Continued Thromboxane A2 Formation Despite Administration of a Platelet Glycoprotein IIb/IIIa Antagonist in Patients Undergoing Coronary Angioplasty
Abstract Experimental data suggest that formation of thromboxane A2 may be suppressed during administration of a glycoprotein IIb/IIIa antagonist. We determined the dose of one such compound, fradafiban, required to provide >80% occupancy of the platelet glycoprotein IIb/IIIa and examined its effects on thromboxane A2 formation in patients undergoing PTCA. The dose response to fradafiban and additional effects of aspirin were explored initially in patients with stable coronary artery disease. Fradafiban induced a dose-dependent inhibition of platelet aggregation that correlated with fibrinogen receptor occupancy and plasma drug concentration. Addition of aspirin 300 mg had no effect on these parameters. At the highest dose, mean fibrinogen receptor occupancy was 89.7±1.2% (n=3) at 4 hours and platelet aggregation had decreased by 93.4±2.7%. Eighteen patients undergoing coronary angioplasty were randomized to receive either aspirin 330 mg or that dose of fradafiban producing >80% fibrinogen receptor occupancy. Platelet aggregation was suppressed throughout the infusion of fradafiban to a greater extent than with aspirin. However, there was a marked increase in urinary excretion of 11-dehydrothromboxane B2 in patients treated with fradafiban: from 1973±889 to a peak of 9760±3509 pg/mg creatinine (P=.0046). Despite this evidence of continued platelet activation in vivo, there were no cases of coronary thrombosis. In conclusion, fradafiban suppresses platelet aggregation and may be a useful alternative to aspirin in the prevention of thrombotic events in patients undergoing PTCA. However, there is continued formation of thromboxane A2, which may continue to exert its effects as a potent vasoconstrictor and vascular smooth muscle mitogen.
- Received May 6, 1997.
- Accepted July 29, 1997.
One of the earliest events in the development of arterial thrombosis is the adhesion and aggregation of platelets at a site of vascular injury. The accumulated platelet mass expresses binding sites for coagulant proteins including factors V and VIII, and acts to localize procoagulant activity and the generation of thrombin.1,2 Platelet aggregation and to some extent platelet adhesion are mediated through the binding of fibrinogen to an integrin on the platelet surface.3 Integrins are adhesion receptors consisting of an heterodimeric complex of two glycoproteins (α and β). In the case of the platelet fibrinogen receptor these subunits are αIIb (IIb) and β3 (IIIa).4 Under resting conditions the platelet glycoprotein (GP) IIb/IIIa has a low affinity for soluble fibrinogen. However, upon activation of the cell, the receptor undergoes a conformational change and expresses a high affinity for several ligands including fibrinogen and von Willebrand factor.5,6 Expression of a fibrinogen receptor is a common response to most platelet agonists including thrombin, thromboxane A2 (TxA2), and ADP, all of which have been implicated in the development of arterial thrombosis in vivo.7–9 Consequently, compounds that antagonize this receptor and prevent platelet aggregation would have a broader spectrum of activity than both aspirin or ticlopidine, which inhibit thromboxane A2 and ADP, respectively.
Several classes of platelet fibrinogen receptor antagonists have been developed10 and this approach has been shown to be effective in preventing thrombosis and restenosis when combined with aspirin during coronary angioplasty.11,12 It is not clear, however, whether there is a need to coadminister aspirin with GPIIb/IIIa antagonists. Suppression of platelet thromboxane A2 may add to the antithrombotic activity of these compounds.13 Moreover, thromboxane A2 is a potent vasoconstrictor14 and vascular smooth muscle mitogen.15 Consequently, suppression of thromboxane A2 biosynthesis may be desirable particularly in the setting of coronary angioplasty. Thromboxane A2 formation is stimulated by most platelet agonists through transmembrane receptors that activate phospholipases either directly or as a consequence of raised intracellular Ca2+. However, thromboxane A2 formation is also dependent on secondary signaling that occurs following fibrinogen-GPIIb/IIIa interactions and subsequent platelet aggregation.16 Indeed, there is evidence that at least some antagonists of the platelet GPIIb/IIIa suppress thromboxane A2 formation in vivo,17 potentially obviating the need for aspirin.
We have examined the pharmacology of a novel, synthetic antagonist of the platelet GPIIb/IIIa receptor (fradafiban) in patients with coronary artery disease. Fradafiban has both a prolonged half-life and oral bioavailability so that it may be suitable for chronic administration.18 The purpose of the study was to characterize the relationship between occupancy of the platelet fibrinogen receptor and platelet aggregation, to identify a dose of fradafiban that would result in a >80% occupancy of the fibrinogen receptor and to examine the generation of thromboxane A2 in the presence of fradafiban alone in patients undergoing coronary angioplasty.
Fradafiban (3S,5S)-5-[(4′-amidino-4-biphenylyl)oxymethyl]-3-carboxymethyl-2-pyrrolidinone) was supplied by Boehringer Ingelheim UK Ltd. The study was approved by the Ethics Committees of Beaumont, Blackrock, and St. James hospitals. Each patient gave written, informed consent before treatment.
Dose Finding Study
Initially 10 patients with stable coronary artery disease (7 males and 3 females, mean age 56±2.7 yr) were evaluated in a dose-ranging study. Those with a diagnosis of unstable angina or recent (within 14 days) acute myocardial infarction were excluded. No patient had contraindications for antiplatelet therapy and baseline tests of hematological, renal, and hepatic function were normal. Before study commencement, all subjects were required to avoid nonsteroidal anti-inflammatory drugs, including aspirin, for at least 10 days. No patient was receiving regular anticoagulant therapy.
The patients received one of three doses of fradafiban: 8.8 mg, 17.7 mg, or 26.5 mg by intravenous infusion. The drug was prepared in a total volume of 500 mL of 5% dextrose. A bolus of 5 mg, 10 mg, or 15 mg, respectively, was given over 30 minutes with the remainder given over 6 hours. Four hours into the infusion, aspirin 300 mg was administered orally to assess whether it had any additive antiplatelet effect. Samples were obtained and bleeding times performed at baseline and 4, 6, and 24 hours after commencement of infusion. The blood was drawn from an indwelling catheter in the arm opposite to the one used for the drug infusion and the catheter was flushed with 5 mL of 0.9% saline. A discard of 10 mL was taken before the sample collection.
Fradafiban Compared With Aspirin During PTCA
In a second study, patients undergoing percutaneous transluminal coronary angioplasty (PTCA) were randomized in a double-blind, placebo controlled fashion to receive either fradafiban or aspirin. Patients received fradafiban+heparin+ aspirin placebo or intravenous placebo fradafiban+heparin+oral aspirin 330 mg. The ratio of active to placebo fradafiban treatments was 4:1. Eighteen patients were enrolled at the 2 hospitals (15 males and 3 females, mean age 56±1.9 years). Two patients started on fradafiban were withdrawn from the study without completing the protocol, in one because the angioplasty failed and in a second due to coronary artery dissection. The latter underwent coronary artery bypass grafting 24 hours later, without any adverse hemostatic events. Their data are not included in the analysis.
Based on the findings of the dose-ranging study, to achieve >80% occupancy of platelet fibrinogen receptors, fradafiban was administered as a bolus dose of 15 mg over 30 minutes followed by a continuous infusion rate of 2.09 mg/h for 23.5 hours. Infusion of drug commenced at least 1 hour before the start of PTCA. Patients also received acetylsalicylic acid 330 mg or placebo orally immediately before commencement of placebo/fradafiban infusion. At the beginning of the procedure a bolus dose of heparin (10 000 IU) was administered followed by a continuous infusion for a minimum of 6 hours. The heparin infusion was adjusted to achieve a therapeutic response as measured by a PPT. Bleeding times and blood samples for platelet studies and drug levels were obtained at timed intervals for up to 72 hours after drug commencement. In addition, timed urine collections were obtained for measurement of urinary 11-dehydro thromboxane B2, the major enzymatic metabolite of thromboxane.19
Platelet aggregation was measured ex vivo using an optical aggregometer (Biodata PAP 4, Biodata Corporation, Horesham, Pa, USA). Samples were collected into syringes containing 3.8% sodium citrate at a final dilution of 1:10. Platelet-rich plasma (PRP) was immediately prepared by centrifugation at 150g for 10 minutes. Following removal of the PRP, the remaining plasma was centrifuged at 1500g for 10 minutes to obtain platelet-poor plasma (PPP). This was then used for calibration of the aggregometer. Platelet aggregation was measured in response to thrombin receptor activator peptide (TRAP): Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp 20 μmol/L (Dr Brian Walker, Queen’s University, Belfast), collagen 2 μg/mL (Helena Laboratories, Beaumont, TX, USA), or a combination of both. Agonists were prepared at 10X concentration and stored on ice during use. Platelet responses were measured as percent aggregation at 4 minutes and are reported as such in the text. Percent inhibition was determined by expressing the platelet aggregation on drug as a percent of the baseline aggregation and subtracting that from 100.
Fibrinogen Receptor Occupancy
Fibrinogen receptor occupancy was measured by competitive assay using 3H fradafiban (Dr. Hans Weisenberger, Dr Karl Thomae GmbH). Platelet-rich plasma was prepared and 200 μL of this was mixed with 10 μL of 14C-sucrose (2.7 kBq) and 10 μL of 3H-fradafiban (5 nmol/L final concentration). The mixture was incubated at room temperature for 20 minutes, centrifuged at 2000g for 5 minutes with subsequent removal of the supernatant. A 100 μL aliquot of this was counted for free ligand. The platelet pellet was dissolved in 200 μL of 0.2N NaOH and 180 μL of this mixed with 10 μL 5N HCl and counted in 2 mL of scintillation cocktail. As 14C-sucrose does not enter cells, and the ratio of 3H to 14C is constant, the amount of free 3H fradafiban could be calculated. The amount of fradafiban bound in the pretreatment samples of each patient was taken as 100% binding for that patient, with lower binding in subsequent samples expressed as a percentage of this. Binding was unaffected by heparin or aspirin.
Ligand-Induced Binding Site (LIBS) Expression
LIBS expression was assessed ex vivo by flow cytometric analysis (FACScan, Becton Dickenson, Oxford, UK). Samples were analyzed for platelet associated fluorescence following incubation with a monoclonal antibody to the D3 epitope (kind gift of Dr. L. Jennings, University of Tennessee, Memphis). Results were presented as a percentage of maximal EDTA induction of LIBS. Samples were collected in 3.8% sodium citrate (1:10 dilution) and three aliquots of 200 μL of PRP prepared. EDTA (5 mmol/L final concentration) was added to the first and an equal volume of vehicle (phosphate buffered saline) to the other two. Samples were incubated at 37°C for 30 minutes and fixed in an equal volume of formaldehyde 2%. The samples were stored at 4°C before the assay. The fixed samples were washed in PBS containing 1% BSA and resuspended in 1 mL of PBS/BSA. Aliquots (100 μL) were incubated with 4 μL of the D3 monoclonal antibody (4 ng/mL) for 30 minutes at room temperature. The samples were washed and r resuspended on 100 μL of fluorescein isothiocyanate goat anti-mouse IgG (FITC GαM) (Becton-Dickinson, Oxford, UK) for a further 30 minutes at room temperature. For each time point, a control sample was incubated with FITC GαM) alone to determine background fluorescence. Flow cytometric analysis was performed with a Becton-Dickinson FACScan and the data analyzed by Becton-Dickinson Lysis II.
Ex vivo and in vitro expression of GMP140 was determined also by flow cytometry. Briefly, aliquots of PRP (50 μL) were incubated with 4 μL of anti-CD62 monoclonal antibody (Immunotech SA, Marseilles, FR) for 20 minutes at room temperature. The samples were washed and resuspended in 100 μL of FITC GαM. Background fluorescence was determined as described above and the samples analyzed by flow cytometry.
For serum thromboxane B2 measurement, whole blood was placed in nonsiliconized glass vials and incubated at 37°C for 45 minutes. Following centrifugation at 1500g for 10 minutes supernatants were stored at −20°C until analyzed. Thromboxane B2 was derivatized to the trimethylsilyl ether, pentafluorobenzyl ester before quantification by gas chromatography and negative-ion, chemical ionization mass spectrometry (INCOS-XL, Finnigan Mat, Hemel Hempstead). Plasma samples for drug levels were collected in 3.8% sodium citrate onto ice and centrifuged at 1500g for 10 minutes. Supernatants were combined with an equal volume of 0.5 N HCl and stored at −20°C. Plasma drug levels were measured by automated column switching high-performance liquid chromatography. Samples were applied to Bondapak C18/Corasil-filled columns and separation achieved on a column filled with ODS-Hypersil, 5 μmol/L. The mobile phase consisted of an equal volume of solution A (ammonium acetate, SDS, and acetic acid) and solution B (acetonitrile and methanol). Fluorescence detection, with excitation at 310 nm, was used for quantitation. The coefficient of variation was <2% and there was an overall imprecision of approximately 6% for plasma samples.
The bleeding time was measured on the volar aspect of the forearm. A blood pressure cuff was positioned and inflated to 40 mm Hg. An incision, 5 mm in length and 1 mm in depth, was made distally using a Simplate bleeding device (Organon Teknika, Durham, NC, USA). Blood was absorbed onto Whatman filter paper every 30 seconds until the bleeding stopped.
The data are expressed as the mean±SEM. The data were analyzed by Kruskal-Wallis one way analysis of variance with subsequent Mann-Whitney test for comparison between groups. This analysis makes no assumptions about the distribution of the data. Paired data, where appropriate, were compared by t test.
In Vitro Studies
To explore whether fradafiban exhibited any agonist activity, platelet-rich plasma from 2 subjects was incubated at increasing concentrations of fradafiban (0.33 ng/ml to 330 μg/mL) for 10 minutes and serum thromboxane B2 determined. There was no change in either subject (0.21 and 0.24 ng/mL to 0.26 and 0.19 ng/mL, respectively, at the highest concentration of drug). In one subject, GMP140 expression was assessed and fell from 13% of cells to 9% following incubation for 10 minutes at the highest concentration of drug, suggesting that in vitro there was no agonist activity.
Dose Finding Study
No serious adverse effects were reported in the dose-finding group. One patient receiving the highest dose of fradafiban had evidence of localized gingival bleeding at 3 hours of infusion that resolved within 30 minutes. No other evidence of bleeding was noted. There was no significant change in platelet counts (x109/L) in the 9 subjects at 6 hours (250±18) or 12 hours (250±18) of infusion compared with baseline (268±18).
Fibrinogen Receptor Occupancy and Platelet Aggregation
Plasma fradafiban concentration was similar at 4 hours (121±18, 232±14, and 319±37 ng/mL) and at 6 hours (114±16, 228±11.5, and 301±44 ng/mL), with low, intermediate, and high doses, respectively, of fradafiban. There was a dose-dependent increase in fibrinogen receptor occupancy (Table⇓). A primary end point of the study was to identify a dose of fradafiban capable of achieving greater than 80% fibrinogen receptor occupancy. The highest dose achieved this criterion in the 3 subjects studied, with an average fibrinogen receptor occupancy of 89.7±1.2% at 4 hours. Fibrinogen receptor occupancy was unchanged (88.6±1.4%) following the administration of aspirin (Table⇓).
Fradafiban also induced a dose-dependent inhibition of platelet aggregation to the thrombin receptor activator peptide (TRAP) or to a combination of TRAP and collagen in the dose-ranging group. Platelet aggregation to the combination of collagen and TRAP at baseline was 79±2.6% and fell at 4 hours of infusion to 18±7% (P=.0081), 14±7.6% (P=.007), and 4.7±2.2% (P=.017) at low, intermediate, and high doses of fradafiban, respectively. The addition of aspirin had no further effect on platelet aggregation, which at 6 hours was 23.3±1.1%, 9.3±5.6%, and 5.3±0.8% at low, intermediate, and high doses of fradafiban, respectively. Inhibition of platelet aggregation and fibrinogen receptor occupancy were closely related to the plasma concentration of fradafiban (Fig 1⇓). Inhibition of platelet aggregation occurred only at a fibrinogen receptor occupancy of >20% and was maximal at a receptor occupancy of >80%. At 24 hours, 17.5 hours after discontinuation of the drug infusion, fibrinogen receptor occupancy was 50.9±3.6% in the highest dose group, and platelet aggregation had recovered to 74±1% of baseline.
Effect of Fradafiban on Bleeding Time
Bleeding time at 4 hours of infusion before the administration of aspirin was prolonged to >30 minutes in 9 of 10 patients in the dose-finding study. Bleeding times were also >30 minutes after the administration of aspirin. In 1 patient receiving the lowest dose, bleeding time increased to 17 minutes at 4 hours. The bleeding time returned to within 20% of baseline at 24 hours in 7 patients and by 28 hours in the remainder.
Effects of Fradafiban and Aspirin on Serum Thromboxane B2
Fradafiban infusion had no significant effect on serum thromboxane B2 levels at 4 hours compared to baseline in the dose-ranging study: 359±38 ng/mL versus 423±17 ng/mL (P=NS). In contrast, aspirin suppressed serum thromboxane B2 by 99%, to 2.5±0.4 ng/mL.
Fradifiban Compared With Aspirin During Coronary Angioplasty
Sixteen of the 18 patients completed the study. Eleven had proximal left anterior descending (LAD) coronary artery lesions, 4 had mid LAD lesions, and 1 had mid right coronary artery disease. Thirteen patients were receiving calcium channel blockers, 6 patients were receiving beta blockers, 2 received angiotensin converting enzyme inhibitors, and 1 patient was on a diuretic. Three patients developed small hematomas at the femoral puncture site, 2 had received fradafiban and 1 had received aspirin. Transient oozing was noted at femoral and/or subclavian line sites during drug infusion in 9 patients (8 fradafiban and 1 placebo). The most serious bleed occurred in a patient treated with aspirin only, who had an episode of blood loss at the time of removal of the femoral sheath, estimated at 200 to 300 mL. There was a fall in hemoglobin (Hb) of 2.8 g/dL in this patient over the study period, which did not require treatment, and the patient completed the study protocol without further complication. One patient receiving active fradafiban suffered a significant fall in hemoglobin over the study period, a decrease of 3.6 gr/dL (15.0 gr/dL to 11.4 gr/dL). This was associated with a moderate femoral hematoma and oozing at catheter sites at 4 and 6 hours. No other sites of bleeding were noted and no blood transfusion was required. One patient treated with fradafiban also had mild bleeding from the buccal mucosa after 10 hours of infusion. This lasted for 1 hour and resolved without treatment. There were no acute thrombotic events.
There was no significant change in platelet count (x109/L) in either the aspirin-treated patients (236±1, 214±14, 204±11, and 207±11 at baseline, 6 hours, 24 hours, and 72 hours, respectively) or in the patients receiving fradafiban (253±15, 231±15, 224±11, and 231±17, at baseline, 6 hours, 24 hours, and 72 hours, respectively).
Administration of fradafiban resulted in a steady state plasma concentration of 295±38 ng/mL, whereas none was detected in patients treated with aspirin alone. Peak fibrinogen receptor occupancy was 95±0.5% (n=16) and at 24 hours (end of infusion) was 87±1.2% (n=16). Note that in all cases, fibrinogen receptor occupancy exceeded 80% throughout the fradafiban infusion (Fig 2⇓). In contrast, fibrinogen receptor occupancy reached a peak of 13.3±14% in those patients receiving aspirin, reflecting a high, possibly aberrant value in 1 patient, as it had returned to normal at 1.5 hours. As for the patients in the dose-finding study, fibrinogen receptor occupancy correlated with plasma drug level (Fig 3⇓). Platelet aggregation was markedly suppressed in the patients receiving fradafiban and again this was closely related to fibrinogen receptor occupancy. Baseline aggregation to TRAP/collagen was 77.5±2.8% and fell to 7.2±1.8% (P=.001) at 6 hours and 6.3±1.7% (P<.0039) at 24 hours. In contrast, platelet aggregation was inhibited only marginally in patients treated with aspirin (77±1.2% to 60±11.7% at 6 hours). Inhibition of platelet aggregation by fradafiban was maximum at 80% receptor occupancy and had a threshold of 20% to 30% receptor occupancy (Fig 3⇓). Following discontinuation of fradafiban, platelet aggregation to the combination of TRAP and collagen recovered over the next 24 hours as fibrinogen receptor occupancy decreased (Fig 2⇓). The delayed recovery of fibrinogen receptor occupancy was due to the long-terminal half-life of the drug (mean 14 hours, range 10.2 to 20.2 hours). In the patients receiving fradafiban, bleeding times increased significantly, to 19.9±2.2 minutes from a baseline of 4.9±0.5 minutes. (P=.0017). Bleeding times had returned to less than 10 minutes in all patients 24 hours after discontinuing the infusion, with a mean of 7.2±0.4 minutes. (Fig 4⇓). In the placebo group receiving aspirin, baseline and peak bleeding times were 4.2±0.2 and 5.5±1.6 minutes (P=NS), respectively.
Patients undergoing coronary angioplasty who received fradafiban and no aspirin had a significant increase in 11-dehydrothromboxane B2 levels (P=0.0046) during the study period (Fig 5⇓). In contrast, urinary 11-dehydrothromboxane B2 was suppressed in the patients treated with aspirin (data not shown). These findings demonstrate persistent platelet activation in the patients receiving fradafiban. Fradafiban did not induce expression of the D3 epitope during administration compared to baseline in either arm of the study. In the dose-ranging study baseline (measured as % maximal EDTA expression) was 8.2±1.3% and was 7.1±2.5% during the fradafiban infusion. In the PTCA group, those receiving fradafiban had a pretreatment expression of 9.4±1.6% and a level 11.5±1.8% during the fradafiban infusion, compared to 4.3±2.6% and 8.3±2.4%, respectively, in those receiving placebo. Similarly, ex vivo GMP140 expression on resting platelets was unaltered in 3 patients receiving fradafiban (3.7±1.2% of platelets before treatment versus 3±0.8% during the fradafiban infusion).
Antagonism of the Platelet GPIIb/IIIa
Several novel antagonists of platelet GPIIb/IIIa are being developed as potential antiplatelet agents. Large-scale clinical trials have been completed with two of these agents, the chimeric antibody 7E311,12 and the peptide antagonist integrelin.20 Whereas the chimeric antibody was effective in preventing events in high-risk angioplasty, integrelin had little protective effect in patients with unstable angina undergoing the same procedure.20 It is possible that this reflected different levels of receptor antagonism by the two agents. 7E3 was administered in a dose that achieved >80% receptor occupancy21 and complete suppression of platelet aggregation. With a lower dose of 7E3, suppression of platelet aggregation was incomplete and no benefit was seen. These data suggest that the level of receptor occupancy is critical for clinical effect, at least in the setting of coronary angioplasty. It is possible that the dose of integrelin was inadequate, as complete inhibition of aggregation has been shown only at higher doses.22
Based on the findings of EPIC, namely that a dose of 7E3 that occupied 80% of receptors was effective in preventing acute thrombotic events during coronary angioplasty, we performed a dose-escalating study to identify a dose of fradafiban that would provide complete suppression of platelet aggregation and >80% occupancy of the platelet GPIIb/IIIa. Fradafiban induced a dose-dependent fibrinogen receptor occupancy and inhibition of platelet aggregation ex vivo. Both fibrinogen receptor occupancy and inhibition of aggregation correlated closely with the plasma concentration of the drug. Moreover, the degree of receptor occupancy was a major determinant of the platelet response. Thus, the antiplatelet activity of fradafiban is explained by its known biological activity.
Although there was a good correlation between platelet aggregation and receptor occupancy, a minimum of 20% fibrinogen receptor occupancy was required before an effect was seen. Consequently, during the washout phase platelet aggregation had recovered considerably at 24 hours despite continued fibrinogen receptor occupancy and detectable plasma drug levels. There was a similar discrepancy with the bleeding time, which recovered rapidly following discontinuation of the drug. A similar disparity between recovery of bleeding time and platelet aggregation following discontinuation of integrelin has been reported.23 Indeed, there are data suggesting that a minimum of 70% to 80% fibrinogen receptor occupancy is required before bleeding time is altered.24
Thromboxane A2 Formation and GPIIb/IIIa Antagonism
During coronary angioplasty there was a marked increase in thromboxane A2 biosynthesis measured as excretion of its enzymatic metabolite, 11-dehydrothromboxane B2, despite substantial suppression of platelet aggregation with fradafiban. One explanation, that fradafiban is acting as a partial agonist and therefore stimulating platelet thromboxane formation, is unlikely for several reasons. Although for ethical reasons there was no “untreated” group for comparison, the magnitude of increase in thromboxane biosynthesis was similar to that reported previously in aspirin-sensitive subjects receiving no antiplatelet therapy during coronary angioplasty.25 Moreover, fradafiban did not induce thromboxane A2 formation in vitro or the expression of LIBS or GMP140 in vitro or ex vivo. The more likely explanation is that platelets continue to be activated by agonists acting on transmembrane receptors linked to phospholipases via G-proteins. Although fibrinogen-GPIIb/IIIa interactions and platelet aggregation provides additional thromboxane A2, this mechanism appears to be important only for weak agonists, such as epinephrine.16
In conclusion, fradafiban suppresses platelet aggregation and may be a useful alternative to aspirin in the prevention of thrombotic events in patients undergoing PTCA. However, even at high (>80%) receptor occupancy and suppression of platelet aggregation, there is continued formation of thromboxane A2, a potent vasoconstrictor and vascular smooth muscle mitogen.
Supported in part by grants from the Wellcome Trust and the Irish Heart Foundation. Dr Byrne is a Health Research Board of Ireland Research Fellow.
Swords NA, Mann KG. The assembly of the prothrombinase complex on adherent platelets. Arterioscler Thromb.. 1993;13:1602–1612.
Gawaz MP, Loftus JC, Bajt ML, Fromjovic MM, Plow EF, Ginsberg MH. Ligand bridging mediates integrin αIIbβ3(platelet GPIIb/IIIa) dependent homotypic and heterotypic cell-cell interactions. J Biol Chem.. 1991;88:1128.
Pytela R, Pierschbacher MD, Ginsberg MH, Plow EF, Ruoslaahti E. Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp-specific adhesion receptors. Science.. 1986;231:1559–1562.
Sims PJ, Ginsberg MH, Plow EF, Shatill SJ. Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex. J Biol Chem.. 1991;266:7345–7352.
Hantgan RR, Nichols WL, Ruggeri ZM. von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets. Blood.. 1990;75:889–894.
Fitzgerald DJ, FitzGerald GA. Role of thrombin and thromboxane A2 in reocclusion following coronary thrombolysis with tissue-type plasminogen activator. Proc Natl Acad Sci U S A.. 1989;86:7585–7589.
Humphries RG, Robertson MJ, Leff P. A novel series of P2T purinoreceptor antagonists: definition of the role of ADP in arterial thrombosis. TIPS.. 1995;16:179–181.
Suehiro K, Smith JW, Plow EF. The ligand recognition specificity of β3 integrins. J Biol Chem.. 1996;271:10365–10371.
Topol EJ, Califf RM, Weisman HF, Ellis SG, Tcheng JE, Worley S, Ivanhoe R, George BS, Fintel D, Weston M, Sigmon K, Anderson KM, Lee KL, Willerson JT. Randomised trial of coronary intervention with antibody against platelet IIb/IIIa integrin for reduction of clinical restenosis: results at six months. Lancet.. 1994;343:881–886.
Dorn GW, Sens D, Chaikhouni A, Mais D, Halushka PV. Cultured vascular smooth muscle cells with functional thromboxane A2 receptors: measurement of U46619-induced calcium efflux. Circ Res.. 1987;60:952–956.
Hanasaki K, Nakano T, Arita H. Receptor-mediated effect of thromboxane A2 in vascular smooth cells. Biochem Pharmacol. 1990;2535–2542:.
Shattil SJ, Haimovich B, Cunningham M, Lipfert L, Parsons JT, Ginsberg MH, and Brugge JS. Tyrosine of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors. J Biol Chem.. 1994;269:14738–14745.
Murphy N, Jennings L, Pratico D, Doyle C, Fitzgerald DJ. Functional relevance of LIBS expression in the response to platelet glycoprotein IIb/IIIa antagonists in vivo. Thromb Haemostas.. 1995;73:1314. Abstract.
Muller TH, Weisenberger H, Brickl R, Kirchner M, Narjes H, Himmelsbach F, Guth B, Krause J, Thomas K. Pharmaco-dynamics and -kinetics of BIBU 52, a platelet glycoprotein (GP) IIb/IIIa antagonist, and its orally active prodrug BIBU 104 in man. Thromb Haemost.. 1995;17:1445. Abstract.
Fitzgerald DJ, Catella F, Roy L, FitzGerald GA. Marked platelet activation in vivo after intravenous streptokinase in patients with acute myocardial infarction. Circulation.. 1988;77:142–150.
Cavagnaro J, Serabian MA, Shealy DJ, Coller BS, Iuliucci JD. Pharmacokinetic analysis of murine monoclonal antibody 7E3 F(ab′)2 in monkeys. Blood. 1987;70(Suppl 1):349a. Abstract.
Delanty N, Catella F, Jennings L, FitzGerald GA, Fitzgerald DJ. Antagonism of the platelet glycoprotein IIb/IIIa receptor in stable angina: effects on platelet activity and receptor conformation. Circulation.. 1993;88:1–318. Abstract.
Anders RJ, Alexander JC, Hantsbarger GL, Burns DM, Oliver SD, Cole G, Fitzgerald DJ. Demonstration of potent inhibition of platelet aggregation with an orally active GPIIb/IIIa receptor antagonist. J Am Coll Cardiol. 1995;117A:Abstract.
Braden GA, Knapp HR, FitzGerald GA. Suppression of eicosanoid biosynthesis during coronary angioplasty by fish oil and aspirin. Circulation.. 1991;84:679–685.