This Article Abstract Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cox, D. Articles by Fitzgerald, D. J. Search for Related Content PubMed PubMed Citation Articles by Cox, D. Articles by Fitzgerald, D. J.

CLINICAL STUDY

Evidence of platelet activation during treatment with a GPIIb/IIIa antagonist in patients presenting with acute coronary syndromes

Dermot Cox, BSc, PhD^*, Richard Smith, BSc, PhD^*, Martin Quinn, MD^*, Pierre Theroux, MD, Peter Crean, MD and Desmond J. Fitzgerald, MD^*

^* Centre for Cardiovascular Science, Royal College of Surgeons in Ireland, Dublin, Ireland
Montreal Heart Institute, Montreal, Quebec, Canada
Department of Cardiology, St. James Hospital, Dublin, Ireland

Manuscript received January 13, 2000; revised manuscript received April 26, 2000, accepted June 26, 2000.

Reprint requests and correspondence: Dr. Dermot Cox, Department of Clinical Pharmacology, Royal College of Surgeons, 123 St. Stephen’s Green, Dublin 2, Ireland.
dcox{at}rcsi.ie

	Abstract

Top Abstract Methods In vitro studies Statistical analysis Results Discussion References

 
OBJECTIVES

The study was done to determine the role of partial agonist activity in the lack of effectiveness of the oral GPIIb/IIIa antagonist orbofiban.

BACKGROUND

Orbofiban, an oral GPIIb/IIIa antagonist, was found to increase the mortality of patients with acute coronary syndromes (ACS) in the OPUS-TIMI-16 trial, despite the fact that it is a very potent anti-platelet agent and that IV agents have proven very effective.

METHODS

Patients (n = 520) with ACS were randomized to orbofiban 30 mg, 40 mg or 50 mg twice daily or 50 mg once daily or placebo. Platelet activity was assessed in 175 patients by examining GPIIb/IIIa receptor conformation, expression of CD63 antigen, and platelet aggregation.

RESULTS

Plasma concentrations of orbofiban at the highest dose (74 ± 6 ng/ml peak, 61 ± 5 ng/ml trough) exceeded the IC₅₀ for platelet aggregation to adenosine diphosphate (ADP) (29 ± 6 ng/ml) and thrombin-activating peptide (61 ± 18 ng/ml). Orbofiban induced a conformational change in GPIIb/IIIa detected as the displacement of the monoclonal antibody mAb2; such conformational changes have been linked to partial agonist activity. Consistent with this, platelet expression of CD63 ex vivo was significantly increased at five time points during the study. In vitro, orbofiban increased platelet aggregation to a submaximal concentration of epinephrine (67 ± 19% vs. 27 ± 9%, n = 5) and increased thromboxane formation when the platelet GPIIb/IIIa were clustered using monoclonal antibodies to the receptor.

CONCLUSIONS

Orbofiban is both an antagonist and a partial agonist of platelet GPIIb/IIIa. At low concentrations of the drug, this partial agonist activity may enhance platelet aggregation. Along with suboptimal plasma drug levels, these findings may help explain the lack of efficacy seen with orbofiban in patients with ACS.

Abbreviations and Acronyms   ADP = adenosine diphosphate   ANOVA = analysis of variance   FSC = forward scatter   GPIIb/IIIa = platelet fibrinogen receptor   IV = intravenous   MI = myocardial infarction   PBS = phosphate buffered saline   PRP = platelet-rich plasma   SSC = side scatter   TRAP = thrombin receptor activating peptide

One factor that may limit the effectiveness of GPIIb/IIIa antagonists is their propensity to act as partial agonists. GPIIb/IIIa is one of a family of receptors, the integrins, that act as receptors for adhesive proteins (1). Upon ligand binding, integrins transmit signals into cells, which in the case of GPIIb/IIIa amplify platelet activation (9–12). Although this is most evident with large ligands such as fibrinogen, small ligands designed as integrin antagonists trigger similar signals in vitro (13,14), particularly if they induce extensive conformational changes in the receptor (15,16). We examined the effects of orbofiban on platelet function when administered for 84 days in patients presenting initially with unstable angina and MI. In addition, we looked for evidence of partial agonist activity in vitro.

	Methods

Top Abstract Methods In vitro studies Statistical analysis Results Discussion References

 
Clinical study.   The study was established as a Phase II dose-finding study of orbofiban. The trial was approved by the Irish Medicines Board, by the Drugs Directorate, Health Canada, and the institutional review bodies of the participating hospitals. All patients gave written, informed consent. Patients were considered eligible for the study if they had unstable angina (<120 h from last chest pain) or a recent MI (<120 h). Patients were randomized to placebo (n = 101) twice daily, orbofiban 30 mg twice daily (n = 109), orbofiban 40 mg twice daily (n = 104), orbofiban 50 mg twice daily (n = 104) or orbofiban 50 mg once daily with a matching placebo in place of the second dose each day (n = 102). Treatment was continued for 84 days. Detailed platelet studies were performed in 175 subjects: placebo (n = 36); orbofiban 30 mg twice daily (n = 34); orbofiban 40 mg twice daily (n = 34); orbofiban 50 mg twice daily (n = 37); and orbofiban 50 mg once daily (n = 34). These patients were all the patients enrolled in all participating Irish and Canadian sites. In addition to the study medication, patients received 162 mg aspirin daily. Samples were taken pre-dose and 4 h and 6 h post-dose on days 1, 28, and 84 (last day) of treatment.

Platelet aggregation.   Citrated (3.8%) whole blood was centrifuged at 850 x g for 3 min to produce platelet-rich plasma (PRP). The remaining blood was centrifuged for 2 min at 2500 x g to prepare platelet-poor plasma. After 1 min pre-incubation at 37°C, platelet aggregation was induced by 20 µmol/liter adenosine diphosphate (ADP) or 5 µmol/liter thrombin receptor activating peptide (TRAP). The extent of platelet aggregation was determined after 2 min by light transmittance (PAP-4 platelet aggregometer, BioData, Horsham, Pennsylvania).

GPIIb/IIIa receptor number and conformation.   The GPIIb/IIIa receptor number and conformation were analyzed by quantifying the binding of two monoclonal antibodies to GPIIb/IIIa, mAb1 and mAb2 (BioCytex, Marseille, France), that recognize distinct epitopes on the GPIIb/IIIa receptor (17). The total number of GPIIb/IIIa receptors can be determined by mAb1, whereas mAb2 recognizes only the unbound GPIIb/IIIa. In the presence of GPIIb/IIIa antagonists, the mAb2 epitope on the receptor disappears as a result of a ligand-induced change in the conformation of GPIIb/IIIa. PRP (20 µl) was incubated with 45 µg/ml of mAb1 or mAb2 for 20 min at room temperature. Samples and a tube containing calibration beads were stained with 20 µl of fluorescein isothiocyanate-labeled anti-mouse antibody for 10 min and transferred to gel tubes containing 1 ml of 0.1% paraformaldehyde for 10 min. These gel tubes consisted of a small tube with a V-shaped bottom containing a small amount of Sephadex gel. The gel tubes were centrifuged at 850 x g for 10 min, which allowed the platelets to penetrate the gel. The supernatant was poured off, and the gel was washed twice in phosphate buffered saline (PBS). The tubes were filled with PBS and stored at 4°C until analyzed (within two weeks). These gel tubes enable the platelets to be washed and also provide a suitable storage medium for them. Prior to analyses, the tubes were centrifuged at 850 x g for 10 min, and the supernatant was poured off. The gel-platelet mix was then re-suspended in the buffer and analyzed by flow cytometry as described below.

Marker of platelet activation.   Platelet activation was monitored by the expression of the protein CD63 antigen, which is released to the platelet surface after activation (18). PRP (20 µl) was incubated with CD63 (BioCytex, Marseille, France) for 20 min at room temperature. A similar tube was also prepared with the addition of TRAP (5 µmol/liter) 10 min prior to the addition of CD63, which was used to gate for CD63-positive platelets. Samples were then stained with a secondary antibody and analyzed by flow cytometry.

Quantitative fluorescence cytometry.   Samples were collected using log forward scatter (FSC), side scatter (SSC) and fluorescence. A region was drawn around the peak density of platelets in the FSC versus SSC plot, and this was used as a gate for analysis of the platelet population. A histogram marker was drawn around the TRAP-activated CD63-stained platelets so as to include 95% of all positive platelets. This was defined as the positive population. Events from the sample population falling in this region were considered positive. Calibration beads (BioCytex, Marseille, France) were also analyzed, and a log plot of mean fluorescence intensity versus molecules of antibody was prepared. Flow cytometry was performed on a Becton Dickinson FACSCalibur flow cytometer and analyzed using CellQuest software (B & D, Oxford, United Kingdom).

Plasma concentration.   The plasma concentration of orbofiban was measured by high-performance liquid chromatography/mass spectrometry.

	In vitro studies

Top Abstract Methods In vitro studies Statistical analysis Results Discussion References

 
Platelet aggregation.   Platelet aggregation was induced with epinephrine, and a concentration that induced <35% platelet aggregation was identified. Platelet aggregation was performed in the absence and presence of orbofiban at a concentration (60 nmol/liter, 20 ng/ml) that alone did not inhibit ADP-induced aggregation (IC₅₀ 130 ± 10 nmol/liter, 43 ± 3 ng/ml).

Thromboxane production.   Signaling through integrins is accommodated by prior clustering of the receptor (12,19). Platelet GPIIb/IIIa receptors were clustered using antibody-coated magnetic beads (20). Briefly, gel-filtered platelets (5 x 10⁶ platelets) were incubated with 2 x 10⁷ beads (Dynal MPC-E, Oslo, Norway) coated with CD41, a monoclonal antibody to GPIIIa, in the presence of orbofiban, abciximab or vehicle for 45 min at room temperature. They were then magnetized for 2 min, snap-frozen, and stored until the thromboxane B₂ levels were measured by ELISA (R&D Systems, Oxon, United Kingdom). Alternatively, platelets were incubated with 4F8 (BioCytex, Marseille, France) without beads, followed by orbofiban, snap-frozen, and thromboxane B₂ levels measured. Note that CD41 and 4F8 are directed against the GPIIIa subunit, and as they are not directed at the ligand-binding site, they do not interfere with the binding of orbofiban or abciximab.

	Statistical analysis

Top Abstract Methods In vitro studies Statistical analysis Results Discussion References

 
Clinical studies.   The data were analyzed by two-way analysis of variance (ANOVA) of the log transformed data with a Bonferroni-Dunn post hoc analysis.

In vitro studies.   Samples were compared with the control values using a paired t test.

Materials.   Orbofiban was a gift from Dr. Bob Anders, GD Searle, Skokie, Illinois. The TRAP was from Dr. Pat Harriott, Queens University, Belfast, UK. All monoclonal antibodies and gel tubes were from BioCytex, Marseille, France.

	Results

Top Abstract Methods In vitro studies Statistical analysis Results Discussion References

 
Clinical studies.   Clinical outcomes
The patient characteristics are shown in Table 1. In the patients with Q wave MI, 58% had their most recent MI in the inferior wall, 11% anterior, 8% anterolateral, 6% posterior, 14% were in another location, and 3% were unknown. There were no differences in baseline characteristics between the treatment groups. The numbers of patients completing the study were 76 of 101 on placebo, 73 of 109 on orbofiban 30 mg bid, 70 of 104 on 40 mg bid, 67 of 104 on 50 mg bid, and 73 of 102 on orbofiban 50 mg qd. The clinical outcomes of the study are shown in Table 2. Again, there were no statistical differences in the outcomes of the different groups, although MI occurred only in patients receiving orbofiban 50 mg twice daily or 50 mg daily.

View larger version (22K): [in this window] [in a new window]   Figure 1 Percent platelet aggregation (± SEM) to ADP (20 µmol/liter) and TRAP (5 µmol/liter) in patients on placebo (n = 24–31) (diamond); orbofiban 30 mg bid (n = 17–29) (solid square); 40 mg bid (n = 21–29) (diamond); 50 mg qd (n = 16–29) (open square); and 50 mg bid (n = 18–31) (open circle).

View larger version (17K): [in this window] [in a new window]   Figure 2 The effect of placebo (solid square) and 50 mg bid orbofiban (solid triangle) on receptor occupancy measured as the ratio of mAb2 to mAb1 binding (n = 4–6).

Thromboxane production
Clustering of GPIIb/IIIa receptors with CD41 in the absence of receptor occupancy induced minimal thromboxane production (1.6 ± 2.9 ng/ml, n = 6). The addition of orbofiban at a concentration that will saturate the receptors (3.3 µg/ml) resulted in an increase in thromboxane to 6.6 ± 3.6 ng/ml (n = 6, p = 0.03). By contrast, abciximab, a monoclonal antibody that blocks the ligand binding site, had no significant effect (2.7 ± 0.8 ng/ml, n = 3, p > 0.05). More marked changes were seen using 4F8 to cluster the GPIIb/IIIa (Fig. 3), and the increase in thromboxane induced by orbofiban was blocked by abciximab (p < 0.05), suggesting that orbofiban must bind to GPIIb/IIIa to induce platelet activation. Neither the addition of abciximab to 4F8-bound platelets nor the addition of orbofiban without 4F8 increased thromboxane generation (data not shown).

View larger version (10K): [in this window] [in a new window]   Figure 3 The effect of orbofiban and abciximab+orbofiban on 4F8-induced thromboxane formation.

	Discussion

Top Abstract Methods In vitro studies Statistical analysis Results Discussion References

GPIIb/IIIa-mediated signaling.   We also addressed the possibility that orbofiban acted as a partial agonist in vivo and increased platelet activity. Binding of fibrinogen to GPIIb/IIIa triggers several intracellular events, so-called outside-in signaling. These include an increase in intracellular Ca²⁺ (13,21), tyrosine phosphorylation (11) and thromboxane A₂ formation (10,21). Outside-in signaling is dependent on clustering of the receptor (22) (feasible only with ligands with multiple binding sites such as fibrinogen) and linkage of the cytoplasmic tail to intracellular proteins (10). Ligand binding appears to be an additional requirement. Thus, small fragments of laminin can induce PGE₂ formation if the laminin receptor is first clustered (19).

Ligand-induced conformational changes.   How ligand binding induces outside-in signaling is unknown, but it may involve a change in receptor conformation. Many GPIIb/IIIa antagonists induce conformational changes that are detected as the appearance of new epitopes (23,24) or, as in the case of mAb2, the disappearance of an epitope (17). We show here that orbofiban reduced the binding of mAb2, although it had no effect on the total number of receptors, measured as the binding of mAb1. The antibody mAb2 recognizes a site remote from the ligand binding site. This site disappears upon binding of small ligands, although mAb2 does not compete directly with the ligand (17). The findings suggest that mAb2 detects a region that becomes obscured as a consequence of a ligand-induced change in conformation. Thus, the disappearance of mAb2 binding in patients on orbofiban is evidence that the platelet GPIIb/IIIa has undergone a change in conformation. Ligand-induced changes in the conformation of GPIIb/IIIa have been implicated in the partial agonist activity of GPIIb/IIIa antagonists. For example, Honda and colleagues (13) found that only compounds inducing conformational changes in the GPIIIa subunit enhanced the rise in platelet calcium and aggregation response to platelet agonists.

Platelet activation.   We also looked for evidence of increased platelet activation ex vivo. Detecting subtle changes in platelet activation ex vivo is difficult. Possibly the best marker is thromboxane A₂ formation (4), which was not possible in this study, because all of the patients were on aspirin. As an alternative we examined the expression of CD63 antigen, which is a protein present in the lysosomes that appears on the surface upon platelet activation (18). Unlike CD62 antigen, CD63 antigen is highly stable once expressed (25). Expression of the CD63 antigen was significantly increased throughout the treatment period in patients receiving orbofiban.

Orbofiban as a partial agonist.   Together with the evidence of a change in conformation, the data suggest that orbofiban enhanced platelet activity in vivo. This hypothesis was supported by in vitro experiments demonstrating that orbofiban acted as a partial agonist. Thus, orbofiban enhanced the platelet response to epinephrine. Low concentrations of orbofiban insufficient to block all of the platelet GPIIb/IIIa receptors were used to avoid masking an effect on platelet aggregation. In a second model, the receptor was first clustered using monoclonal antibodies to mimic one of the requirements for signaling via natural ligands. The addition of the antagonist to clustered receptors resulted in an increase in thromboxane, a marker of platelet activation. It may be argued that this type of clustering does not mimic what happens in vivo. Nevertheless, the experiment demonstrates the potential of orbofiban to induce strong signaling. The increase in thromboxane in the presence of 4F8 was prevented by the monoclonal antibody abciximab, which binds to the ligand recognition site of GPIIb/IIIa and prevents orbofiban binding (17). This finding demonstrates that orbofiban-induced platelet activation is receptor mediated. However, a recent report found that orbofiban can induce apoptosis in rat cardiomyocytes (26), although it is not known if this can occur in human platelets.

Clinical significance.   Whether the increased platelet activity detected in patients on orbofiban is clinically relevant and contributed to the poor outcome of OPUS-TIMI-16 is unclear. The ability of orbofiban to enhance platelet aggregation may be important, particularly during the trough period prior to dosing, when there is insufficient drug to block platelet aggregation effectively. Platelet activation is enhanced and plays a major role in the pathogenesis of acute coronary syndromes, including unstable angina and MI (4). Platelet microemboli have also been seen in coronary microvessels of patients suffering sudden cardiac death (7). Increased platelet activation has also been reported in a phase II study of sibrafiban, another oral GPIIb/IIIa antagonist (27), and a recent report has confirmed the increase in platelet activation in the phase III orbofiban study (28). It is possible that in the setting of low-level platelet activation seen in coronary artery disease, additional activation through the GPIIb/IIIa receptor may provoke rather than prevent thrombosis, especially when drug levels are at trough.

Although there is evidence that abciximab (14,29) and eptifibatide (30), both of which are clinically effective, also activate platelets in vitro, these drugs are administered intravenously and at doses that achieve high levels of receptor occupancy. Moreover, they are combined with anticoagulants, which have been shown to enhance the response to a GPIIb/IIIa antagonist in an experimental model of coronary thrombosis (31). Thus, partial agonism may not be a problem with IV compounds, because they maintain stable plasma levels with a high level of blockade, are supplemented with anticoagulants, and are administered for only a short period of time.

Conversely, partial agonism combined with either low dosing or a low trough can lead to an increase in events in patients treated with an oral GPIIb/IIIa antagonist. The longer duration of treatment also increases the likelihood of a patient’s forgetting to take medication, thereby increasing the chance of a low trough.

In conclusion, the relatively low plasma drug levels and suboptimal inhibition of platelet aggregation along with a partial agonist effect may have contributed to the poor clinical efficacy of orbofiban.

	Footnotes

 
This work was funded by Searle and Company, Skokie, Illinois, and supported by grants from the Health Research Board and the Higher Education Authority of Ireland.

	References

Top Abstract Methods In vitro studies Statistical analysis Results Discussion References

 

1. Cox D, Aoki T, Seki J, Motoyama Y, Yoshida K. The pharmacology of the integrins. Med Res Rev. 1994;14:195–228[CrossRef][Medline]
2. Manoharan G, Maynard S, Adgey J. The therapeutic use of glycoprotein IIb/IIIa inhibitors in acute coronary syndromes. Expert Opin Invest Drugs. 1999;8:555–566[CrossRef]
3. Kong D, Califf R, Miller D, et al. Clinical outcomes of therapeutic agents that block the platelet glycoprotein IIb/IIIa integrin in ischemic heart disease. Circulation. 1998;98:2829–2835[Abstract/Free Full Text]
4. Fitzgerald D, Roy L, Catella F, Fitzgerald G. Platelet activation in unstable coronary disease. N Engl J Med. 1986;315:983–989[Abstract]
5. Fitzgerald D, Catella F, Roy L, Fitzgerald G. Marked platelet activation in vivo after intravenous streptokinase in patients with acute myocardial infarction. Circulation. 1988;77:142–150[Abstract/Free Full Text]
6. Falk E. Unstable angina with fatal outcome: dynamic coronary thrombosis leading to infarction and/or sudden death. Autopsy evidence of recurrent mural thrombosis with peripheral embolization culminating in total vascular occlusion. Circulation. 1985;71:699–708[Abstract/Free Full Text]
7. Davies M, Thomas A, Knapman P, Hangartner J. Intramyocardial platelet aggregation in patients with unstable angina suffering sudden ischemic cardiac death. Circulation. 1986;73:418–427[Abstract/Free Full Text]
8. Cannon CP, McCabe CH, Wilcox RG, et al. Oral glycoprotein IIb/IIIa inhibition with orbofiban in patients with unstable coronary syndrome (OPUS-TIMI 16) trial. Circulation. 2000;102:149–156[Abstract/Free Full Text]
9. Du X, Ginsberg M. Integrin _IIbß₃ and platelet function. Thromb Haemost. 1997;78:96–100[Medline]
10. Stephens G, O’Luanaigh N, Reilly D, et al. A sequence within the cytoplasmic tail of GPIIb independently activates platelet aggregation and thromboxane synthesis. J Biol Chem. 1998;273:20317–20322[Abstract/Free Full Text]
11. Shattil S, Gao J, Kashiwagi H. Not just another pretty face: regulation of platelet function at the cytoplasmic face of integrin _IIbß₃. Thromb Haemost. 1997;78:220–225[Medline]
12. Schoenwaelder S, Burridge K. Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol. 1999;11:274–286[CrossRef][Medline]
13. Honda S, Tomiyama Y, Aoki T, et al. Association between ligand-induced conformational changes of integrin _IIbß₃ and _IIbß₃-mediated intracellular Ca²⁺ signaling. Blood. 1998;92:3675–3683[Abstract/Free Full Text]
14. Peter K, Schwartz M, Ylanne J, et al. Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (_IIbß₃) inhibitors. Blood. 1998;92:3240–3249[Abstract/Free Full Text]
15. Du X, Plow E, Frelinger A, O’Toole T, Loftus J, Ginsberg M. Ligands "activate" integrin _IIbß₃ (platelet GPIIb/IIIa). Thromb Haemost. 1991;68:731–736
16. Cox D, Aoki T, Seki J, Motoyama Y, Yoshida K. Pentamidine: a specific, non-peptide GPIIb/IIIa antagonist. Thromb Haemost. 1996;75:503–509[Medline]
17. Quinn M, Deering A, Stewart M, Cox D, Foley B, Fitzgerald D. Quantifying GPIIb/IIIa receptor binding using two monoclonal antibodies. Discriminating abciximab and small molecular weight antagonists. Circulation. 1999;99:2231–2238[Abstract/Free Full Text]
18. Hamamoto K, Ohga S, Nomura S, Yasunaga K. Cellular distribution of CD63 antigen in platelets and in three megakaryocytic cell lines. Histochem J. 1994;26:367–375[CrossRef][Medline]
19. Corcoran M, Kibbey M, Kleinman H, Wahl L. Laminin SIKVAV peptide induction of monocyte/macrophage prostaglandin E2 and matrix metalloproteinases. J Biol Chem. 1995;270:10365–10368[Abstract/Free Full Text]
20. Schmidt C, Pommerenke H, Frieda D, Nebe B, Rychly J. Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins. J Biol Chem. 1998;273:5081–5085[Abstract/Free Full Text]
21. Aoki T, Tomiyama Y, Honda S, et al. Difference of (Ca²⁺)i movements in platelets stimulated by thrombin and TRAP: the involvement of _IIbß₃-mediated TXA₂ synthesis. Thromb Haemost. 1998;79:1184–1190[Medline]
22. Hato T, Pampori N, Shattil S. Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin _IIbß₃. J Cell Biol. 1998;14:1685–1695
23. Frelinger A, Cohen I, Plow EF, et al. Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers. J Biol Chem. 1990;265:6346–6352[Abstract/Free Full Text]
24. Frelinger A, Du XP, Plow EF, Ginsberg MH. Monoclonal antibodies to ligand-occupied conformers of integrin _IIbß₃ (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function. J Biol Chem. 1991;266:17106–17111[Abstract/Free Full Text]
25. Kostelijk E, Fijnheer R, Nieuwenhuis H, Gouwerok C, de Korte D. Soluble P-selectin as parameter for platelet activation during storage. Thromb Haemost. 1996;76:1086–1089[Medline]
26. Adderley S, Fitzgerald D. Glycoprotein IIb/IIIa antagonists induce apoptosis in rat cardiomyocytes by caspase-3 activation. J Biol Chem. 2000;275:5760–5766[Abstract/Free Full Text]
27. Ault K, Cannon C, Mitchell J, et al. Platelet activation in patients after an acute coronary syndrome: results from the TIMI-12 trial. Thrombolysis in Myocardial Infarction. J Am Coll Cardiol. 1999;33:634–639[Abstract/Free Full Text]
28. Holmes M, Sobel B, Cannon C, Schneider D. Increased platelet reactivity in patients given orbofiban after an acute coronary syndrome: an OPUS-TIMI-16 substudy. Orbofiban in Patients with Unstable coronary syndromes. Thrombolysis In Myocardial Infarction. Am J Cardiol 2000;85:491–3, A10.
29. Peter K, Straub A, Kohler B, et al. Platelet activation as a potential mechanism of GP IIb/IIIa inhibitor-induced thrombocytopenia. Am J Cardiol. 1999;84:519–524[CrossRef][Medline]
30. Holmes M, Sobel B, Schneider D. Variable responses to inhibition of fibrinogen binding induced by tirofiban and eptifibatide in blood from healthy subjects. Am J Cardiol. 1999;84:203–207[CrossRef][Medline]
31. Pratico D, Murphy N, Fitzgerald D. Interaction of a thrombin inhibitor and a platelet GPIIb/IIIa antagonist in vivo: evidence that thrombin mediates platelet aggregation and subsequent thromboxane A₂ formation during coronary thrombolysis. J Pharmacol Exp Ther. 1997;281:1178–1185[Abstract/Free Full Text]

This article has been cited by other articles:

				R. Blue, M. Murcia, C. Karan, M. Jirouskova, and B. S. Coller Application of high-throughput screening to identify a novel {alpha}IIb-specific small- molecule inhibitor of {alpha}IIb{beta}3-mediated platelet interaction with fibrinogen Blood, February 1, 2008; 111(3): 1248 - 1256. [Abstract] [Full Text] [PDF]

				S. R. Steinhubl, J. J. Badimon, D. L. Bhatt, J.-M. Herbert, and T. F. Luscher Clinical evidence for anti-inflammatory effects of antiplatelet therapy in patients with atherothrombotic disease Vascular Medicine, May 1, 2007; 12(2): 113 - 122. [Abstract] [PDF]

				N. Bassler, C. Loeffler, P. Mangin, Y. Yuan, M. Schwarz, C. E. Hagemeyer, S. U. Eisenhardt, I. Ahrens, C. Bode, S. P. Jackson, et al. A Mechanistic Model for Paradoxical Platelet Activation by Ligand-Mimetic {alpha}IIb{beta}3 (GPIIb/IIIa) Antagonists Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): E9 - E15. [Abstract] [Full Text] [PDF]

				J. E. Freedman Molecular Regulation of Platelet-Dependent Thrombosis Circulation, October 25, 2005; 112(17): 2725 - 2734. [Abstract] [Full Text] [PDF]

				R. H. Aster Immune Thrombocytopenia Caused by Glycoprotein IIb/IIIa Inhibitors Chest, February 1, 2005; 127(2_suppl): 53S - 59S. [Abstract] [Full Text] [PDF]

				C. Patrono, B. Coller, G. A. FitzGerald, J. Hirsh, and G. Roth Platelet-Active Drugs: The Relationships Among Dose, Effectiveness, and Side Effects: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy Chest, September 1, 2004; 126(3_suppl): 234S - 264S. [Abstract] [Full Text] [PDF]

				Z. Xiao and P. Theroux Clopidogrel inhibits platelet-leukocyte interactions and thrombin receptor agonist peptide-induced platelet activation in patients with an acute coronary syndrome J. Am. Coll. Cardiol., June 2, 2004; 43(11): 1982 - 1988. [Abstract] [Full Text] [PDF]

				M. Schwarz, Y. Katagiri, M. Kotani, N. Bassler, C. Loeffler, C. Bode, and K. Peter Reversibility versus Persistence of GPIIb/IIIa Blocker-Induced Conformational Change of GPIIb/IIIa ({alpha}IIb{beta}3, CD41/CD61) J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 1002 - 1011. [Abstract] [Full Text] [PDF]

				M. Dalby, G. Montalescot, C. B. d. Sollier, E. Vicaut, T. Soulat, J.-P. Collet, R. Choussat, V. Gallois, G. Drobinski, L. Drouet, et al. Eptifibatide provides additional platelet inhibition in Non-ST-Elevation myocardial infarction patients already treated with aspirin and clopidogrel: Results of the platelet activity extinction in Non-Q-Wave myocardial infarction with aspirin, clopidogrel, and eptifibatide (PEACE) study J. Am. Coll. Cardiol., January 21, 2004; 43(2): 162 - 168. [Abstract] [Full Text] [PDF]

				C. Patrono, F. Bachmann, C. Baigent, C. Bode, R. De Caterina, B. Charbonnier, D. Fitzgerald, J. Hirsh, S. Husted, J. Kvasnicka, et al. Expert Consensus Document on the Use of Antiplatelet Agents: The Task Force on the Use of Antiplatelet Agents in Patients with Atherosclerotic Cardiovascular Disease of the European Society of Cardiology Eur. Heart J., January 2, 2004; 25(2): 166 - 181. [Full Text] [PDF]

				C. J Knight Antiplatelet treatment in stable coronary artery disease Heart, October 1, 2003; 89(10): 1273 - 1278. [Full Text] [PDF]

				M. J. Quinn, T. V. Byzova, J. Qin, E. J. Topol, and E. F. Plow Integrin {alpha}IIb{beta}3 and Its Antagonism Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 945 - 952. [Abstract] [Full Text] [PDF]

				J. A. de Lemos, D. A. Morrow, M. S. Sabatine, S. A. Murphy, C. M. Gibson, E. M. Antman, C. H. McCabe, C. P. Cannon, and E. Braunwald Association Between Plasma Levels of Monocyte Chemoattractant Protein-1 and Long-Term Clinical Outcomes in Patients With Acute Coronary Syndromes Circulation, February 11, 2003; 107(5): 690 - 695. [Abstract] [Full Text] [PDF]

				M. Mirabet, D. Garcia-Dorado, J. Inserte, J. A. Barrabes, R.-M. Lidon, B. Soriano, M. Azevedo, F. Padilla, L. Agullo, M. Ruiz-Meana, et al. Platelets activated by transient coronary occlusion exacerbate ischemia-reperfusion injury in rat hearts Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1134 - H1141. [Abstract] [Full Text] [PDF]

				M. J. Quinn, E. F. Plow, and E. J. Topol Platelet Glycoprotein IIb/IIIa Inhibitors: Recognition of a Two-Edged Sword? Circulation, July 16, 2002; 106(3): 379 - 385. [Full Text] [PDF]

				K. Peter, M. Schwarz, C. Bode, A. L. Frelinger III, M. I. Furman, L. A. Krueger, M. R. Barnard, and A. D. Michelson Activating Effects of GPIIb/IIIa Blockers: An Intrinsic Consequence of Ligand- Mimetic Properties * Response Circulation, May 28, 2002; 105 (21): e180 - e181. [Full Text] [PDF]

				G. W. Stone, D. J. Moliterno, M. Bertrand, F.-J. Neumann, H. C. Herrmann, E. R. Powers, C. L. Grines, J. W. Moses, D. J. Cohen, E. A. Cohen, et al. Impact of Clinical Syndrome Acuity on the Differential Response to 2 Glycoprotein IIb/IIIa Inhibitors in Patients Undergoing Coronary Stenting: The TARGET Trial* Circulation, May 21, 2002; 105(20): 2347 - 2354. [Abstract] [Full Text] [PDF]

				F.W.G. Leebeek, E. Boersma, C.P. Cannon, F.J.J. van de Werf, and M.L. Simoons Oral glycoprotein IIb/IIIa receptor inhibitors in patients with cardiovascular disease: why were the results so unfavourable Eur. Heart J., March 2, 2002; 23(6): 444 - 457. [Full Text] [PDF]

				C. Patrono Antiplatelet strategies Eur. Heart J. Suppl., February 1, 2002; 4(suppl_A): A42 - A47. [Abstract] [PDF]

				F. F. O'Connor, D. C. Shields, A. Fitzgerald, C. P. Cannon, E. Braunwald, and D. J. Fitzgerald Genetic variation in glycoprotein IIb/IIIa (GPIIb/IIIa) as a determinant of the responses to an oral GPIIb/IIIa antagonist in patients with unstable coronary syndromes Blood, December 1, 2001; 98(12): 3256 - 3260. [Abstract] [Full Text] [PDF]

				J. Vermylen, M. Hoylaerts, J. Arnout, D. P. Chew, D. L. Bhatt, E. J. Topol, and S. Sapp Increased Mortality With Long-Term Platelet Glycoprotein IIb/IIIa Antagonists: An Explanation? Response Circulation, November 13, 2001; 104 (20): e109 - e109. [Full Text] [PDF]