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Articles

Triflavin Inhibits Platelet-Induced Vasoconstriction in De-endothelialized Aorta

J.-R. Sheu, M. H. Yen, W. C. Hung, Y. M. Lee, C. H. Su, T. F. Huang
https://doi.org/10.1161/01.ATV.17.12.3461
Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3461-3468
Originally published December 1, 1997
J.-R. Sheu
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M. H. Yen
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W. C. Hung
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Y. M. Lee
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C. H. Su
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T. F. Huang
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Abstract

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 5×107 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.

  • RGD-containing peptides
  • triflavin
  • de-endothelialization
  • serotonin
  • thromboxanes
  • Received October 22, 1996.
  • Accepted April 15, 1997.

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 α- and β-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; αIIbβ3 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.

Methods

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 (5×107 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 (3×108 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 (3×108 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 (3×108 cells per milliliter) were incubated with various concentrations of the peptides or anti-integrin α2β1 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.

Results

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 5×107 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 5×107 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 (5×107 cells per milliliter) of human platelets to characterize the inhibitory effect of RGD-containing peptides on this reaction.

Figure 1.
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Figure 1.

A, Time course of the tension response of de-endothelialized rat thoracic aorta to the addition of various concentrations of human platelet suspensions. ○, 1×106 cells per milliliter; •, 1×107 cells per milliliter; ▿, 5×107 cells per milliliter; ▾, 1×108 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 (5×107 cells per milliliter). Control (○, without RGD-containing peptides in the organ chamber); GRGDS (•, 1 mmol/L); trigramin (▿, 10 μmol/L); triflavin (▾, 0.5 μmol/L; □, 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 (5×107 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 (5×107 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).

Figure 2.
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Figure 2.

Dose-response curves of (A) triflavin (•) and trigramin (○) and (B) GRGDS (○), GRGDSPK (•), and GRGDF (▿) in aggregating platelets (5×107 cells per milliliter)-induced vasoconstriction in de-endothelialized thoracic aorta. 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=4). *P<.01; **P<.001, significant difference compared with trigramin.

Figure 3.
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Figure 3.

Traces of isometric tension recordings of three rings from the same rat thoracic aorta in which the endothelium had been removed. The platelet suspensions (5×107 cells per milliliter) were preincubated without triflavin (a) or with triflavin (2 μmol/L; b) or trigramin (10 μmol/L; c) for 5 minutes followed by the addition of platelet suspensions (↓) 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⇓).

Figure 4.
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Figure 4.

Effect of RGD-containing peptides on the release of (A) TxB2 and (B) 5-hydroxytryptamine (serotonin) from human platelets, as determined by an EIA kit assay of samples withdrawn from the organ bath after the addition of platelets (5×107 cells per milliliter). Cells were incubated with GRGDS, GRGDF, and GRGDSPK (all at 2 mmol/L); trigramin (10 μmol/L); and triflavin (2 μmol/L). Data are presented as mean±SEM (n=4). *P<.05, significant difference compared with the control.

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 (5×107 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.

Figure 5.
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Figure 5.

Scanning electron micrograph of de-endothelialized rat thoracic aorta after the addition of platelets (5×107 cells per milliliter) into the organ chamber without (A) or with (B) triflavin (2 μmol/L) (magnification 4500).

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.

Figure 6.
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Figure 6.

Effect of RGD-containing peptides on collagen-activated platelet adhesion to Fn (A), Vn (B), vWF (C), and Lm (D) substrates. Platelets were washed, labeled, activated with collagen (10 μg/mL), and incubated with various concentrations of the peptides for 20 minutes and then transferred to plates coated with Fn, Vn, vWF, and Lm (all at 1 μg per well). Adhesion after 30 minutes was measured. Results are shown for triflavin (1 μmol/L), trigramin (2 μmol/L [hatched bar]; 8 μmol/L [open bar]), GRGDS (2 mmol/L), GRGDF (2 mmol/L), GRGDSPK (2 mmol/L), and GRGES (2 mmol/L). The extent of adhesion is expressed as a percentage of platelets initially added to the plates. Data are presented as mean±SEM (n=4). *P<.001, significant difference compared with GRGES.

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 α2β1 (a collagen receptor on platelet or other cells),26 markedly inhibited the adhesion of resting platelets to type IV collagen (69±8%).

Figure 7.
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Figure 7.

Effect of RGD-containing peptides on resting platelet adhesion to type IV collagen. Platelets were washed, labeled, and incubated with various concentrations of the peptides or anti-integrin α2β1 monoclonal antibody for 20 minutes and then transferred to plates coated with type I collagen (at 5 μg per well). Adhesion after 30 minutes was measured. Results are shown for triflavin (10 μmol/L), trigramin (20 μmol/L [hatched bar]; 50 μmol/L [open bar]), GRGDS (2 mmol/L), GRGDF (2 mmol/L), GRGDSPK (2 mmol/L), GRGES (2 mmol/L), and anti-integrin α2β1 monoclonal antibody (10 μmol/L). The extent of adhesion is expressed as a percentage of platelets initially added to the plates. Data are presented as mean±SEM (n=4). *P<.001, significant difference compared with GRGES.

Discussion

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 5×107 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.

Selected Abbreviations and Acronyms

Fn=fibronectin
Lm=laminin
PE=phenylephrine
Tx=thromboxane
Vn=vitronectin
vWF=von Willebrand factor

Acknowledgments

This work was supported by a grant from the National Science Council of Taiwan (NSC85 to 2331-B-38 to 026).

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Arteriosclerosis, Thrombosis, and Vascular Biology
December 1997, Volume 17, Issue 12
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    Triflavin Inhibits Platelet-Induced Vasoconstriction in De-endothelialized Aorta
    J.-R. Sheu, M. H. Yen, W. C. Hung, Y. M. Lee, C. H. Su and T. F. Huang
    Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3461-3468, originally published December 1, 1997
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    Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3461-3468, originally published December 1, 1997
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