(Circulation. 2000;101:324.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Flavone-8-Acetic Acid (Flavonoid) Profoundly Reduces Platelet-Dependent Thrombosis and Vasoconstriction After Deep Arterial Injury In Vivo
From Buchanan County Cardiology, PC (J.S.M.), St. Joseph, Mo; Green Lane Hospital (M.W.I.W.), Epsom, Auckland, New Zealand; Hospital Clinic I Provincial de Barcelona (M.H.), Spain; Department of Oncology and Comprehensive Cancer Center (J.M.R.), and Department of Biostatistics (D.E.G.), Mayo Clinic and Mayo Foundation, Rochester, Minn; and Cardiovascular Institute (J.H.C.), Mount Sinai Medical Center, New York.
Correspondence to James H. Chesebro, MD, Cardiovascular Institute, Box 1030, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029-6574.
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Abstract |
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Background—Flavone-8-acetic acid (FAA; [Flavonoid]), an adjuvant antitumor drug, inhibits ristocetin-induced aggregation of human platelets. The effect of FAA on platelet-dependent thrombosis was studied in vivo in the porcine carotid artery after deep arterial injury by balloon angioplasty.
Methods and Results—111In-labeled autologous platelet and 125I-labeled porcine fibrin(ogen) deposition, and the incidence of macroscopic mural thrombosis onto deeply injured artery (tunica media) were compared in 20 pigs (40±1 kg [mean±SEM], body surface area=1.0±0.1 m2), randomized to FAA bolus (n=10) of 5.5g/m2, followed by an infusion at 0.14g · m-2 · min-1 or placebo (n=10). Vasoconstriction was measured immediately beyond the dilated segment using quantitative angiography. Platelet deposition (x106/cm2 of carotid artery) was reduced over 12-fold in pigs treated with FAA (13±3 versus 164±51, P=0.001) compared with placebo. Fibrin(ogen) deposition (x1012 molecules/cm2 of carotid artery) did not significantly differ in FAA-treated pigs versus placebo (40±8 versus 140±69, P=0.08). Large mural thrombi were present in 100% of placebo-treated pigs versus very small thrombi in 40% of FAA-treated pigs (P=0.005). Vasoconstriction was reduced from 46±6% in the placebo group to 15±3% in the FAA group (P<0.001). Plasma level of FAA before angioplasty was 515±23 µg/mL. The activated partial thromboplastin time was unchanged. The bleeding time was >2SD above the normal mean in 4 of 5 treated pigs (increased from 157±29 to 522±123 s).
Conclusions—FAA markedly reduced platelet deposition, mural
thrombi, and injury-induced vasoconstriction after deep
arterial injury, suggesting that a major inhibition of
platelet glycoprotein Ib
may be beneficial therapy.
Key Words: angioplasty • platelets • platelet aggregation inhibitors • thrombus • vasoconstriction
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Introduction |
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Von Willebrand (vWF) factor is necessary for normal platelet adhesion over an area of damaged vessel wall as well as for platelet-platelet interaction (aggregation) under high-shear flow conditions.1 2 3 4 5 6 7 8 9 These interactions involve the platelet membrane glycoprotein (GP) complexes Ib-
and IIb-IIIa, and also fibrinogen, fibronectin, and
vitronectin.1 2 3 4 5 6 7 8 9 Flavone-8-acetic acid (FAA; Flavonoid)10 11 is an adjuvant antitumor drug which inhibits implantation of solid tumors in the mouse but also inhibits ristocetin-induced, vWF-dependent platelet aggregation in humans.12 This may cause a profound reduction in platelet-rich arterial thrombosis after deep arterial injury. In Phase II clinical studies in humans, no clinically significant toxicity was observed. Thus the effect of FAA, at the maximal dose tolerated by humans,10 11 12 on platelet-dependent thrombosis was studied in vivo in the deeply injured porcine carotid artery produced by balloon angioplasty as a model of mainly GP Ib inhibition.
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Methods |
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Twenty normal pigs of Babcock 4-way cross stock (a mixture of Landrace, Yorkshire, Hampshire, and Durock breeds),
4 months old
with a mean weight of 40±1 kg (1 m2 body
surface area),13 were obtained from local farmers. They
were randomly assigned to treatment with either placebo (0.9% saline)
or FAA (National Cancer Institute), administered as a bolus of
5.5 g/m2 followed immediately by an infusion at
0.14g · m-2 ·
min-1. Loading dose and maintenance
infusion were calculated on the basis of preliminary pharmacokinetic
experiments in pigs. Monoexponential declines in plasma
concentrations of FAA were fitted to the equation
C=Ae-t, where A is the intercept at t=0, and
is the elimination rate constant. A weighting factor of 1/C, where
C is the plasma concentration of FAA at time t, was employed. Drug administration during the balloon dilatation procedure was not blinded, but all subsequent tissue and sample analysis was performed without knowledge of the treatment administered. This study was approved by the Mayo Clinic Animal Care Committee and conformed to American Heart Association guidelines.
Experimental Protocol
The model of deep arterial injury in the porcine
carotid artery has been described in detail
previously.14 15 16 Autologous platelets were labeled
with 300 µCu of 111In-tropolone and reinjected
together with 250 µCu of 125I-labeled porcine
fibrinogen on the day before the procedure.15 16 17 On the
day of surgery, the pigs were sedated with 1g intramuscular
ketamine (Ketaset, Bristol Laboratories), intubated and
mechanically ventilated with room air (Harvard respirator, Harvard
Apparatus). Anesthesia was maintained with a
continuous infusion of etomidate (Abbott Laboratories, North Chicago)
40 mg/L, fentanyl (Elkins-Sinn, Inc) 10 mg/L, and ketamine 1000
mg/L, at about 5 mL/min. The ECG and intra-arterial
pressure were continuously monitored throughout the procedure.
The left femoral vein and artery and the right femoral vein were dissected. A 9F sheath was placed in the left femoral artery, and 14-gauge angiocaths were inserted in the left and right femoral veins. Blood for platelet count, fibrinogen, hematocrit, activated partial thromboplastin time (aPTT), and FAA levels was obtained from the right femoral vein. All the lines for blood sampling were continuously flushed with 0.9% saline. After all the lines were in place, a basal bleeding time was performed in the ear using a standardized method.18 The normal saline treatment bolus, or FAA was then given and followed immediately by the infusion administered via the left femoral vein with a Harvard pump (Harvard Apparatus) at the rate of 0.8 mL/min. Thirty minutes after starting the infusions, another bleeding time was performed.
An 8-mmx3-cm polyethylene angioplasty catheter (Blue Max, Medi-tech) was advanced under fluoroscopic guidance to the left and then to the right common carotid arteries for arterial dilatation. Angioplasty was performed 30 minutes after starting treatment, between the first and third cervical vertebrae, using a standardized procedure (5 inflations of 30 s duration at 7 atm, with 60 s between inflations).14 15 16 Carotid angiography was performed by injecting 6 mL of ionic contrast material (Renografin 76, Squibb) just before carotid dilatation, using a catheter (8F) with a metal ring of known dimension (Cordis Corporation). Spot films were also taken during balloon dilatation. After the series of 5 inflations, the balloon catheter was withdrawn to the proximal carotid artery and angiography was repeated.
Fifteen minutes after dilatation of the right carotid artery, 120 mL of 0.5% Evans blue in 0.9% saline was injected into the descending aorta to demarcate the extent of arterial injury. Animals then received an overdose of pentobarbital and were euthanized. The proximal descending aorta was immediately cannulated and the carotid arteries perfused with buffered 0.9% saline until eluent from the external jugular veins was clear. The vessels were then perfused with buffered 2% glutaraldehyde for 15 minutes. All perfusions were at physiological pressure. After fixation in situ, the carotid arteries were harvested and cleaned of all adventitia. The dilated portions were divided into 2 equal segments and 2 similar-sized segments were taken proximal and distal to the dilated areas.14 15 16
Tissue Analysis
Platelet and fibrin(ogen) deposition on the
arterial segments were quantified by the method of Dewanjee
et al.17 19 20 Counting for 111In
was performed on the day of surgery and for 125I,
2 to 3 weeks later after the 111In had
decayed.
The extent of deep arterial injury (defined as a tear through the internal elastic lamina into the arterial media) in the dilated portion of the vessel was assessed histologically as previously described.14 15 16 Each segment was cut open, pinned, and color photographed. Computer-assisted planimetric measurements of the area of deep injury and the total segment area were then obtained. The presence of macroscopic mural thrombosis was assessed using a 2-fold magnifying glass.
Vasoconstriction
The severity of localized vasoconstriction was determined
immediately distal to the dilatation site from angiograms of the common
carotid arteries obtained before and after the dilatation procedure.
Computer-assisted planimetry was used to measure the mean maximal
narrowing in lumen diameter before and after the procedure, expressed
as a percentage of the respective arterial dimension before
dilatation.16
Laboratory Tests
All blood samples were collected with the 2-syringe technique
(0.13 mol/L trisodium citrate as anticoagulant;
anticoagulant:blood=1:10). Samples for platelet count, fibrinogen,
hematocrit, aPTT, and FAA concentration were drawn before drug
administration, 30 minutes after starting the infusions, and
immediately before euthanasia. Platelet count, hematocrit, aPTT,
and fibrinogen were determined using standard laboratory methods. Blood
for FAA levels was mixed 9 parts to 1 with 0.13 mol/L trisodium citrate
solution, centrifuged to obtain plasma, and stored at -70°C.
Assays were performed as a single batch. The method of determination of
FAA in plasma was that of L. Malspeis (written communication;
1987). Briefly, plasma (0.25 to 0.50 mL) was diluted to 1 mL
with 0.5 mL of 0.5 mol/L sodium acetate (pH 3.0) and normal saline.
After addition of diethylether (4 mL), tubes were shaken on a
mechanical shaker for 15 minutes. Following low speed
centrifugation, the ether phase was evaporated to
dryness under a stream of nitrogen and the residue dissolved in mobile
solvent before high-performance liquid
chromatography (HPLC) analysis. Samples were
analyzed by reversed-phase HPLC on an IBM
C18 (10 µm) column with a mobile solvent
of methanol/water (60/40) containing 20 mL glacial acetic acid per
liter of the methanol/water mixture. Detection was by UV absorbance at
254 nm. Standard curves were prepared by adding known amounts of FAA to
blood blank plasma and analyzing as described above. Concentrations of
FAA were determined by fitting unknown sample peak areas to equations
derived from standard curves.
Statistical Methods
Results are presented as mean±SEM. Two dilated segments
per artery per animal were analyzed. Because of the variability
of platelet and fibrin(ogen) deposition and to use the animal as
the unit of study (because all segments in the pig were exposed to the
same treatment), analysis was performed on the natural
logarithm of these values (per cm2 of total area)
averaged over all deeply injured segments. Treated and control groups
were then compared using the Student’s t test for
continuous variables. Pearson’s 
2 test
was used to test for a difference between groups in the incidence of
mural thrombus.
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Results |
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Platelet and Fibrin(ogen) Deposition
Deep arterial injury occurred in 70% of segments in the dilated region, the remainder had subendothelial injury. Platelet deposition in deeply injured segments in animals treated with FAA was >12-fold lower than those treated with placebo (13±3 versus 164±51x106/cm2, P=0.001). Fibrin(ogen) deposition was similar but slightly less in treated animals (40±8 versus 140±69x1012 molecules/cm2, P=0.08; Figure 1
).
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Mural Thrombus
Large macroscopic mural thrombi were present in all pigs
treated with placebo. FAA produced a reduction in the incidence and
size of thrombus formation. Very small mural thrombi occurred in 40%
of treated pigs (P=0.005). There were large thrombi in 85%
of the deeply injured segments in the placebo group and very small
thrombi in 30% of the treated group.
Vasoconstriction
Vasoconstriction immediately distal to the area of dilatation was
significantly greater in the placebo group than in FAA-treated animals
(46±6% versus 15±3%, P<0.001; Figures 1 through 3
).
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FAA Pharmacokinetics
Plasma elimination of FAA in 2 animals administered with an
intravenous bolus dose of 1 g/m2 was
fit to a 1-compartment open model. Plasma half-life and plasma
clearance values were 27.9 minutes and 279 mL ·
min-1 · m-2,
respectively. The intravenous bolus and continuous infusion
doses to maintain a plasma concentration of 500 µg/mL calculated from
these values were 5.5 g/m2 and 140 mg ·
min-1 · m-2,
respectively.
Laboratory Tests
The plasma level of FAA before angioplasty was 515±23 µg/mL; at
the end of the procedure, it was 575±36 µg/mL. The aPTT was only
slightly increased in the treated animals (1.0 to 1.2 times baseline),
but the bleeding time in the 5 animals in which it was measured
increased from 157±29 to 522±123 s. In 4 of the animals the bleeding
time was prolonged >210 s, (2 SD above the mean laboratory value)
after the administration of FAA.
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Discussion |
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This study demonstrates that platelet-dependent thrombus formation following deep arterial injury by balloon dilatation is profoundly reduced by FAA (Flavonoid) which appears to block vWF platelet glycoprotein Ib
-dependent
platelet aggregation. This suggests that this mechanism of
antithrombotic therapy may be clinically useful. We evaluated a dosage
of FAA in the upper therapeutic range in humans as assessed by plasma
concentrations.10 11 12 Platelet deposition and the
incidence of mural thrombosis in pigs treated with FAA was
significantly lower than those treated with placebo. Fibrin(ogen)
deposition was similar and not significantly decreased by FAA compared
with placebo. FAA is an adjuvant antitumor agent that inhibits implantation and causes necrosis of solid tumors in mice by an unknown mechanism.10 11 12 Necrosis of solid tumors by FAA triggers intravascular coagulation21 22 23 24 25 26 and thus, reduced tumor blood flow. Prolonged treatment causes reduced tumor blood flow, which may lead to hemorrhagic necrosis of these tumors.21 22 These changes were not seen in normal tissue and are thought to be secondary to necrosis in the solid tumors.
Rubin et al found that FAA administered to patients with cancer
inhibited ristocetin-induced platelet aggregation (vWF-GP
Ib-dependent aggregation) and prolonged the bleeding
time.12 Ex vivo and in vitro platelet aggregation
studies with human platelet-rich plasma showed that in the presence
of FAA, aggregation induced by adenosine diphosphate (ADP),
collagen, arachidonic acid, and adrenalin was not
inhibited.12 Plasma ristocetin cofactor activity was
unchanged.12
vWF interacts with human platelets through 2 different
mechanisms.1 2 5 6 Under high-shear flow conditions, the
vessel-wall bound vWF binds to the platelet GPIb- in the early
phases of hemostasis (platelet adhesion), a process independent of
ADP and induced by ristocetin. The other interaction, of soluble vWF
with platelets involves glycoprotein IIb-IIIa complex
exposed on activated platelets (platelet aggregation).
This process requires ADP and Ca++ and is not
induced by ristocetin, in common with other adhesive proteins like
fibrinogen, fibronectin, and probably with thrombospondin. Interactions
of the glycoprotein IIb-IIIa complex, a member of a large
family of related molecules known as integrins, with adhesive proteins
involves the RDG (Arg-Gly-Asp) amino acid recognition sequence,
is necessary for platelet-platelet adhesion
(platelet aggregation).1 2 3 4 5 6 7 8 27 Interaction and binding
of proteins to the glycoprotein Ib- does not involve the
RDG recognition sequence.1 2 The antithrombotic mode of
action of FAA (Flavonoid) remains unknown, but aggregation studies with
ristocetin and prolongation of the bleeding time in humans and in our
study suggest that FAA interferes with platelets in the formation
of the initial platelet hemostatic plug. This is probably achieved
by inhibition of binding of vWF to its binding site on the platelet
GP Ib-.
It was recently discovered that thrombin binds with high affinity to
platelet GP Ib.28 29 Thrombin binding site on GP
Ib is distinct from, but in close proximity to, that involved in
binding of the adhesive protein vWF.28 29 The proposed
role of GP Ib in thrombin binding includes acting as high-affinity
receptor for bringing thrombin near the platelet
surface.30 It was suggested that initiating event in
thrombin-induced platelet activation occurs via the GP
Ib.31 FAA binding to vWF site on GP Ib, owing to
proximity, could partially cover high-affinity binding site for
thrombin on GP Ib. This could be another plausible explanation for
our findings. FAA significantly decreased platelet deposition and
macroscopic thrombosis (antiplatelet effect, solid-phase
platelet GP Ib), but did not have a significant effect on
fibrin(ogen) deposition or prolongation of aPTT (no anticoagulant
activity; there was no inhibitory effect on the action of
thrombin on the soluble-phase fibrinogen).
During administration of FAA, vasoconstriction just distal to the site of dilatation was significantly reduced. We previously showed that the degree of platelet deposition directly correlates with the degree of arterial vasoconstriction.16 Whether the current reduced vasoconstriction relates mainly to platelet deposition or to a direct action of FAA on the vessel wall, the endothelium, increased nitric oxide (which increases guanosine 3',5'- cGMP levels in vascular tissue [similar to other flavonoids]), or to some other mechanism, is unclear.16 32 33 34
In conclusion, complex mechanisms are involved in the formation of
arterial thrombi. At dosages used in clinical practice FAA
(Flavonoid) is an effective agent for reducing platelet-dependent
thrombosis in vivo over areas of deep arterial injury.
There may be a role for interaction of vWF and GP Ib in acute
ischemic coronary syndromes.35 36 37 FAA
also reduces the vasoconstriction associated with arterial
balloon angioplasty probably related to the reduction in platelet
deposition.16
Potentially, FAA could be used for short periods (it also has short plasma half-life) during vascular interventions, in combination with other antithrombotics/anticoagulants, for primary prevention of platelet dependent thrombosis in the areas of deep arterial injury.
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Acknowledgments |
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Dr Mruk was the recipient of a fellowship from NIH-HL (O7111/14T); Dr Webster was the recipient of a Fellowship from the National Heart Foundation of New Zealand and the Fogarty Center, National Institutes of Health; and Dr Heras was the recipient of a research grant from CIRIT-Generalitat de Catalunya, Spain.
Received April 9, 1999; revision received July 21, 1999; accepted July 29, 1999.
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