol/L) (Fig 6B).
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Binding of 125I rLAPP to Collagen Type III, vWF, or
Blood Platelets
To improve our understanding of the mechanism of action of rLAPP,
rLAPP was radiolabeled, and binding studies with coated collagen type
III were performed. As shown in Fig 7, rLAPP bound well
to collagen type III. Saturation was achieved with 8 µg rLAPP/mL
within 30 minutes of incubation. A mean Kd of
118±8.2 nM (mean±SEM, n=3) was calculated. The binding of
125I rLAPP to collagen type III was a reversible binding;
the binding of 125I rLAPP was displaced by unlabeled rLAPP
or by dilution (not shown). However, the binding of 125I
rLAPP to collagen type III was independent of the
simultaneous presence of vWF. Even a 100 times excess
(molar base) vWF did not affect the binding of rLAPP to the
collagen.
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125I rLAPP did not bind to vWF coated to microtiter wells. To rule out the possibility that rLAPP prevents the binding of vWF to collagen by binding to free vWF, 81.8 µg/mL 125I rLAPP was preincubated with 30 µg/mL vWF for 2 hours at room temperature. 125I rLAPP that was not bound to vWF was separated from vWF by gel filtration. The vWF-associated peak (fractions 9 to 12) contained 15 µg vWF and 50 ng 125I rLAPP. This small amount of rLAPP had no effect on vWF binding to collagen type III–coated wells. 125I rLAPP did not bind to platelets. Only 0.1% of radiolabeled rLAPP that was incubated with platelets was detected in the platelet pellet. This amount was not decreased by an excess of unlabeled rLAPP.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the current study we investigated a novel protein, isolated from the salivary glands of the leech Haementeria officinalis7 and cloned and expressed in yeast.8 This protein (rLAPP) is unique in that it specifically blocks both collagen-induced platelet aggregation and platelet adhesion to collagen. In this study we concentrated on the effect of rLAPP on platelet deposition to collagen and collagen-rich surfaces under flow conditions.
Platelet deposition on sprayed surfaces of collagen types I, III, and IV was inhibited by rLAPP in a concentration-dependent manner. The inhibitory effect of rLAPP at a high sr was much greater than at a low sr, which suggests that rLAPP may affect platelet-collagen interactions in the arterial system. Collagen type V is also present in the vessel wall but was not studied because platelets do not adhere to collagen type V under flow conditions in vitro.6 24 Platelet adhesion to collagen type VI was also tested because it has been shown that collagen type VI is a binding site for vWF in the vessel wall.25 Platelet adhesion to collagen type VI was only partly inhibited at an rLAPP concentration of 3 µmol/L, a concentration of rLAPP that completely inhibited platelet adhesion to collagen types I, III, and IV. The inhibition of platelet adhesion to collagen type VI was independent of sr, although absolute platelet coverage was higher at low sr. Platelet adhesion to other adhesive proteins, fibronectin and vWF, were not affected by rLAPP at a concentration that completely inhibited platelet adhesion to collagen types I, III, and IV.
The poor platelet adhesion to collagen types I and IV at high sr in blood from some donors could be due to low plasma magnesium. We recently studied the effect of plasma magnesium on platelet adhesion to collagen (unpublished results) and found that low magnesium concentrations in the physiological range resulted in impaired platelet adhesion to collagen types I and IV at high sr.
Platelet adhesion to the ECM of endothelial cells was also not inhibited by rLAPP. At present, the reason for this lack of an effect is unknown. The relative insensitivity of platelet adhesion to collagen type VI to rLAPP inhibition could possibly explain the absence of platelet inhibition of the ECM of endothelial cells. If collagen type VI were the most important collagen in the endothelial cell matrix, the effect of rLAPP would not be visible. Alternatively, collagen in the endothelial cell matrix may play only a minor role in the total amount of detected platelet adhesion in this system.26
Previous studies from our laboratory16 demonstrated increased platelet deposition on the connective tissue of atherosclerotic plaque compared with the normal intima, using 6-µm-thick cross sections of human coronary arteries mounted on glass coverslips. rLAPP caused a pronounced inhibition of platelet deposition on cross sections of atherosclerotic coronary arteries, demonstrating the involvement of collagen in the increased platelet deposition to the atherosclerotic plaque. The degree of inhibition was, as with the sprayed collagens, dependent on the blood donor, but also on the vessel wall used, suggesting that different types of collagen could be responsible for increased platelet deposition. It was striking that platelet aggregates on the luminal side of the intima and on the adventitia were less inhibited than platelet aggregates on the atherosclerotic plaque. This observation suggests the intriguing possibility that rLAPP may be able to block the thrombotic reaction on the atherosclerotic plaque without impairing normal hemostasis.
Since rLAPP is a more potent inhibitor of platelet adhesion to collagen at high sr than at lower sr and was not able to inhibit platelet adhesion to the ECM, we hypothesized that rLAPP could inhibit platelet adhesion to collagen by interacting with the binding of vWF to collagen. Preincubation of sprayed collagen type III with 10 µg vWF/mL for 1 hour at room temperature had no effect on the efficacy of rLAPP. Data obtained in binding studies indicated that rLAPP binds to collagen and prevents the binding of 125I vWF to collagen. It has now become clear that rLAPP will also displace vWF that is already bound to collagen. We cannot exclude the possibility that rLAPP has other mechanisms to inhibit platelet adhesion to collagen. Up to now, there is no evidence that vWF plays a role in platelet adhesion to collagen under static conditions, and rLAPP inhibits platelet adhesion to collagen under static conditions in a purified plasma-free system.8
rLAPP was also able to inhibit the binding of 125I fibronectin to collagen, but this inhibition was much less than for vWF. Cockburn et al27 showed that fibronectin binding to collagen was independent of vWF. The inhibition of fibronectin binding to collagen by rLAPP could easily be overcome by increasing the amount of fibronectin, suggesting that rLAPP and fibronectin competed for the same binding site on collagen type III. Since fibronectin is present in normal plasma at a concentration of 300 µg/mL, it is not plausible that the interaction of rLAPP with the binding of fibronectin to collagen plays a role in the inhibition of platelet adhesion. The binding of vWF to collagen type III does not show simple binding kinetics, probably because vWF consists of multimers with different binding affinities. rLAPP caused a shift in this affinity of vWF for collagen, indicating that it works at least partly as a competitive inhibitor.
At present almost no drugs exist that are able to inhibit platelet adhesion to the vessel wall. Currently, VCL-peptide,28 aurin tricarboxylic acid,29 30 31 and RGD-peptides are the most promising inhibitors. The RGDS and dRGDW-peptides inhibit partially and only at high sr.32
rLAPP is a potent platelet adhesion inhibitor under arterial conditions. A study in baboons at a relatively low sr of 500 s-1 showed no effect on platelet deposition to an arteriovenous shunt.33 Because of its properties described above, in vivo studies at high srs appear justified.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the Netherlands Heart Foundation (88.066). We wish to thank Drs E. Dale Lehman and Robert Maigetter for isolating the recombinant LAPP and Drs C. Klöpping and N. de Jonge (Department of Cardiology) for the coronary arteries from patients who were undergoing cardiac transplantation. We wish to acknowledge the technical assistance of Annemarie M. van de Hoeven in culturing the cells and the Red Cross Bloodbank Utrecht for blood supply.
Received December 1, 1994; accepted June 28, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Hovig T. Release of a platelet aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline extract of tendons. Thromb Diath Haemorrh. 1963;9:264-278.
2. Stemerman MB. Vascular intima components: precursor in thrombosis. Prog Hemost Thromb. 1974;2:1-47. [Medline] [Order article via Infotrieve]
3. Santoro SA. Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen. Cell. 1986;46:913-920. [Medline] [Order article via Infotrieve]
4.
Kunicki TJ, Nugent DJ, Staats SJ, Orchekowski RP,
Wayner EA, Carter WG. The human fibroblast class II
extracellular matrix receptor mediates platelet adhesion to
collagen and is identical to the platelet glycoprotein
Ia/IIa complex. J Biol Chem. 1988;263:4516-4519.
5.
Coller BS, Beer JH, Scudder LE, Steinberg MH.
Collagen-platelet interactions: evidence for a direct
interaction of collagen with platelet Gp Ia/IIa and an indirect
interaction with platelet Gp IIb/IIIa mediated by adhesive
proteins. Blood. 1989;74:182-192.
6.
Saelman EUM, Nieuwenhuis HK, Hese KM, de Groot PG,
Hejihen HF, Sage EH, Williams S, McKeown L, Gralnick HR, Sixma JJ.
Platelet adhesion to collagen types I through VIII under
conditions of stasis and flow is mediated by GpIa/IIa
(
2ß1-integrin). Blood. 1994;83:1244-1250.
7.
Connolly TM, Jacobs JW, Condra C. An
inhibitor of collagen-stimulated platelet
activation from the salivary glands of the Haementeria officinalis
leech, I: identification, isolation, and characterization.
J Biol Chem. 1992;267:6893-6898.
8.
Keller PM, Schultz LD, Condra C, Karczewski J,
Connolly TM. An inhibitor of collagen-stimulated
platelet activation from the salivary glands of the Haementeria
officinalis leech, II: cloning of the cDNA and expression.
J Biol Chem. 1992;267:6899-6904.
9. Houdijk WPM, Sakariassen KS, Nievelstein PFEM, Sixma JJ. Role of factor VIII-von Willebrand factor and fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar collagen types I and III. J Clin Invest. 1985;75:531-540.
10.
Engvall E, Ruoslahti E, Miller EJ. Affinity of
fibronectin to collagens of different genetic types and to
fibrinogen. J Exp Med. 1978;147:1584-1595.
11. Klebe RJ, Bentley KL, Sasser PJ, Schoen RC. Elution of fibronectin from collagen with chaotropic agents. Exp Cell Res. 1980;130:111-117. [Medline] [Order article via Infotrieve]
12.
Sodetz JM, Pizzo SV, McKee PA. Relationship of
sialic acid to function and in vivo survival of human factor VIII/von
Willebrand factor protein. J Biol
Chem. 1977;252:5538-5546.
13. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest. 1973;52:2745-2756.
14. Willems C, Astaldi GCB, de Groot PG, Janssen MC, Gonsalves MD, Zeijlemaker WP, Van Mourik JA, Van Aken WG. Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells. Exp Cell Res. 1982;139:191-197. [Medline] [Order article via Infotrieve]
15.
Houdijk WPM, de Groot PG, Nievelstein PFEM, Sakariassen
KS, Sixma JJ. Subendothelial proteins and
platelet adhesion: von Willebrand factor and fibronectin,
not thrombospondin, are involved in platelet adhesion to
extracellular matrix of human vascular endothelial
cells. Arteriosclerosis. 1986;6:24-33.
16. Van Zanten GH, de Graaf S, Slootweg PJ, Heijnen HFG, Connolly TM, de Groot PG, Sixma JJ. Increased platelet deposition on atherosclerotic coronary arteries. J Clin Invest. 1994;93:615-632.
17.
Beumer S, IJsseldijk MJW, de Groot PG, Sixma JJ.
Platelet adhesion to fibronectin in flow: dependence on
surface concentration and shear rate, role of platelet membrane
glycoproteins Gp IIb/IIIa and VLA-5, and inhibition by
heparin. Blood. 1994;84:3724-3733.
18. Sakariassen KS, Aarts PAMM, de Groot PG, Houdijk WPM, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med. 1983;102:522-535. [Medline] [Order article via Infotrieve]
19.
Nievelstein PFEM, D'Alessio PA, Sixma JJ.
Fibronectin in platelet adhesion to human collagen types I
and III: use of nonfibrillar collagen in flowing blood studies.
Arteriosclerosis. 1988;8:200-206.
20. Weiss HJ, Turitto VT, Baumgartner HR. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood: shear rate dependent decrease of adhesion in von Willebrand's disease and the Bernard-Soulier syndrome. J Lab Clin Med. 1978;92:750-764. [Medline] [Order article via Infotrieve]
21.
Nievelstein PFEM, De Groot PG, D'Alessio PA, Heijnen
HFG, Orlando E, Sixma JJ. Platelet adhesion to vascular
cells: the role of exogenous von Willebrand factor in
platelet adhesion.
Arteriosclerosis. 1990;10:462-469.
22. Adams JC. Heavy metal intensification of DAB-based HRP reaction product. J Histochem Cytochem. 1981;29:775. Letter. [Medline] [Order article via Infotrieve]
23. Condra C, Karczewski J, Lehman ED, Maigetter RZ, Manno PD, Gould RJ, Connolly TM. The mechanism of inhibition of collagen-stimulated platelet activation by the leech antiplatelet protein. Circulation. 1992;86(suppl I):I-408. Abstract.
24. Parsons TJ, Haycraft DL, Hoak JC, Sage H. Interaction of platelets and purified collagens in a laminar flow model. Thromb Res. 1986;43:435-443.[Medline] [Order article via Infotrieve]
25. Rand JH, Patel ND, Schwartz E, Zhou SL, Potter BJ. 150-kD von Willebrand factor binding protein extracted from human vascular subendothelium is type VI collagen. J Clin Invest. 1991;88:253-259.
26. Hindriks GA, Sixma JJ, De Groot PhG. Ascorbic acid increases the thrombogenicity of cellular matrices. Thromb Haemost. 1991;66:505-509. [Medline] [Order article via Infotrieve]
27. Cockburn CG, Fitzsimmons CM, Barnes MJ. The interaction of fibronectin (fn) with native, polymeric collagen (collagen fibres): comparison with von Willebrand factor (vWF)-binding by collagen. Thromb Haemost. 1989;61:378-385. [Medline] [Order article via Infotrieve]
28.
Gralnick HR, Williams S, McKeown L, Kramer W, Krutzsch
H, Gorecki M, Pinet A, Garfinkel LI. A monomeric von
Willebrand factor fragment, Leu-504-Lys-728, inhibits vWF
interaction with glycoprotein Ib-IX. Proc
Natl Acad Sci U S A. 1992;89:7880-7884.
29.
Strony J, Phillips M, Brands D, Moake J, Adelman B.
Aurintricarboxylic acid in a canine model of coronary
artery thrombosis. Circulation. 1990;81:1106-1114.
30.
Weinstein M, Vosburgh E, Philips M, Turner N,
Chute-Rose L, Moake J. Isolation from commercial
aurintricarboxylic acid of the most effective polymeric
inhibitors of von Willebrand factor interaction
with Gp Ib: comparison with other polyanionic and polyaromatic
polymers. Blood. 1991;78:2291-2298.
31. Girma JP, Fressinaud E, Christophe O, Rouault C, Obert B, Takahashi Y, Meyer D. Aurin tricarboxylic acid inhibits platelet adhesion to collagen by binding to the 509-695 disulphide loop of von Willebrand factor and competing with glycoprotein Ib. Thromb Haemost. 1992;68:707-713. [Medline] [Order article via Infotrieve]
32.
Hantgan RR, Endenburg SC, Cavero I, Marguerie G, Uzan
A, Sixma JJ, de Groot PG. Inhibition of platelet adhesion to
fibrin(ogen) in flowing whole blood by Arg-Gly-Asp and fibrinogen
-chain carboxy terminal peptides. Thromb
Haemost. 1992;68:694-700. [Medline]
[Order article via Infotrieve]
33.
Schaffer LW, Davidson JT, Siegl PKS, Gould RJ, Nutt RF,
Brady SF, Connolly TM. Recombinant leech antiplatelet
protein prevents collagen-mediated platelet aggregation but not
collagen graft thrombosis in baboons.
Arterioscler Thromb. 1993;13:1593-1601.
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