Triflavin Inhibits Platelet-Induced Vasoconstriction in De-endothelialized Aorta

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.

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.

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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).

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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.

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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⇓).

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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.

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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.

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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%).

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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.