© 1997 American Heart Association, Inc.
Articles |
Triflavin Inhibits Platelet-Induced Vasoconstriction in De-endothelialized Aorta
From the Graduate Institute of Medical Sciences, Taipei Medical College (J.-R.S., W.C.H., C.H.S.), the Department of Pharmacology, National Defense Medical Center (M.H.Y., Y.M.L.), and the Institute of Pharmacology, Medical College, National Taiwan University (T.F.H.), Taipei, Taiwan.
Correspondence to Dr Joen-Rong Sheu, Graduate Institute of Medical Sciences, Taipei Medical College, No. 250, Wu-Hsing St, Taipei, Taiwan.
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Abstract |
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Abstract Triflavin, a 7.5-kD cysteine-rich polypeptide purified from Trimeresurus favoviridis snake venom, belongs to a family of Arg-Gly-Asp-(RGD)–containing peptides, termed disintegrins. In this study, aggregating human platelets dose- dependently induced vasoconstriction in de-endothelialized isolated rat thoracic aortas. At 5x107 cells per milliliter, platelets induced a peak tension averaging 65±7.2% of the tension induced by phenylephrine (10 µmol/L). The relative effectiveness of RGD-containing peptides (including venom peptides triflavin and trigramin, small RGD synthetic peptides Gly-Arg-Gly-Asp-Ser [GRGDS], Gly-Arg-Gly-Asp-Phe [GRGDF], and Gly-Arg-Gly-Asp-Ser-Pro-Lys [GRGDSPK]) was examined by testing the inhibitory effect on aggregating platelet-induced vasoconstriction in de-endothelialized aorta. Triflavin (1 µmol/L) significantly inhibited the platelet-induced vasoconstriction, whereas neither trigramin (10 µmol/L) nor small RGD peptides (2 mmol/L) (ie, GRGDS, GRGDF, and GRGDSPK) showed any significant effect. The release of serotonin and the formation of thromboxane A2 from aggregating platelets were both significantly inhibited by triflavin (2 µmol/L), whereas trigramin and small RGD-containing peptides showed no significant effect. On scanning electron micrographs of de-endothelialized aorta, aggregating platelets adhered to the subendothelium, with loss of their discoid shape, to form irregular spheres with pseudopod extensions. Triflavin (2 µmol/L) markedly reduced the adhesion of platelets to the subendothelium in the same aorta. Furthermore, RGD-containing peptides (including triflavin, trigramin, and small RGD-containing peptides) inhibited the adhesion of 10 µg/mL collagen-activated platelets to extracellular matrices (ie, fibronectin, vitronectin, and von Willebrand factor). It is concluded that the marked ability of triflavin to inhibit aggregating platelet-induced vasoconstriction in de-endothelialized aorta compared with other RGD-containing peptides (including trigramin), may be due at least partly to triflavin's efficiently preventing the activation of platelets subsequent to inhibition of serotonin release and thromboxane A2 formation. However, the different abilities of triflavin compared with other RGD-containing peptides was not related to the ability to inhibit adhesion of platelets to extracellular matrices. Therefore, from the results of this study, it appears that triflavin may be a useful therapeutic agent for the treatment of thromboembolism and its associated angiospasm.
Key Words: RGD-containing peptides • triflavin • de-endothelialization • serotonin • thromboxanes
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Introduction |
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Platelets play an important role in the regulation of vascular tone.1 2 3 When platelets aggregate, they release a number of substances, including 5-hydroxytryptamine and TxA2, both of which can cause contraction of vascular smooth muscle cells.4 It has been reported that aggregating platelets cause the contraction of de-endothelialized vascular segments and also the relaxation of blood vessels with intact endothelium.1 5 For example, isolated canine coronary arteries denuded of endothelium contract in the presence of aggregating canine platelets. In contrast, platelets cause an endothelium-dependent relaxation of the same arteries.6 Platelet-induced relaxation of endothelium-intact blood vessels is thought to be mediated by the release of endothelium-derived relaxing factor, whereas platelet-induced contraction of de-endothelialized blood vessels is related to the release of TxA2 and serotonin.1 3 Shimokawa and Vanhoutte2 reported that aggregating platelets cause the contraction of porcine coronary arteries subjected to temporary total occlusion, despite the presence of intact endothelium, indicating a dysfunctional endothelium. These observations imply that endothelial cells play an important role in the regulation of vascular tone when exposed to aggregating platelets.
Recently, many trigramin-like antiplatelet peptides (or
disintegrins) have been reported.7 8 9 10 11 12 These
peptides all contain RGD, are rich in cysteine, and bind with high
affinity to integrins on the cell surface. A family of cell
surface–adhesion receptors termed "integrins" has been
described.13 The integrins comprise a superfamily
of transmembrane receptors that participate in cell-cell and
cell-substrata interactions. Integrin receptors are membrane-spanning
heterodimers consisting of noncovalently associated 
- and
ß-subunits.14 15 Trigramin, an RGD-containing
peptide purified from the venom of Trimeresurus gramineus,
is a specific fibrinogen receptor antagonist with a high
binding affinity (Kd, 20 nmol/L) for the
activated platelet fibrinogen receptor
(glycoprotein IIb/IIIa;
IIbß3
integrin).16 17 Triflavin (also known as
flavoridin) is a trigramin-like antiplatelet peptide purified from
Trimeresurus flavoviridis snake venom that is more potent
than trigramin.12 18 19 Its primary structure
consists of 70 amino acid residues, including 12 cysteines, with an RGD
sequence at position 49 to 51.20 We previously
reported that triflavin inhibits platelet aggregation by
interfering with the interaction of fibrinogen with the
glycoprotein IIb/IIIa complex.20 21
Binding of fibrinogen to the glycoprotein IIb/IIIa complex
appears to be the final common pathway for platelet aggregation.
The present study was designed to determine the inhibitory effect of triflavin and other RGD-containing peptides (including naturally occurring venom peptide, trigramin, and the synthetic RGD peptides, GRGDS, GRGDF, and GRGDSPK) on the response of de-endothelialized aorta to aggregating platelets and to compare the activity of these RGD-containing peptides.
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Methods |
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Preparation of Rat Aortic Rings
Sprague-Dawley rats of both sexes, 270 to 350 g, were used. The animals were anesthetized with urethane (1.2 g/kg IP). The thoracic aorta was isolated and the excess fat and connective tissue were removed. Vessels were cut into rings about 4 mm in length and mounted in organ baths containing Krebs solution of the following composition (mmol/L): NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgCl2 1.2, CaCl2 2.5, and glucose 11. The tissue bath solution was maintained at 37°C and gassed with 95% O2/5% CO2. The aortic rings were denuded of endothelium by inserting a cotton ball into the lumen and gently rolling the ring back and forth. Two stainless steel hooks, one fixed and the other connected to a transducer (Grass FT03), were inserted into the aortic lumen in tissue-organ baths containing 10 mL Krebs solution. The aorta was equilibrated in the medium for 90 minutes with three changes of Krebs solution. Each ring was progressively stretched to the optimal point on its length-tension curve as determined by the active tension developed to PE (10 µmol/L) and maintained under an optimal resting tension before specific experimental processes were initiated. Vasoconstrictions were recorded isometrically via a force-displacement transducer connected to a Grass model 7D polygraph. The procedure for de-endothelializing the aorta did not alter significantly the maximum tension the vessels could produce when contracted with PE (10 µmol/L; average increase in tension, 3.2±0.4 and 2.9±0.2 g in rings with and without endothelium, respectively). Rings from which the endothelium had been removed showed no change in tension on the addition of acetylcholine, and this was taken to indicate that the vessels had been denuded completely.
Preparation of Human Platelet Suspensions
Blood was collected from healthy human volunteers who had not
taken any medicine during the preceding 2 weeks and mixed with 3.8%
(wt/vol) sodium citrate (9:1, vol/vol). Citrated blood was immediately
centrifuged at 120g for 10 minutes at 25°C to
obtain platelet-rich plasma. Platelet-rich plasma was
supplemented with prostaglandin E1
(0.5 µmol/L) and heparin (6.4 IU/mL), incubated for 10 minutes
at 37°C, and centrifuged again at 1000g for 20
minutes at 4°C. The supernatant was discarded, and the pellet was
washed with tris(hydroxymethyl)aminomethane
(Tris)-sodium-glucose (TSG) buffer, pH 7.4 containing (mmol/L) Tris-HCl
25, EDTA 0.01, NaCl 134, glucose 5 and suspended in TSG buffer at a
final concentration of 109 cells per
milliliter.
Experimental Protocols
De-endothelialized rings were contracted with
platelets, and the resulting platelet concentration in the
organ chamber was 106 to
108 cells per milliliter. When added to the organ
chambers, the platelets aggregated on exposure to glass, the
collagen of the cut vessel surfaces, and the calcium in the Krebs
buffer, while being stirred with bubbling by the gas mixture.
Completion of aggregation was evidenced in all cases by clearing of the
initially turbid solution and the formation of visible platelet
clumps and strands. In other experiments, the platelet suspensions
(5x107 cells per milliliter) were preincubated
with various concentrations of RGD-containing peptides (including
naturally occurring venom peptides triflavin and trigramin or the
synthetic small peptides GRGDS, GRGDF, and GRGDSPK) for 5 minutes at
room temperature before adding to the organ chambers.
TxB2 Determination
One-milliliter aliquots of the fluid were collected 10 minutes
after the addition of platelets and frozen at -70°C until
analysis. The TxB2 levels of the fluid
were measured using an EIA kit (Cayman Chem) according to the
instructions of the manufacturer.
Serotonin Determination
Samples of fluid were withdrawn from the organ bath 12 minutes
after the addition of platelets. Five hundred aliquots of the fluid
were frozen at -70°C until analysis. For analysis,
the fluid was filtered through a
polytetrafluoroethylene membrane (Cameo 25
F syringe filter, 0.45-µm pore size). The serotonin in
the resulting fluid was quantitated by using an enzyme immunoassay
(EIA) kit (Immunotech) according to the instructions of the
manufacturer.
Histological Examination
For scanning electron microscopy,
de-endothelialized aortic rings were fixed in 4%
glutaraldehyde and postfixed with 1% osmium tetroxide
(OsO4) in 0.1 mol/L cacodylate buffer solution
(pH 7.4) at room temperature for 1.5 hours. After several washes in
cacodylate buffer, the tissues were dehydrated in graded alcohols.
Specimens were then critical point dried in a critical point dryer
(Hitachi, HCP-2). Under a dissecting microscope, tissues were then cut
longitudinally with a razor blade to enable full exposure of the
luminal surface. Tissues were then coated with gold-palladium (Hitachi,
IB2). All specimens were examined by a scanning
electron microscope (Hitachi, S2400).
Preparation of BCECF-AM–Labeled Human Washed Platelet
Suspensions
Blood was collected from healthy human volunteers and mixed with
acid/citrate/dextrose (9:1, vol/vol). After
centrifugation for 10 minutes at 120g at
room temperature, the supernatant (platelet-rich plasma) was
supplemented with prostaglandin E1
(0.5 µmol/L) and heparin (6.4 U/mL), incubated for 10 minutes at
37°C, and centrifuged at 500g for 10 minutes. The
platelet pellet was suspended in 5 mL of
Ca2+-free Tyrode's solution (pH 7.3), and then
apyrase (1 U/mL), prostaglandin E1
(0.5 µmol/L), heparin (6.4 U/mL) and BCECF-AM (5 µmol/L)
were added, followed by incubation for 40 minutes at 37°C. After
centrifugation of the suspension at 500g for
6 minutes, the washing procedure was repeated. The washed platelets
were finally suspended in Ca2+-free Tyrode's
solution (3x108 cells per milliliter),
containing 1 µmol/L prostaglandin
E1.
Preparation of Matrix Coating
Substrates for platelet adhesion studies were prepared in
plates (96-well; Costar). Fifty microliters of Fn, Vn, vWF, Lm (all at
1 µg per well in phosphate-buffered saline), or type IV collagen (5
µg per well) was added to the wells, and the plates were incubated at
room temperature for 4 hours. After incubation, the solutions were
aspirated and the wells filled with buffer containing bovine serum
albumin (0.5%). Control wells were filled with bovine serum
albumin only.
Adhesion Assays
Platelet adhesion assays were performed as described by
Haverstick et al.22 Equal volumes of platelet
suspensions and peptides were mixed and collagen was added to a final
concentration of 10 µg/mL. The mixture was incubated for 20 minutes
at room temperature without shaking. Fifty-microliter aliquots of
collagen-activated platelet suspensions
(3x108 cells per milliliter) were then
transferred to the wells coated with the matrices and incubated at room
temperature for 30 minutes without shaking. In some experiments,
resting platelet suspensions (3x108 cells
per milliliter) were incubated with various concentrations of the
peptides or anti-integrin
2ß1 monoclonal
antibody for 20 minutes at room temperature and then transferred to the
wells coated with type IV collagen without shaking. Nonadherent
platelets were removed by aspiration, and the platelets were
gently washed three times with Ca2+-free
Tyrode's solution. The extent of binding was determined with a
CytoFluor 2300 fluorescence plate reader (Millipore).
Statistical Analysis
In each group of experiments, n refers to the number of rats
from which vessels were taken. In vessels contracted with PE (10
µmol/L), the changes in tension induced by platelets are
expressed as a percentage of the PE-induced tension. The experimental
results are expressed as the mean±SEM and accompanied by the number of
observations. Data were assessed by ANOVA. If this analysis
indicated significant differences among the group means, each group was
then compared by the Newman-Keuls method. A value of P<.05
was considered significant.
Materials
Trimeresurus flavoviridis venom and
Trimeresurus gramineus venom were purchased from Latoxan and
stored at -20°C. Triflavin and trigramin were purified from the
venom of T. flavoviridis and T. gramineus,
respectively, as previously described.17 18
Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Asp-Ser-Pro-Lys (GRGDSPK)
were purchased from Peninsula Laboratories. Gly-Arg-Gly-Asp-Phe (GRGDF)
was synthesized by the Chiron Mimotopes Pty Ltd. Sodium citrate, PE,
heparin, prostaglandin E1, apyrase
(grade III), acetylcholine, EDTA, glucose, Fn (from bovine plasma),
Tris-HCl, Vn (from human plasma), type IV collagen (from mouse
sarcoma), and Lm (from the basement membrane of mouse sarcoma) were
obtained from Sigma Chemical Co. BCECF-AM was purchased from Molecular
Probes, Inc; vWF (from human plasma) was purchased from Calbiochem;
methiothepin maleate was purchased from ICN Pharma, Inc; SQ 29548 was
purchased from Biomol; and MCA 743 monoclonal antibody was obtained
from Serotec.
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Results |
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Platelet-Induced Vasoconstriction in De-endothelialized Thoracic Aortic Rings
As shown in Fig 1A
, aggregating
platelets dose dependently induced vasoconstriction in
de-endothelialized vessels. At
5x107 cells per milliliter, aggregating
platelets induced a peak tension that averaged 65±7.2% of the
tension induced by PE (10 µmol/L) (Fig 1A). Peak tension was
reached at an average of 9 minutes after the addition of the
platelets and was then followed by a gradual decline in tension. An
equal volume of supernatant, prepared by centrifugation
(15 000g for 10 minutes) of platelet suspensions, added
to the organ chamber produced no significant effects on vessel tension
(data not shown). In some experiments, autologous rat blood was used.
The results with human or rat platelets were similar in all
experiments (ie, rat platelets at 5x107
cells per milliliter induced a peak tension about 60±6% of the
tension induced by PE (10 µmol/L; data not shown). Because a
large amount of blood was needed to obtain washed platelets and the
platelet yield from rat blood was inadequate, this study was
performed with human blood. Moreover, aggregation was evidenced by the
gradual clearing of the initially turbid solution, visible platelet
clumping, and platelet aggregates on the blood vessel surface
observed under scanning electron microscopy. Furthermore, we also
examined the extent of platelet aggregation in the organ chambers
by using a cell counter (Coulter Counter, ZM). We found that the number
of platelets in the bath fluid was <104
cells per milliliter 9 minutes after the addition of platelets into
the organ chamber (106 to
108 cells per milliliter), indicating that the
platelets in the organ chamber were significantly aggregated. In
subsequent experiments, we used an appropriate concentration
(5x107 cells per milliliter) of human
platelets to characterize the inhibitory effect of
RGD-containing peptides on this reaction.
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,
1x106 cells per milliliter; 
, 1x107 cells
per milliliter; 
, 5x107 cells per milliliter; 
,
1x108 cells per milliliter. B, Time course of the tension
response of de-endothelialized rat thoracic aorta to
the addition of platelets in the presence of RGD-containing
peptides. The aorta was preincubated with RGD-containing peptides
(including naturally occurring venom peptides triflavin and trigramin
or small RGD-containing peptides GRGDS, GRGDF, or GRGDSPK) for 5
minutes followed by the addition of platelet suspensions
(5x107 cells per milliliter). Control (
, 2 µmol/L). Changes in tension are expressed as a
percentage of the contraction of the rings to PE (10 µmol/L).
Data are presented as mean±SEM (n=5). *P<.01;
**P<.001, significant difference compared with the
control.
Effects of RGD-Containing Peptides on Platelet-Induced
Vasoconstriction in De-endothelialized Aortic
Vessels
The effect of RGD-containing peptides on platelet-induced
vasoconstriction in de-endothelialized aortic vessels
was explored at fixed concentrations of platelets
(5x107 cells per milliliter) and time point (9
minutes after the addition of platelets). By treating the
platelets with RGD-containing peptides (including the naturally
occurring venom peptides triflavin and trigramin or the synthetic
peptides GRGDS, GRGDF, and GRGDSPK) in an organ bath for 5 minutes, the
relative effectiveness of these RGD-containing peptides could be more
carefully examined by testing the inhibitory effect of
aggregating platelet-induced vasoconstriction in aortic rings
without endothelium over a wide range of peptide
concentrations. The results are shown in Figs 1B
and 2. At 1 µmol/L, triflavin caused a
significantly greater inhibition of the platelet-induced
contraction than either trigramin (10 µmol/L) or the synthetic
RGD-containing peptides (2 mmol/L) (ie, GRGDS, GRGDF, and GRGDSPK)
(Fig 2). At 2 µmol/L, triflavin showed a maximal
inhibitory effect of about 45±6%. There were no marked
differences (P>.05) in the abilities of trigramin and
RGD-containing synthetic peptides to inhibit the vasoconstriction
induced by aggregating platelets (Figs 2 and 3). On the other hand,
TxA2 receptor antagonist SQ 29548
(1 µmol/L)23 or the serotonin
receptor antagonist methiothepin (1
µmol/L)24 present in the bath fluid
significantly inhibited the aggregating platelet
(5x107 cells per milliliter)-induced
vasoconstriction by about 48±4% and 39±6%, respectively. Moreover,
the combination of SQ 29548 (1 µmol/L) and methiothepin (1
µmol/L) caused a greater inhibition of platelet-induced
vasoconstriction (78±9%) than either SQ 29548 or methiothepin alone
(data not shown).
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) into the organ chambers. Peak
tensions (a, 2.6 g; b, 0.9 g; c, 2.4 g) were
obtained at 9 minutes after the addition of the platelets,
respectively.
Assay of TxB2
TxB2 levels measured in the bath fluid were
8.9±1.1 ng/mL 10 minutes after the addition of platelets. The
release of TxB2 was affected by the presence of
triflavin (5.8±0.8 ng/mL; Fig 4A), but
it was not significantly reduced by incubation with other
RGD-containing peptides, including trigramin or synthetic peptides (ie,
GRGDS, GRGDF, and GRGDSPK) (Fig 4A).
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Assay of Serotonin
In this study, we measured the amount of serotonin in
the bath fluid by using a serotonin EIA kit instead of
conventional isotope-labeled or high-performance liquid
chromatography assays. Aggregating platelets
(5x107 cells per milliliter) released
serotonin into the bath fluid at a concentration of
22.9±3.5 ng/mL, measured 12 minutes after the addition of the
platelets (Fig 4B). The release of serotonin was
significantly inhibited by triflavin (2 µmol/L; 13.6±2 ng/mL)
but was not inhibited by either the presence of trigramin (10
µmol/L) or other RGD small peptides in the bath fluid (Fig 4B).
Scanning Electron Microscopy of De-endothelialized
Aortic Vessels
Fig 5A is a scanning electron
micrograph of de-endothelialized aortic vessels to
which platelets have adhered. The endothelium was
completely denuded and platelets adhered to the
subendothelium. A change from the normal discoid shape
to that of irregular spheres and the extension of pseudopods were
observed. On the other hand, triflavin (2 µmol/L) markedly
reduced the adhesion of platelets to the
subendothelium in the same rat aorta (Fig 5B). This
result was also found in the presence of trigramin (10 µmol/L)
and synthetic RGD peptides (data not shown), suggesting that
RGD-containing peptides may inhibit the adhesion of platelets to
the subendothelium of the aorta.
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Effect of RGD-Containing Peptides on Platelet Adhesion to
Vascular Subendothelial Matrices
Platelets adhere to and are activated by contact with
the vascular subendothelial layer, which consists of a
meshwork of connective-tissue proteins including collagen, Fn, Vn, Lm,
and vWF, synthesized by the endothelial cells. In this
study, the relative effectiveness of the RGD-containing peptides was
more carefully examined by studying the adhesion of collagen (10
µg/mL)-activated platelets to Fn, Vn, vWF, and Lm over a
wide range of peptide concentrations. BCECF-AM has previously been used
in a fluorescence-based viability assessment in adherent cell
cultures.25 In this study, we evaluated the
extent of collagen-activated platelet adhesion to plates
coated with vascular subendothelium proteins by using
BCECF-labeled platelets instead of conventional isotope-labeled
platelets. The results are shown in Fig 6. At 1 µmol/L, triflavin
effectively inhibited the adhesion of platelets to Fn, Vn, and vWF
by about 92%, 84%, and 76%, respectively. Moreover, trigramin (2 and
8 µmol/L) also dose dependently inhibited the adhesion of
platelets to Fn, Vn, and vWF (Fig 6). On a molar basis, triflavin
was about eight times more potent than trigramin at inhibiting the
adhesion of platelets to Fn, Vn, and vWF. To determine whether
activated-platelet adhesion was RGD dependent,
RGD-containing synthetic peptides were tested. At 2 mmol/L, all of
the RGD-containing synthetic peptides significantly inhibited the
adhesion of activated platelets to Fn, Vn, and vWF, but
less effectively than triflavin or trigramin. In contrast, triflavin
and other RGD-containing peptides did not significantly suppress the
adhesion of activated platelets to Lm (Fig 6D), suggesting
that the adhesion of platelets to immobilized Lm may be
via a non–RGD-dependent pathway. The control peptide GRGES (2
mmol/L) had no significant effect on cell adhesion (Fig 6), indicating
that RGD-containing peptides may interrupt the adhesion of
activated platelets to Fn, Vn, and vWF, but not to Lm.
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In addition, Fig 7 showed that triflavin
(10 µmol/L), trigramin (20 and 50 µmol/L), and other
RGD-containing synthetic peptides (all at 2 mmol/L) did not
significantly inhibit the adhesion of resting platelets to
immobilized type IV collagen, whereas MCA 743 monoclonal
antibody (10 µmol/L) directed to a functional epitope on
integrin 2ß1 (a
collagen receptor on platelet or other
cells),26 markedly inhibited the adhesion of
resting platelets to type IV collagen (69±8%).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This study shows that human platelets trigger the contraction of the thoracic aorta from which the endothelium has been removed. Two substances are released by aggregating platelets that are known contractile agents in certain vascular smooth muscle preparations: 5-hydroxytryptamine (serotonin) and TxA2.4 27 28 This present study demonstrated that both serotonin and TxA2 are released during the aggregation of platelets under the experimental conditions imposed and that both contribute to the vasocontraction. During the separation of the platelets from whole blood, some activation of platelets may have occurred, with some release of TxA2, serotonin, and platelet-activating factor, which may account for the influence on vascular tone. However, the supernatants from the platelet suspensions had no effect on vascular tone, suggesting that significant amounts of vasoconstrictor material or materials were released only during platelet–vessel wall interaction.
This study shows also that human platelets dose dependently induce
vasoconstriction in isolated rat thoracic aorta. Maximal tension was
reached about 9 minutes after the addition of platelets, and about
5x107 cells per milliliter of platelets
induced the maximal vasoconstriction under these experimental
conditions (Fig 1).
In our previous studies,18 19 triflavin was found
to inhibit human platelet aggregation stimulated by thrombin,
collagen, ADP, and U46619, not only in washed human platelets but
also in platelet-rich plasma and whole blood. We concluded that
triflavin inhibits platelet aggregation by interfering with
fibrinogen binding to its specific receptor associated with the
glycoprotein IIb/IIIa complex on the platelet surface
membrane.20 21 Furthermore, disintegrins do not
inhibit platelet release unless it follows platelet adhesion or
clumping.29 Recently, we further demonstrated
that triflavin is an effective antithrombotic agent in preventing
thromboembolism in in vivo and ex vivo
models.30 31 32 The series of experiments described
in this report were performed to examine whether or not peptides
containing the RGD sequence could effectively inhibit the aggregating
platelet-induced vasoconstriction. The results revealed that
triflavin dose dependently inhibited platelet-induced
vasoconstriction in de-endothelialized rat thoracic
aorta. At 2 µmol/L, triflavin showed a maximal
inhibitory effect (about 45%), whereas trigramin had no
significant inhibitory effect on this reaction, even at
concentrations up to 10 µmol/L. We previously reported that
triflavin was threefold more efficient than trigramin at inhibiting
platelet aggregation in vitro and ex
vivo.30 33 It has also been reported that the
synthetic peptide RGDF is fourfold to fivefold more efficient than RGDX
at inhibiting fibrinogen binding.34 This finding
has also been supported by studies in which triflavin (containing RGDF)
was found to be significantly more efficient than trigramin (containing
RGDD) in platelet aggregation.18 However, in
this study, alterations in the sequence of amino acid residues within
these small peptides, by transposition of amino acids, did not
significantly inhibit the platelet-induced vasoconstriction in
de-endothelialized aorta. In particular, GRGDF was no
more efficient than GRGDS or GRGDSPK (Fig 2B).
When endothelial continuity is disrupted, platelets
rapidly adhere to the subendothelial components that
are exposed. This adhesion is accomplished by an initial attachment
followed by the spreading of platelets. Several evidences strongly
suggest that platelet glycoprotein IIb/IIIa complex and
vWF in plasma and/or in subendothelium mediate initial
attachment.35 36 Platelet
glycoprotein IIb/IIIa complex provides binding sites for
fibrinogen,37 38 Fn,39
vWF,40 and also for Vn.41
All these ligands react with platelet glycoprotein
IIb/IIIa complex only after platelet activation in a divalent
cation–dependent manner, and the binding is inhibited by peptides
containing an Arg-Gly-Asp sequence. The precise mechanism and ligand(s)
involved in platelet adhesion to the subendothelium
through the glycoprotein IIb/IIIa complex mechanism,
however, remain to be elucidated. In this study, we speculated that the
obvious greater ability of triflavin to inhibit platelet-induced
de-endothelialized vasoconstriction compared with other
RGD-containing peptides (including trigramin) may be due to
triflavin's greater ability to inhibit the adhesion of platelets
to de-endothelialized vessels or
subendothelial matrices. As shown in Fig 6, we found
that triflavin and trigramin, as well as small RGD peptides,
significantly inhibited the adhesion of platelets to extracellular
matrices (ie, Fn, Vn, and vWF), except for Lm. At the same
concentrations, these peptides did not inhibit platelet-induced
vasoconstriction, whereas triflavin did (Fig 2). On the other hand, we
also found that all RGD-containing peptides employed in this study did
not significantly inhibit the adhesion of resting platelets to type
IV collagen (Fig 7). Taken together, these results indicate that the
different abilities of triflavin, compared with trigramin or other
synthetic peptides, at inhibiting platelet-induced vasoconstriction
at least in part are not related to the ability to inhibit the adhesion
of platelets to extracellular matrices.
Thus, whether or not RGD-containing peptides influence a variety of
vasoactive substances (ie, serotonin,
TxA2) released from aggregating platelets can
be identified. There were, however, interesting differences observed in
that only triflavin significantly inhibited serotonin
release and TxA2 formation in aggregating
platelets, whereas trigramin and the other three small RGD peptides
did not significantly affect these reactions. This result is reflected
in Fig 2, which shows that only triflavin inhibits platelet-induced
vasoconstriction in de-endothelialized vessels. In a
previous report,20 we found that triflavin bound
to the glycoprotein IIb/IIIa complex of resting and
activated platelets with a similar binding affinity,
whereas trigramin and small RGD synthetic peptides (ie, GRGDS) bound
with a much lower affinity to resting than to activated
platelets.16 In this study, platelet
suspensions were preincubated with RGD-containing peptides for 5
minutes followed by addition to the organ chamber, indicating that
triflavin had bound to the glycoprotein IIb/IIIa complex of
resting platelets before its addition to the organ chamber.
Therefore, we speculated that the obvious inhibitory effect
of triflavin on aggregating platelet-induced vasoconstriction may
be due at least partly to the fact that triflavin but not trigramin or
synthetic RGD-containing peptides has a higher binding efficacy toward
the glycoprotein IIb/IIIa complex of the resting
platelet membrane before addition to the organ chamber, thereby
leading to the prevention of platelet activation and reduced
serotonin release and TxA2 formation
from platelets, resulting finally in reduced vasoconstriction.
However, the exact mechanism is still unclear and requires further
characterization. In this study, small RGD-containing peptides did not
have any significant effects on this vasoconstriction model. Although
we used doses of these small peptides that were much greater than that
of triflavin, serotonin release and
TxA2 formation were no different from those of
the control. We previously reported that Arg-Gly-Asp represents
the active site of triflavin;20 in vitro studies,
however, have shown that triflavin is at least 800 to 1000 times more
potent at inhibiting platelet aggregation and fibrinogen binding to
platelets than GRGDS.19 20 Therefore, the
negative results with small RGD peptides in this vasoconstriction model
may be related to a lower efficacy than that of triflavin or to their
nonspecific properties, resulting in an inability to inhibit
serotonin release and TxA2
formation.
In conclusion, in this study, we demonstrated that triflavin inhibits platelet-induced vasoconstriction in de-endothelialized aorta. Under the same conditions, trigramin and small RGD peptides, even at much higher doses, appeared ineffective. The finding that triflavin causes the inhibition of platelet-induced vasoconstriction may be of benefit in the treatment of thrombotic disorders caused by endothelial cell damage that results in platelet adherence to the disrupted surface, followed by platelet aggregation initiated at the point of blood vessel–damaged endothelial cells, and finally the triggering of angiospasm. Therefore, it appears that triflavin has a potent antithrombotic activity that not only inhibits platelet aggregation and the adhesion of activated platelets to extracellular matrices but also may inhibit arterial thromboembolism and its associated angiospasm.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a grant from the National Science Council of Taiwan (NSC85 to 2331-B-38 to 026).
Received October 22, 1996; accepted April 15, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Houston DS, Shepherd JT, Vanhoutte PM. Aggregating human platelets cause direct contraction and endothelium-dependent relaxation of isolated canine coronary arteries. J Clin Invest. 1986;78:539–544.
2. Shimokawa H, Vanhoutte PM. Angiographic demonstration of hyperconstriction induced by serotonin and aggregating platelets in porcine coronary arteries with regenerated endothelium. J Am Coll Cardiol. 1991;17:1197–1202.[Abstract]
3. Yang BC, Chandna VK, Lawson DL, Mehta JL. Platelets enhance anoxic contraction of rat aortic rings via PAF-dependent mechanism. J Cardiovasc Pharmacol. 1993;21:249–255.[Medline] [Order article via Infotrieve]
4.
Ellis EF, Oelz O, Roberts LJ II, Payne NA, Sweetman
BJ, Nies AS, Oates JA. Coronary arterial smooth
muscle contraction by a substance released from platelets: evidence
that it is thromboxane A2.
Science. 1976;193:1135–1137.
5. Raymenants EB, Yang BC, Nicolini FA, Behrens P, Lawson DL, Mehta JL. Verapamil and aspirin modulate the platelet-mediated vasomotion in arterial segments with intact or disrupted endothelium. J Am Coll Cardiol. 1993;22:684–689.[Abstract]
6.
Cohen RA, Shepherd JT, Vanhoutte PM.
Inhibitory role of the endothelium in the
response of isolated coronary arteries to platelets.
Science. 1983;221:273–274.
7.
Gan ZR, Gould RJ, Jacobs JW, Friedman PA, Polokoff MA.
Echistatin, a potent platelet aggregation inhibitor
from the venom of the viper, Echis carinatus. J
Biol Chem. 1988;263:19827–19832.
8.
Chao BH, Jakubowski JA, Savage B, Chow EP, Marzec LM,
Harkeri LA, Maraganore JM. Agkistrodon piscivorus
platelet aggregation inhibitor: a potent
inhibitor of platelet activation. Proc Natl Acad
Sci U S A. 1989;86:8050–8054.
9.
Shebuski RJ, Ramjit DR, Bencen GH, Polokoff MA.
Characterization and platelet inhibitory activity of
bitistatin, a potent arginine-glycine-aspartic acid-containing peptide
from the venom of the viper Bitis arietans. J
Biol Chem. 1989;264:21550–21556.
10. Williams JA, Rucinski B, Holt JC, Niewiarowski S. Elegantin, and albolabrin purified peptides from viper venoms: homologies with the RGDS domain of fibrinogen and von Willebrand factor. Biochim Biophys Acta. 1990;1039:81–89.[Medline] [Order article via Infotrieve]
11. Huang TF, Liu CZ, Ouyang C, Teng CM. Halysin, an Arg-Gly-Asp-containing snake venom peptide, inhibits platelet aggregation by acting as fibrinogen receptor antagonist. Biochem Pharmacol. 1991;42:1209–1219.[Medline] [Order article via Infotrieve]
12. Gould RJ, Polokoff MA, Friedman PA, Huang TF, Holt JC, Cook JJ, Niewiarowski S. Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc Soc Exp Biol Med. 1990;195:168–171.[Medline] [Order article via Infotrieve]
13. Hynes RO. Integrins: a family of cell surface receptors. Cell. 1987;48:549–554.[Medline] [Order article via Infotrieve]
14.
Hemler ME, Thang C, Schwartz L. The VLA protein family:
characterization of five distinct cell surface heterodimers each with a
common 130 000 molecular weight ß subunit. J Biol
Chem. 1987;262:3300–3309.
15.
Hemler ME, Crouse C, Takada M, Sonnenberg A.
Association of the VLA-6 subunit with a novel protein. J
Biol Chem. 1989;264:6529–6535.
16.
Huang TF, Holt JC, Lukasiewicz H, Niewiarowski S.
Trigramin, a low molecular weight peptide inhibiting fibrinogen
interaction with platelet receptors expressed on
glycoprotein IIb-IIIa complex. J Biol Chem. 1987;262:16157–16163.
17. Huang TF, Holt JC, Kirby EPH, Niewiarowski S. Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets. Biochemistry. 1989;28:661–666.[Medline] [Order article via Infotrieve]
18. Huang TF, Sheu JR, Teng CM. A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom. Biochem J. 1991;277:351–357.
19. Huang TF, Sheu JR, Teng CM. Mechanism of action of a potent antiplatelet peptide, triflavin from Trimeresurus flavoviridis snake venom. Thromb Haemost. 1991;66:489–493.[Medline] [Order article via Infotrieve]
20. Huang TF, Sheu JR, Teng CM, Chen SW, Liu CS. Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb/IIIa complex. J Biochem. 1991;109:329–334.
21. Sheu JR, Teng CM, Huang TF. Triflavin, an RGD-containing antiplatelet peptide, binds to GPIIIa of ADP-stimulated platelets. Biochem Biophys Res Commun. 1992;189:1236–1242.[Medline] [Order article via Infotrieve]
22.
Haverstick DM, Cowan JF, Yamada KM, Santoro SA.
Inhibition of platelet adhesion to fibronectin, fibrinogen, and von
Willebrand factor substrates by a synthetic tetrapeptide
derived from the cell-binding domain of fibrinogen. Blood. 1985;66:946–952.
23.
Ogletree ML, Harris DN, Greenberg R, Haslanger MF,
Nakane M. Pharmacological actions of SQ29548, a novel selective
thromboxane antagonist. J Pharmacol
Exp Ther. 1985;234:435–441.
24. Engel G, Gothert M, Muller-Schweinitzer E, Schlicker E, Sistonen L, Stadler PA. Evidence for common pharmacological properties of 3H-5-hydroxytryptamine binding sites, presynaptic 5-hydroxytryptamine autoreceptors in CNS and inhibitory presynaptics 5-hydroxytryptamine receptors on sympathetic nerves. Naunyn Schmiedebergs Arch Pharmacol. 1983;324:116–124.[Medline] [Order article via Infotrieve]
25. Vaporciyan AA, Jones ML, Ward PA. Rapid analysis of leukocyte-endothelial adhesion. J Immunol Methods. 1993;195:93–100.
26.
Santoro SA, Zutter MM. The
2ß1 integrin: a
collagen receptor on platelets and other cells. Thromb
Haemost. 1995;74:813–821.[Medline]
[Order article via Infotrieve]
27. Brazenor RM, Angus JA. Actions of serotonin antagonists on dog coronary artery. Eur J Pharmacol. 1982;81:569–576.[Medline] [Order article via Infotrieve]
28. Cohen RA, Shepherd JT, Vanhoutte PM. 5-Hydroxytryptamine can mediate endothelium-dependent relaxation of coronary arteries. Am J Physiol. 1983;245:H1077–H1080.
29.
Musial JM, Niewiarowski S, Rucinski B, Stewart GJ, Cook
JJ, Williams JA, Edmunds LH. Inhibition of platelet adhesion to
surfaces of extracorporeal circuits by disintegrins, RGD-containing
peptides from viper venom. Circulation. 1990;82:261–273.
30. Sheu JR, Huang TF. Ex-vivo and in vivo antithrombotic effect of triflavin, an RGD-containing peptide. J Pharm Pharmacol. 1994;46:58–62.[Medline] [Order article via Infotrieve]
31. Sheu JR, Chao SH, Yen MH, Huang TF. In vivo antithrombotic effect of triflavin, an Arg-Gly-Asp containing peptide on platelet plug formation in mesenteric microvessels of mice. Thromb Haemost. 1994;72:617–621.[Medline] [Order article via Infotrieve]
32. Sheu JR, Yen MH, Peng HC, Chang MG, Huang TF. Triflavin, an Arg-Gly-Asp-containing peptide, prevents platelet plug formation in in vivo experiments. Eur J Pharmacol. 1995;294:231–238.[Medline] [Order article via Infotrieve]
33. Sheu JR, Lin CH, Peng HC, Huang TF. Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell substratum adhesion through an RGD-dependent mechanism. Peptides. 1994;15:1391–1398.[Medline] [Order article via Infotrieve]
34.
Plow EE, Pierschbacher MD, Ruoslahti E, Marguerie G,
Ginsberg MH. Arginyl-Glycyl-Aspartic acid sequences and fibrinogen
binding to platelets. Blood. 1987;70:110–115.
35.
Turitto VT, Weiss HJ, Zimmerman T, Sussman I. Factor
VIII/von Willebrand factor in subendothelium
mediates platelet adhesion. Blood. 1985;65:823–831.
36. Sakariassen KS, Nievelstein PFEM, Coller BS, Sixma JJ. The role of platelet membrane glycoprotein Ib and IIb/IIIa in platelet adherence of human artery subendothelium. Br J Haematol. 1986;63:681–691.[Medline] [Order article via Infotrieve]
37.
Gogstad GO, Brosstad F, Krutness MB, Hagen I, Solum NO.
Fibrinogen-binding properties of the human platelet
glycoprotein IIb/IIIa complex: a study using cross
radioimmunoelectrophoresis. Blood. 1982;60:663–671.
38. Nachman RL, Leung LLK. Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen. J Clin Invest. 1982;69:263–269.
39. Gardner JM, Hynes RO. Interaction of fibronectin with its receptor on platelets. Cell. 1985;42:439–448.[Medline] [Order article via Infotrieve]
40. Ruggeri ZM, Demarco L, Gatti L, Bader R, Montgomery RR. Platelets have more than one binding site for von Willebrand factor. J Clin Invest. 1983;72:1–12.
41.
Pytela R, Pierschbacher MD, Ginsberg MH, Plow EF,
Ruoslahti E. Platelet membrane glycoprotein IIb/IIIa:
member of a family of Arg-Gly-Asp-specific adhesion receptors.
Science. 1986;231:1559–1562.
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