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Effects of Ioxaglate on Cultured Microvascular Endothelial Cells
Letter to the Editor
EFFECTS OF IOXAGLATE ON CULTURED MICROVASCULAR ENDOTHELIAL CELLS: DO ALL IN VITRO STUDIES ACTUALLY REFLECT CLINICAL SITUATIONS? From: Jean-Marc Ide´e, PharmD Philippe Prigent, PhD Claire Corot, PharmD, PhD Laboratoire Guerbet Department of Pharmacology, BP50400 Roissy-Charles-de-Gaulle Cedex F-95943 France
Editor: Facts do not cease to exist because one ignores them. Aldous Huxley
Furuta et al (1) recently investigated the effects of the nonionic monomeric contrast medium iohexol and the low-osmolar ionic agent ioxaglate on cultured microvascular cells by measuring the efflux of lactate dehydrogenase (LDH) into the extracellular medium. Iodine concentrations were 100 and 150 mg of iodine per milliliter, and exposure times were 10, 30, and 60 minutes. The authors report that ioxaglate, but not iohexol, induced time- and concentration-dependent morphologic degeneration and that ioxaglate increased the LDH signal to 18.9% at 150 mg of iodine per milliliter and after 60 minutes of incubation. They concluded that this cytotoxic action of ioxaglate may have clinical consequences associated with the occurrence of phlebitis, pain, and thrombosis. This interesting article deserves comment. Methods The concentrations Furuta et al selected for contrast media were 100 and 150 mg of iodine per milliliter, and the exposure times ranged from 10 – 60 minutes. These experimental conditions seem far removed from the phar-
98
macokinetic behavior of contrast media. Contrast medium is rapidly diffused in the human body; it does not remain at the concentrated levels outlined by Furuta et al for extended periods of time. In fact, about 70% of the dose is cleared from the plasma compartment within 2-5 minutes after injection (2). The clinical relevance of the reported data thus seems subject to discussion, since continuous contact for 1 hour between a highly concentrated solution of contrast agent and endothelial cells is unlikely. The contrast media were diluted with Krebs-Ringer buffer. We believe that a dilution of the growth medium containing fetal calf serum would have been more relevant and less aggressive to the endothelial cells. Apparently, the authors failed to check for a possible analytic interference between the test solutions and the reagents of the LDH colorimetric detection kit. Given the high concentrations of contrast media, this would have been a wise precaution. An important part of the reported data relies on morphologic observations. Unfortunately, the investigators were apparently not blinded with regard to the contrast agent being tested. Data Analysis What is the authors’ definition of a “severe degeneration of cells” (1)? The quantitative criteria that allow such a qualitative allegation are not clearly stated in the Materials and Methods or Results sections. It should be underlined that, although significant morphologic changes were reported after 10 minutes of exposure at the higher concentration, “severe degeneration of cells” and detachment of cells from the culture plate that led to a 20% reduction in the number of cells were achieved only after a 60minute exposure to the higher concentration tested. This 20% reduction in the total number of cells counted is consistent with the 18.9% increase in the LDH signal. In other words, cell death was achieved in only 20% of endothelial cells exposed to ioxaglate in drastic conditions. In the Discussion section, the authors indicate that after a brief exposure to ioxaglate, most cells appeared to survive without specific organelle damage. Put together, these data do not support the conclusion that “the direct cyto-
Academic Radiology, Vol 9, No 1, January 2002
toxic action of ioxaglate on endothelial cells could be an important cause of endothelial cell dysfunction, which is closely related to the occurrence of phlebitis, pain, and thrombosis” (1). Discussion of Data The authors refer to an article published by researchers from their own laboratory. The researchers concluded that ioxaglate, but not iohexol, increased the Evans blue dye extravasation in the rat’s pulmonary vascular bed (3). The intravenous doses at which a statistically significant extravasation of the marker was noticed were 4 and 6 g of iodine per kilogram. This is about six to nine times the maximum dose of contrast medium injected for computed tomographic procedures. At 2 g of iodine per kilogram, the authors did not observe any such effects (3). Here again, the aggressive experimental conditions selected by the authors preclude any extrapolation to clinical conditions. For some unclear reasons, the authors do not discuss their data in light of other studies that deal with the interaction of contrast agents and vascular physiology. In the only clinical study available to date (4), the authors compared the vasomotor responses to ioxaglate, to the nonionic monomer iopromide, or to the nonionic dimer iodixanol at quantitative coronary angiography in patients with or without coronary artery disease. Both nonionic agents exerted statistically significant and different vasomotor effects on angiographically normal coronary segments in patients with coronary artery disease and in patients with normal arteries, whereas ioxaglate had no effect on epicardial coronary dimensions. The nonionic contrast media induced substantial dilation of angiographically normal segments and constriction of segments close to the stenosis. In this article, Limbruno et al (4) question whether an additional vasoconstrictory effect on the site of exposure of thrombogenic material might have thrombotic consequences. This clinical study leads to speculations that are totally at odds with those of Furuta et al. Several studies have shown a better endothelial tolerance for ioxaglate than for nonionic agents. They are not mentioned in the Discussion section of the article by Furuta et al. Just to quote a few, Abeyama et al (5) have shown that the nonionic agents iopamidol and ioversol each produced activation of human endothelial cells with expression of P-selectin, whereas ioxaglate induced a down-regulation of both P- and E-selectin. Furthermore, adhesion of leukocytes to the endothelial cells was signif-
LETTER TO THE EDITOR
icantly greater with ioversol than with ioxaglate. Last but not least, the production of tumor necrosis factor ␣ was greater in the cells with leukocyte adhesion after exposure to a nonionic agent than after exposure to either ioxaglate or a control substance (5). In another study, a high-osmolar ionic agent and nonionic iopamidol stimulated ADPase activity of the aortic endothelium, whereas ioxaglate left this enzyme activity unchanged (6). Furthermore, high-osmolar contrast media have been shown to produce a time- and concentrationdependent alteration in the incorporation of 3H-leucine and 3H-thymidine into macromolecules of human endothelial cells. Low-osmolar agents, either ionic (ioxaglate) or not, had markedly less effect (7). Interestingly, however, there were differences between the various low-osmolar agents, with the nonionic iopromide showing fairly marked effects on the cells. The least effect was achieved with the dimeric agent iotrolan. Interestingly, in this study, LDH release reached significance only for high-osmolar agents and after 2 hours of exposure to undiluted solutions (7). This is not consistent with the data presented by Furuta et al. In conclusion, Furuta et al (1) conclude that the cytotoxicity of ioxaglate may be associated with the occurrence of pain, phlebitis, and thrombosis. At the very least, this conclusion can be considered a misuse of language, and this allegation deserves a brief discussion. Does ioxaglate induce more pain at angiography than iohexol? There is clear-cut clinical evidence that ioxaglate actually causes significantly less pain than iohexol (8 –10). Does ioxaglate cause more thrombophlebitis than iohexol? To the best of our knowledge, there are no clinical data to support this allegation. Bowles et al (11) found no statistically significant difference between the two agents when used for ascending venography in 100 patients with respect to immediate and delayed side effects. Ioxaglate has been shown to induce less thrombophlebitis than high-osmolar contrast agents (12,13). Does ioxaglate induce more thrombosis than iohexol? A large body of in vitro (2,14 –18), animal (19 –21), and clinical (22–27) studies clearly shows that ioxaglate is actually associated with a lower thrombotic risk than iohexol. A recent meta-analysis showed that, compared to nonionic monomers, ioxaglate reduces the rate of coronary abrupt closure by 32% (P ⫽ .03) (28). For a general review of the literature, see reference 29. In other words, all of the clinical speculations raised by Furuta et al on the basis of their in vitro data are refuted by the literature. Thus, the
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IDEE ET AL
conclusion made by Furuta et al may be regarded as unfair and misleading. In vitro models that use cell lines are definitely valuable when performing an in-depth exploration of the mechanisms of action in pharmacology or toxicology. However, the accuracy of their predictive value as exclusive screening tests for toxicologic purposes appears, as discussed above, highly debatable where contrast media are concerned. REFERENCES 1. Furuta W, Sendo T, Kataoka Y, Oishi R. Morphologic degeneration of human microvascular endothelial cells induced by iodinated contrast media. Acad Radiol 2001; 8:158 –161. 2. Eloy R, Corot C, Belleville J. Contrast media for angiography: physicochemical properties, pharmacokinetics and biocompatibility. Clin Materials 1991; 7:89 –197. 3. Sendo T, Kataoka Y, Takeda Y, Furuta W, Oishi R. Nitric oxide protects against contrast media-increased pulmonary vascular permeability in rats. Invest Radiol 2000; 35:472– 478. 4. Limbruno U, Petronio AS, Amoroso G, et al. The impact of coronary artery disease on the coronary vasomotor response to nonionic contrast media. Circulation 2000; 101:491– 497. 5. Abeyama K, Oh S, Kawano K, et al. Nonionic contrast agents produce thrombotic effect by inducing adhesion of leukocytes on human endothelium. Biochem Biophys Res Commun 1995; 212:776 –783. 6. Caprino L, Togna AR, Zappacosta B, Giardina B, Togna G. Modulation of ADPase and t-PA release by radiographic contrast media in bovine aortic endothelium. Pharmacol Res 1997; 35:429 – 433. 7. Morgan DML, Bettmann MA. Effects of X-ray contrast media and radiation on human vascular endothelial cells in vitro. Cardiovasc Intervent Radiol 1989; 12:154 –160. 8. Murphy G, Campbell DR, Fraser DB. Pain in peripheral arteriography: an asessment of conventional versus ionic and nonionic low osmolality contrast agents. Can Assoc Radiol J 1988; 39:103–106. 9. Stiris MG, Laerum F. Iohexol and ioxaglate in peripheral angiography. Acta Radiol 1987; 28:767–770. 10. Smith DC, Yahiku PY, Mahoney MD, Hart KL. Three new low-osmolality contrast agents: a comparative study of patient discomfort. AJNR Am J Neuroradiol 1988; 9:137–139. 11. Bowles JN, Thomas ML, Treweeke PS. A comparative trial of the diagnostic quality of and tolerance for two low concentration low osmolality contrast media for phlebography. Eur J Radiol 1986; 6:301–302. 12. Eliasen B, Horup A, Jensen AR. Thrombosis following phlebography with high-osmolar and low-osmolar contrast media. Eur J Radiol 1983; 3:97–98.
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13. Thomas ML, Briggs GM, Kuan BB. Contrast agent-induced thrombophlebitis following leg phlebography: meglumine ioxaglate versus meglumine iothalamate. Radiology 1983; 147:399 – 400. 14. Chronos NAF, Goodall AH, Wilson DJ, Sigwart U, Buller NP. Profound platelet degranulation is an important side effect of some types of contrast media used in interventional cardiology. Circulation 1993; 88: 2035–2044. 15. Li X, Gabriel DA. Differences between contrast media in the inhibition of platelet activation by specific platelet agonists. Acad Radiol 1997; 4:108 –114. 16. Kopko PM, Smith DC, Bull BS. Thrombin generation in nonclottable mixtures of blood and nonionic contrast agents. Radiology 1990; 174: 459 – 461. 17. Corot C, Chronos N, Sabattier V. In vitro comparison of the effects of contrast media on coagulation and platelet activation. Blood Coagul Fibrinolysis 1996; 7:602– 608. 18. Melton LG, Dehmer GJ, Gabriel DA. Alterations in fibrin detected in coronary sinus blood after heparin and coronary angiography with a nonionic contrast agent (iohexol). Am J Cardiol 1998; 82:295–298. 19. Markou CP, Chronos NAF, Hanson SR. Antithrombotic effects of ionic and non-ionic contrast media in nonhuman primates. Thromb Haemost 2001; 85:488 – 493. 20. Grines CL, Mickelson JK, Diaz C, DeMaria AN. Acute thrombosis in a canine model of arterial injury: effect of ionic versus non-ionic contrast media. J Invasive Cardiol 1991; 3(suppl B):18B–23B. 21. Pislaru S, Pislaru C, Szilard M, Arnout J, Van de Werf F. In vivo effects of contrast media on coronary thrombolysis. J Am Coll Cardiol 1998; 32:1102–1108. 22. Piessens JH, Stammen F, Vrolix MC, et al. Effects of an ionic versus a nonionic low osmolar contrast agent on the thrombotic complications of coronary angioplasty. Cathet Cardiovasc Diagn 1993; 28:99 –105. 23. Qureshi NR, Den Heijer P, Crijns JGM. Percutaneous coronary angioscopic comparison of thrombus formation during percutaneous coronary angioplasty with ionic and nonionic low osmolality contrast media in unstable angina. Am J Cardiol 1997; 80:700 –704. 24. Grines CL, Schreiber TL, Savas V, et al. A randomized trial of low osmolar ionic versus nonionic contrast media in patients with myocardial infarction or unstable angina undergoing percutaneous transluminal coronary angioplasty. J Am Coll Cardiol 1996; 27:1381–1386. 25. Scheller B, Hennen B, Pohl A, Schieffer H, Markwirth T. Acute and subacute stent occlusion: risk-reduction by ionic contrast media. Eur Heart J 2001; 22:385–391. 26. Sato E, Saito I, Tama Contrast Media Study Group. Risk of clot formation with ionic and nonionic contrast media in cerebral angiography. Acad Radiol 1996; 3:925–928. 27. Esplugas E, Cequier A, Jara F, et al. Risk of thrombosis during coronary angioplasty with low osmolality contrast media. Am J Cardiol 1991; 68:1020 –1040. 28. Cucherat M, Leizorovicz A. Effects of non-ionic contrast media on abrupt vessel closure and ischaemic complications after angioplasty: a meta-analysis. Am J Cardiol 1999; 84(suppl 6A):98P. 29. Ide´e JM, Corot C. Thrombotic risk associated with the use of iodinated contrast media in interventional cardiology: pathophysiology and clinical aspects. Fundam Clin Pharmacol 1999; 13:613– 623.
EFFECTS OF IOXAGLATE ON CULTURED MICROVASCULAR ENDOTHELIAL CELLS: DO ALL IN VITRO STUDIES ACTUALLY REFLECT CLINICAL SITUATIONS? From: Jean-Marc Ide´e, PharmD Philippe Prigent, PhD Claire Corot, PharmD, PhD Laboratoire Guerbet Department of Pharmacology, BP50400 Roissy-Charles-de-Gaulle Cedex F-95943 France
Editor: Facts do not cease to exist because one ignores them. Aldous Huxley
Furuta et al (1) recently investigated the effects of the nonionic monomeric contrast medium iohexol and the low-osmolar ionic agent ioxaglate on cultured microvascular cells by measuring the efflux of lactate dehydrogenase (LDH) into the extracellular medium. Iodine concentrations were 100 and 150 mg of iodine per milliliter, and exposure times were 10, 30, and 60 minutes. The authors report that ioxaglate, but not iohexol, induced time- and concentration-dependent morphologic degeneration and that ioxaglate increased the LDH signal to 18.9% at 150 mg of iodine per milliliter and after 60 minutes of incubation. They concluded that this cytotoxic action of ioxaglate may have clinical consequences associated with the occurrence of phlebitis, pain, and thrombosis. This interesting article deserves comment. Methods The concentrations Furuta et al selected for contrast media were 100 and 150 mg of iodine per milliliter, and the exposure times ranged from 10 – 60 minutes. These experimental conditions seem far removed from the phar-
98
macokinetic behavior of contrast media. Contrast medium is rapidly diffused in the human body; it does not remain at the concentrated levels outlined by Furuta et al for extended periods of time. In fact, about 70% of the dose is cleared from the plasma compartment within 2-5 minutes after injection (2). The clinical relevance of the reported data thus seems subject to discussion, since continuous contact for 1 hour between a highly concentrated solution of contrast agent and endothelial cells is unlikely. The contrast media were diluted with Krebs-Ringer buffer. We believe that a dilution of the growth medium containing fetal calf serum would have been more relevant and less aggressive to the endothelial cells. Apparently, the authors failed to check for a possible analytic interference between the test solutions and the reagents of the LDH colorimetric detection kit. Given the high concentrations of contrast media, this would have been a wise precaution. An important part of the reported data relies on morphologic observations. Unfortunately, the investigators were apparently not blinded with regard to the contrast agent being tested. Data Analysis What is the authors’ definition of a “severe degeneration of cells” (1)? The quantitative criteria that allow such a qualitative allegation are not clearly stated in the Materials and Methods or Results sections. It should be underlined that, although significant morphologic changes were reported after 10 minutes of exposure at the higher concentration, “severe degeneration of cells” and detachment of cells from the culture plate that led to a 20% reduction in the number of cells were achieved only after a 60minute exposure to the higher concentration tested. This 20% reduction in the total number of cells counted is consistent with the 18.9% increase in the LDH signal. In other words, cell death was achieved in only 20% of endothelial cells exposed to ioxaglate in drastic conditions. In the Discussion section, the authors indicate that after a brief exposure to ioxaglate, most cells appeared to survive without specific organelle damage. Put together, these data do not support the conclusion that “the direct cyto-
Academic Radiology, Vol 9, No 1, January 2002
toxic action of ioxaglate on endothelial cells could be an important cause of endothelial cell dysfunction, which is closely related to the occurrence of phlebitis, pain, and thrombosis” (1). Discussion of Data The authors refer to an article published by researchers from their own laboratory. The researchers concluded that ioxaglate, but not iohexol, increased the Evans blue dye extravasation in the rat’s pulmonary vascular bed (3). The intravenous doses at which a statistically significant extravasation of the marker was noticed were 4 and 6 g of iodine per kilogram. This is about six to nine times the maximum dose of contrast medium injected for computed tomographic procedures. At 2 g of iodine per kilogram, the authors did not observe any such effects (3). Here again, the aggressive experimental conditions selected by the authors preclude any extrapolation to clinical conditions. For some unclear reasons, the authors do not discuss their data in light of other studies that deal with the interaction of contrast agents and vascular physiology. In the only clinical study available to date (4), the authors compared the vasomotor responses to ioxaglate, to the nonionic monomer iopromide, or to the nonionic dimer iodixanol at quantitative coronary angiography in patients with or without coronary artery disease. Both nonionic agents exerted statistically significant and different vasomotor effects on angiographically normal coronary segments in patients with coronary artery disease and in patients with normal arteries, whereas ioxaglate had no effect on epicardial coronary dimensions. The nonionic contrast media induced substantial dilation of angiographically normal segments and constriction of segments close to the stenosis. In this article, Limbruno et al (4) question whether an additional vasoconstrictory effect on the site of exposure of thrombogenic material might have thrombotic consequences. This clinical study leads to speculations that are totally at odds with those of Furuta et al. Several studies have shown a better endothelial tolerance for ioxaglate than for nonionic agents. They are not mentioned in the Discussion section of the article by Furuta et al. Just to quote a few, Abeyama et al (5) have shown that the nonionic agents iopamidol and ioversol each produced activation of human endothelial cells with expression of P-selectin, whereas ioxaglate induced a down-regulation of both P- and E-selectin. Furthermore, adhesion of leukocytes to the endothelial cells was signif-
LETTER TO THE EDITOR
icantly greater with ioversol than with ioxaglate. Last but not least, the production of tumor necrosis factor ␣ was greater in the cells with leukocyte adhesion after exposure to a nonionic agent than after exposure to either ioxaglate or a control substance (5). In another study, a high-osmolar ionic agent and nonionic iopamidol stimulated ADPase activity of the aortic endothelium, whereas ioxaglate left this enzyme activity unchanged (6). Furthermore, high-osmolar contrast media have been shown to produce a time- and concentrationdependent alteration in the incorporation of 3H-leucine and 3H-thymidine into macromolecules of human endothelial cells. Low-osmolar agents, either ionic (ioxaglate) or not, had markedly less effect (7). Interestingly, however, there were differences between the various low-osmolar agents, with the nonionic iopromide showing fairly marked effects on the cells. The least effect was achieved with the dimeric agent iotrolan. Interestingly, in this study, LDH release reached significance only for high-osmolar agents and after 2 hours of exposure to undiluted solutions (7). This is not consistent with the data presented by Furuta et al. In conclusion, Furuta et al (1) conclude that the cytotoxicity of ioxaglate may be associated with the occurrence of pain, phlebitis, and thrombosis. At the very least, this conclusion can be considered a misuse of language, and this allegation deserves a brief discussion. Does ioxaglate induce more pain at angiography than iohexol? There is clear-cut clinical evidence that ioxaglate actually causes significantly less pain than iohexol (8 –10). Does ioxaglate cause more thrombophlebitis than iohexol? To the best of our knowledge, there are no clinical data to support this allegation. Bowles et al (11) found no statistically significant difference between the two agents when used for ascending venography in 100 patients with respect to immediate and delayed side effects. Ioxaglate has been shown to induce less thrombophlebitis than high-osmolar contrast agents (12,13). Does ioxaglate induce more thrombosis than iohexol? A large body of in vitro (2,14 –18), animal (19 –21), and clinical (22–27) studies clearly shows that ioxaglate is actually associated with a lower thrombotic risk than iohexol. A recent meta-analysis showed that, compared to nonionic monomers, ioxaglate reduces the rate of coronary abrupt closure by 32% (P ⫽ .03) (28). For a general review of the literature, see reference 29. In other words, all of the clinical speculations raised by Furuta et al on the basis of their in vitro data are refuted by the literature. Thus, the
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IDEE ET AL
conclusion made by Furuta et al may be regarded as unfair and misleading. In vitro models that use cell lines are definitely valuable when performing an in-depth exploration of the mechanisms of action in pharmacology or toxicology. However, the accuracy of their predictive value as exclusive screening tests for toxicologic purposes appears, as discussed above, highly debatable where contrast media are concerned. REFERENCES 1. Furuta W, Sendo T, Kataoka Y, Oishi R. Morphologic degeneration of human microvascular endothelial cells induced by iodinated contrast media. Acad Radiol 2001; 8:158 –161. 2. Eloy R, Corot C, Belleville J. Contrast media for angiography: physicochemical properties, pharmacokinetics and biocompatibility. Clin Materials 1991; 7:89 –197. 3. Sendo T, Kataoka Y, Takeda Y, Furuta W, Oishi R. Nitric oxide protects against contrast media-increased pulmonary vascular permeability in rats. Invest Radiol 2000; 35:472– 478. 4. Limbruno U, Petronio AS, Amoroso G, et al. The impact of coronary artery disease on the coronary vasomotor response to nonionic contrast media. Circulation 2000; 101:491– 497. 5. Abeyama K, Oh S, Kawano K, et al. Nonionic contrast agents produce thrombotic effect by inducing adhesion of leukocytes on human endothelium. Biochem Biophys Res Commun 1995; 212:776 –783. 6. Caprino L, Togna AR, Zappacosta B, Giardina B, Togna G. Modulation of ADPase and t-PA release by radiographic contrast media in bovine aortic endothelium. Pharmacol Res 1997; 35:429 – 433. 7. Morgan DML, Bettmann MA. Effects of X-ray contrast media and radiation on human vascular endothelial cells in vitro. Cardiovasc Intervent Radiol 1989; 12:154 –160. 8. Murphy G, Campbell DR, Fraser DB. Pain in peripheral arteriography: an asessment of conventional versus ionic and nonionic low osmolality contrast agents. Can Assoc Radiol J 1988; 39:103–106. 9. Stiris MG, Laerum F. Iohexol and ioxaglate in peripheral angiography. Acta Radiol 1987; 28:767–770. 10. Smith DC, Yahiku PY, Mahoney MD, Hart KL. Three new low-osmolality contrast agents: a comparative study of patient discomfort. AJNR Am J Neuroradiol 1988; 9:137–139. 11. Bowles JN, Thomas ML, Treweeke PS. A comparative trial of the diagnostic quality of and tolerance for two low concentration low osmolality contrast media for phlebography. Eur J Radiol 1986; 6:301–302. 12. Eliasen B, Horup A, Jensen AR. Thrombosis following phlebography with high-osmolar and low-osmolar contrast media. Eur J Radiol 1983; 3:97–98.
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13. Thomas ML, Briggs GM, Kuan BB. Contrast agent-induced thrombophlebitis following leg phlebography: meglumine ioxaglate versus meglumine iothalamate. Radiology 1983; 147:399 – 400. 14. Chronos NAF, Goodall AH, Wilson DJ, Sigwart U, Buller NP. Profound platelet degranulation is an important side effect of some types of contrast media used in interventional cardiology. Circulation 1993; 88: 2035–2044. 15. Li X, Gabriel DA. Differences between contrast media in the inhibition of platelet activation by specific platelet agonists. Acad Radiol 1997; 4:108 –114. 16. Kopko PM, Smith DC, Bull BS. Thrombin generation in nonclottable mixtures of blood and nonionic contrast agents. Radiology 1990; 174: 459 – 461. 17. Corot C, Chronos N, Sabattier V. In vitro comparison of the effects of contrast media on coagulation and platelet activation. Blood Coagul Fibrinolysis 1996; 7:602– 608. 18. Melton LG, Dehmer GJ, Gabriel DA. Alterations in fibrin detected in coronary sinus blood after heparin and coronary angiography with a nonionic contrast agent (iohexol). Am J Cardiol 1998; 82:295–298. 19. Markou CP, Chronos NAF, Hanson SR. Antithrombotic effects of ionic and non-ionic contrast media in nonhuman primates. Thromb Haemost 2001; 85:488 – 493. 20. Grines CL, Mickelson JK, Diaz C, DeMaria AN. Acute thrombosis in a canine model of arterial injury: effect of ionic versus non-ionic contrast media. J Invasive Cardiol 1991; 3(suppl B):18B–23B. 21. Pislaru S, Pislaru C, Szilard M, Arnout J, Van de Werf F. In vivo effects of contrast media on coronary thrombolysis. J Am Coll Cardiol 1998; 32:1102–1108. 22. Piessens JH, Stammen F, Vrolix MC, et al. Effects of an ionic versus a nonionic low osmolar contrast agent on the thrombotic complications of coronary angioplasty. Cathet Cardiovasc Diagn 1993; 28:99 –105. 23. Qureshi NR, Den Heijer P, Crijns JGM. Percutaneous coronary angioscopic comparison of thrombus formation during percutaneous coronary angioplasty with ionic and nonionic low osmolality contrast media in unstable angina. Am J Cardiol 1997; 80:700 –704. 24. Grines CL, Schreiber TL, Savas V, et al. A randomized trial of low osmolar ionic versus nonionic contrast media in patients with myocardial infarction or unstable angina undergoing percutaneous transluminal coronary angioplasty. J Am Coll Cardiol 1996; 27:1381–1386. 25. Scheller B, Hennen B, Pohl A, Schieffer H, Markwirth T. Acute and subacute stent occlusion: risk-reduction by ionic contrast media. Eur Heart J 2001; 22:385–391. 26. Sato E, Saito I, Tama Contrast Media Study Group. Risk of clot formation with ionic and nonionic contrast media in cerebral angiography. Acad Radiol 1996; 3:925–928. 27. Esplugas E, Cequier A, Jara F, et al. Risk of thrombosis during coronary angioplasty with low osmolality contrast media. Am J Cardiol 1991; 68:1020 –1040. 28. Cucherat M, Leizorovicz A. Effects of non-ionic contrast media on abrupt vessel closure and ischaemic complications after angioplasty: a meta-analysis. Am J Cardiol 1999; 84(suppl 6A):98P. 29. Ide´e JM, Corot C. Thrombotic risk associated with the use of iodinated contrast media in interventional cardiology: pathophysiology and clinical aspects. Fundam Clin Pharmacol 1999; 13:613– 623.