Iohexol, platelet activation and thrombosis

Transcription

1 Acta Radiologica ISSN: (Print) (Online) Journal homepage: Iohexol, platelet activation and thrombosis Kjell S. Sakariassen, M. Buchmann, M. J. A. G. Hamers & H. Stormorken To cite this article: Kjell S. Sakariassen, M. Buchmann, M. J. A. G. Hamers & H. Stormorken (1998) Iohexol, platelet activation and thrombosis, Acta Radiologica, 39:4, To link to this article: Published online: 04 Jan Submit your article to this journal Article views: 43 Full Terms & Conditions of access and use can be found at

2 Acta Radiologica 39 (1998) Prinred in Denmark - AN rights reserved Copyright 0 Acra Radiologicu 1998 AC TA R A D 1 0 L 0 G I CA ISSN IOHEXOL, PLATELET ACTIVATION AND THROMBOSIS 11. Iohexol-induced platelet secretion does not affect collageninduced or tissue-factor-induced thrombus formation in blood that is anticoagulated with heparin and aspirin K. S. SAKARIASSEN"~, M. BUCHMANN', M. J. A. G. HAMERS' and H. STORMORKEN' 'Nycomed Imaging AS, Bioreg Research, Oslo; and 'Department of Biology, Division of General Physiology, University of Oslo, Oslo; Norway. Abstract Background: There is a dispute about the potential effects of radiographic contrast media (CM) on thrombogenesis. The nonionic CM iohexol triggers platelet P-thromboglobulin (P-TG) secretion, and thus may activate the platelets and promote thrombosis. We addressed this topic in a study employing a human model of arterial thrombus formation in the presence of aspirin and heparin. This was a follow-up to our initial study (on thrombus formation in native blood) which did not include antithrombotic drugs. The nonionic CM iohexol (monomer) and iodixanol (dimer) and the ionic CM ioxaglate (dimer) were compared. Methods and Results: Thrombus formation was triggered by a surface rich in either collagen or tissue factor, positioned in a parallel-plate perfusion chamber device at an arterial wall shear rate of 2600 s-i. Blood from healthy volunteers, following ingestion of 1 g aspirin, was mixed with 40 vol% CM and 2.0 IU/ml heparin and passed over the surfaces. Thrombus formation in the presence of either CM showed no difference, despite the fact that iohexol triggered a pronounced platelet p-tg secretion; iodixanol or ioxaglate were virtually inert. Conclusion: There was no association between iohexol-induced P-TG secretion and thrombus formation on collagen (platelet-driven) or on tissue factor (thrombin-driven) in the presence of a standard antithrombotic regimen of aspirin and heparin as used in the clinic. The notion of a thrombotic risk due to platelet activation by iohexol was thus not substantiated by this study. Key words: Contrast media, iohexol, iodixanol, ioxaglate; platelet activation; thrombus formation; arterial shear. Correspondence: Kjell S. Sakariassen, Department of Biology, Division of General Physiology, PO. Box 1051, Blindern, N Oslo, Norway. FAX t Accepted for publication 10 December Ionic and nonionic contrast media (CM) inhibit in vitro blood plasma coagulation and blood platelet aggregation in plasma with a high platelet level. It is generally accepted that ionic CM are stronger inhibitors than nonionic CM (2, 24). These differences would appear to be of no significance in vivo in the presence of proper antithrombotic drug administration since the incidence of thrombo-embolic disorders reported for the two types of CM does not differ in procedures of angiography (1, 22) or percutaneous transluminal coronary angioplasty (PTCA) (6, 9). However, recent reports have indicated a slightly increased incidence of thrombosis following the use of the nonionic CM iohexol in PTCA (3, 5), particularly in patients with acute ischemic syndromes (7). Quite reasonably, the discussion has included the in vitro observation that P-thromboglobulin (p- TG) secretion is induced by the nonionic CM iohexol but not by the ionic CM ioxaglate (1). 355

3 K. S. SAKARIASSEN ET AL. Since P-TG is generally used as a specific marker for intravascular platelet activation and thromboembolism (lo), the question naturally arose as to whether iohexol is a platelet activator and as such a prothrombotic. Such a possibility seems remote since iohexol inhibits in vitro platelet aggregation and is therefore by definition no platelet agonist but rather a weak platelet antagonist (24). Indeed, the collagen-induced thrombus formation in native human blood mixed with iohexol in a well characterized thrombus-forming device at arterial shear (20) was no different from the thrombus formed in blood mixed with the ionic ioxaglate or nonionic iodixanol(l9). This hding was observed in spite of the high iohexol-elicited P-TG secretion; ioxaglate or iodixanol did not trigger significant P- TG secretion. Nevertheless, the potential consequences of the iohexol-dependent P-TG secretion for thrombus formation in flowing anticoagulated blood are unknown and demand attention. This prompted us to study the potential effects of iohexol-dependent P-TG secretion on arterial thrombus formation in blood that is anticoagulated with clinically relevant doses of aspirin and heparin. For this purpose, we employed a parallelplate perfusion chamber device in which thrombus formation is triggered by purified human type I11 collagen fibrils or by human tissue factor/phospholipids at high arterial shear (13, 19, 20). Under these experimental conditions, the collagen-induced thrombus formation is independent of coagulation whereas the tissue-factor-induced coagulation is thrombin-driven (4, 12). In this way, the potential effects of CM on collagen-dependent or thrombin-dependent thrombus formation were differentiated and quantified at clinically relevant doses of aspirin and heparin. Also of importance was the fact that previous experience with this model and these thrombus-promoting surfaces had demonstrated an association between the size of the platelet thrombus and the P-TG plasma levels (12-16), e.g. large thrombi resulted in higher P-TG plasma levels than smaller thrombi did. However, our recently performed study on the effect of CM or glucose in native human blood (19) showed no such parallel - on the contrary, thrombus formation at increasing glucose osmolality showed the opposite association: the platelet thrombus volume decreased in parallel with increasing P-TG secretion. Material and Methods Contrast media and controls: The following CM were used: iohexol (Omnipaque, nonionic monomer, 350 mg I/ml, 825 mosmol, Nycomed Amers- ham); iodixanol (Visipaque, nonionic dimer, 320 mg I/ml, 280 mosmol, Nycomed Amersham); and ioxaglate (Hexabrix, ionic dimer, 320 mg Uml, 580 mosmol, Guerbet). A glucose solution of 300 mosmol was used as control. Blood donors: Twenty healthy young nonsmoking men donated (with informed consent) 110 ml blood for two perfusion experiments on the same day. The blood parameters hematocrit, hemoglobin, platelet count, and leukocyte count were within the normal ranges for all donors. All volunteers ingested 1 g aspirin (Novid, Nycomed Pharma) two hours before the first of the two successive perfusions. This aspirin dose completely blocks the formation of the platelet thromboxane A2 by its irreversible acetylation of the cyclo-oxygenase (1 8). In one perfusion, the blood was used to study the potential effects of one of the three CM on thrombus formation. In the other perfusion, the blood was used for the glucose control. Of the first perfusions, half were performed with CM and the other half with glucose. Thrombogenic surfaces: The surfaces used to trigger thrombus formation consisted either of type I11 collagen fibrils purified from human placenta (4, 19, 20) or of a tissue-factor/phospholipid preparation made from human placenta (Thrombore1 S, Behringwerke AS, Marburg, Germany) (12, 13). These preparations have previously been characterized biochemically and used in studies of thrombus formation in flowing native or anticoagulated human blood (4, 12-16, 19, 20). Human ex vivo thrombosis model and perfusions: The parallel-plate perfusion chamber device (20, 21) connected with the mixing device was employed (12, 19). In this system, native blood is drawn directly by an occlusive roller pump from an antecubital vein over the collagen-coated or the tissue-factor/phospholipid-coated cover slip surface positioned in the flow channel of the perfusion chamber. The pump is placed distal to the perfusion chamber. The mixing device, which is positioned proximal to the perfusion chamber, homogeneously mixed CM, unfractionated heparin (Heparin, 5000 IU/ml, Nycomed Pharma) or glucose when pumped into the ex vivo blood stream from the vein. Thus, the introduction of a solution into the blood flow results in homogeneous mixing of solution and blood proximal to the perfusion chamber and the thrombus-promoting surface. All blood-contact surfaces of the mixing device (Carmeda AB, Stockholm, Sweden) were covalently coated with heparin to minimize the activation of platelets and coagulation (12). Blood perfusions with CM or glucose lasted for 5 min at an arterial wall shear rate of 2600 s-*. 356

4 IOHEXOL, PLATELET ACTIVATION AND THROMBOSIS - I1 This shear rate is within the range of those encountered in vessels with moderate atherosclerosis (25). The flow rate was 10 ml/min. In all the perfusion experiments, the CM or glucose in the blood was 40 ~01%. Heparin was infused together with CM or glucose at a ha1 concentration in the diluted blood of 2.0 IU/ml. For the termination of blood perfusion, specimen fixation and epoxy resin embedding, we used the same procedures as described in our previous report (19). Measurement of plasma p-tg: Plasma levels of P-TG (IU/ml) from blood samples drawn distal to the perfusion chamber at 4 min of perfusion (14, 19) were quantified by radio-immunometric assay (Amersham, UK). The upper level of the normal range of plasma P-TG was defined as 40 IU/ml. Thrombus morphometry was performed on 1- pm-thick epoxy-embedded sections as previously described (19, 21). Thrombus parameters for fibrin deposition (% of surface coverage with fibrin), platelet adhesion (YO of surface coverage with platelets), and platelet thrombus volume (pm3/pm2 thrombus volume) were measured. Coagulation assay: Activated partial thromboplastin time was measured with the Cephotest (Nycomed Pharma). Statistical analysis: The results are expressed as mean2sem or mean+sd. Significance for the paired and nonpaired data was calculated by means of the Student's two-tailed t-test. Values of p<0.05 were considered significant B r T N Results Heparin anticoagulation: Heparin introduction into the ex vivo blood stream prolonged the activated partial clotting time from to s (meanksd, n=4). Collagen-induced thrombus formation: Fibrin deposition in presence of CM or the 300 mosmol glucose control covered less than 1% of the surface. Platelet-collagen adhesion (Fig. 1A) did not differ with the various CM or glucose used. The platelet thrombus volume (Fig. 1B) was within the same range, irrespective of the CM used. The average thrombus volume was lower in blood mixed with glucose. The only significant statistical difference (P<O.OOl) in thrombus volume was observed in blood mixed with glucose. Iohexol profoundly increased the P-TG plasma level (P<O.OOl) (Fig. 1C). In contrast, the P-TG plasma levels in blood mixed with iodixanol or ioxaglate were low and within the same range as in the glucose control. Tissue-factor-induced thrombus formation: None " iodlxand glucose iohexol ioxaglate Fig. 1. Thrombus formation elicited by fibrillar collagen in blood containing aspirin and heparin in the presence of CM at a wall shear rate of 2600 s-i. A) Platelet-collagen adhesion (X of surface coverage with platelets). B) Platelet thrombus volume (pm3/pm2). C) P-TG plasma levels (IWml). Mean-CSEM, n=5. Student's t-test *** p<o.ool. 357

5 K. S. SAKARIASSEN ET AL. 201 c B = E \ D T a k cp. E d 1000 h iodixanol glucose lodlxanol glucose iohexol loxaglate iohexol loxaglate Fig. 2. Thrombus formation elicited by tissue-factor in blood containing aspirin and heparin in the presence of CM at a wall shear rate of 2600 s-*. A) Fibrin deposition (% of surface coverage with fibrin). B) Platelet-fibrin adhesion ( 3% of surface coverage with platelets). C) Platelet thrombus volume (pm3/pm2). D) P-TG plasma levels (IU/ml). Mean? SEM, n=5. Student s t-test *** p<o.ool. of the CM affected fibrin deposition (Fig. 2A). The fibrin deposition was within the same range as that observed in blood mixed with glucose. Platelet-fibrin adhesion (Fig. 2B) was not affected by the CM. The adhesion level was within the same range as that in the presence of glucose. The platelet thrombus volume (Fig. 2C) did not differ according to the presence of the various CM. The average thrombus volume was lower in blood mixed with glucose, although not significantly different from those in blood mixed with CM. Representative light-micrographs of tissue-factor-induced thrombi on semi-thin sections were prepared perpendicular to the direction of the blood flow (Fig. 3). Thrombi formed in the presence of the various CM showed no apparent differences in morphology or size (Fig. 3). However, the average thrombus size in the presence of glucose appeared lower than those in the presence of CM (Fig. 3D). The plasma levels of P-TG (Fig. 2D) reached much higher values in the presence of iohexol than in the presence of iodixanol, ioxaglate or glucose (p<o.ool). Discussion Thrombo-embolic events during and shortly after PTCA procedures are much feared. In this regard, the activation of platelets by angiography CM has been a central issue. We have addressed this issue in studies employing a human model of thrombus formation that was previously used to assess the relationship between platelet P-TG secretion and thrombus formation in human blood with or with- 358

6 IOHEXOL, PLATELET ACTIVATION AND THROMBOSIS ~ I1 Fig. 3. Representative light micrographs of tissue-factor-induced thrombi formed in the presence of CM, aspirin and heparin following 5-min perfusions at a wall shear rate of 2600 s-l. The semi-thin sections were prepared perpendicular to the direction of the blood flow. A) Thrombus formed in the presence of 40 vol% iohexol. B) Thrombus formed in the presence of 40 vol% ioxaglate. C) Thrombus formed in the presence of 40 vol% iodixanol. D) Thrombus formed in the presence of 40 vol% 300 mosmol glucose. No differences in thrombus morphology or size were observed in the presence of CM. However, inclusion of glucose reduced the thrombus volume, although not statistically significant. Magnification X 800. out the presence of various platelet antagonists (14-16). In these experiments, it was shown that a significant decrease in thrombus formation resulted from the oral administration of aspirin, clopidogrel or a reversible thromboxane A2 receptor antagonist and was reflected in a concomitant decrease in the plasma levels of P-TG. However, our recent experiments with CM, native blood and this perfusion chamber device demonstrated that this association was not always present since the profound iohexol-induced p-tg secretion was not associated with an enhanced thrombus formation (19). Furthermore, an increasing glucose osmolality which increased the p-tg secretion was paralleled by a decreasing thrombus formation (19). In this current study, we have extended our previous investigation on iohexol-induced P-TG secretion and thrombus formation (19). We employed the same ex vivo thrombosis model at the same high arterial shear, plus two vascular surfaces where thrombus formation is platelet-driven (collagen) or thrombin-driven (tissue factor), and blood that was anticoagulated with clinically relevant doses of aspirin and heparin. No evidence of enhanced thrombus formation elicited by iohexol was observed, despite the fact that iohexol triggered a pronounced secretion of P-TG. Average thrombus volumes in blood mixed with ioxaglate or iodixanol were no different from that elicited by iohexol, although the first two CM did not induce significant P-TG secretion. Thus, the P-TG secretion and the size of the platelet thrombi did not parallel each other as previously observed in a number of studies with platelet antagonists in the same model (14-16). However, the current data accord well with our recently reported findings on iohexol-induced P-TG secretion and a corresponding nonaffected collagen-induced thrombus formation in native blood (19). The iohexol-induced P- TG secretion is apparently not affected by clinical doses of aspirin and heparin. The collagen-induced thrombus formation did not differ according to the CM investigated. Thrombus formation at this wall shear rate of 2600 s-l is independent of coagulant activity (4, 12) and is thus entirely platelet-driven. In spite of the very high plasma levels of P-TG triggered by iohexol, this platelet-dependent thrombus formation was not enhanced. This indicates that iohexol is not a true platelet agonist, at least not in this arterial blood flow condition, since the platelet thrombus volume was not significantly affected. Furthermore, it is well known and generally accepted that iohexol is an antagonist of in vitro induced platelet aggregation (24). This assay is performed at relatively low shear conditions (shear rate of <300 s-'). However, the possibility remains open that iohexol renders platelets less responsive

7 K. S. SAKARIASSEN ET AL. to further agonist stimuli, as has been observed upon repeated stimuli by adenosine diphosphate or collagen (8, 17). Alternatively, the secretion of inhibitory platelet substance(s) (such as a plateletderived growth factor which inhibits thrombin-induced in vitro platelet aggregation) (26) may neutralize the apparent iohexol- or glucose-induced platelet activation. Thus, platelet alpha granules possess both prothrombotic and antithrombotic substances which are secreted upon platelet activation. The tissue-factor-dependent coagulation and fibrin deposition were not inhibited by the CM. This is contrary to in vitro coagulation assays in which ioxaglate appears as the strongest inhibitor of the three CM (2, 23, 24). It is possible that the presence of the antithrombotic drugs had already suppressed coagulation and fibrin deposition so that an additional inhibition by CM was not detected by the morphometric approach. Platelet thrombus formation did not differ in the presence of either of these CM, and can thus be seen as analogous to the apparent lack of CM effect on coagulation. It is noted that on this surface with its high level of tissue factor, platelet thrombus formation is entirely dependent on the deposited fibrin mesh (12), and thus on coagulation. Our present study shows that high shear platelet-driven or thrombin-driven platelet thrombus formation does not differ in the presence of iohex- 01, iodixanol or ioxaglate at clinical doses of aspirin and heparin. Thus the use of this model system, with two relevant thrombus-promoting vascular surfaces and with human blood containing a standard antithrombotic drug regimen as used in PTCA, shows that iohexol does not promote thrombus formation despite its apparent platelet activation. Studies are currently being planned on the potential effects of these CM on thrombus formation at a lower shear, which is also present in PTCA-treated vessels. REFERENCES 1. CHRONOS N. A. F., GOODALL A. H., WILSON D. J., SIGWART U. & BULLER N. l?: Profound platelet degranulation is an important side effect of some types of contrast media used in interventional cardiology. Circulation 88 (1993), ELOY R., COROT C. & BELLEVILLE J.: Contrast media for angiography. Physicochemical properties, pharmacokinetics and biocompatibility. Clin. Mat. 7 (1991), ESPLUGAS E., CEQUIER A,, FRANCISCO J. et al.: Risk of thrombosis during coronary angioplasty with low osmolality contrast media. Am. J. Cardiol. 68 (1991), FRESSINALJD E., SAKARIASSEN K. S., ROTHSCHILD C., BAUMGARTNER H. R. & MEYER D.: Shear rate dependent decrease of thrombus growth on collagen in non-anticoagulated blood from patients suffering from von Willebrand disease or hemophilia A. Blood 80 (1992), GASPERETTI C. M., FELDMAN M. D., BURWELL. R. et al.: Influence of contrast media on thrombus formation during coronary angioplasty. J. Am. Coll. Cardiol. 18 (1991), GERTZ E. W.: Thromboembolic events and non-ionic contrast. Diagn. Imaging 58 (1989), GRINES C. L., SCHREIBER T. L., 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. COIL Cardiol. 27 (1996), HALLAM T. J., RUGGLES P, A., SCRUTTON M. C. & WALLIS R. B.: Desensitisation in human and rabbit platelets. Thromb. Haemost. 47 (1982), JONSON L. W., KRONE R. & THE REGISTRY COMMITTEE OF THE SOCIETY FOR CARDIAC ANGI~GRAPHY AND INTER- VENTION (SCA & I): Cardiac catheterization. Cath. Cardiovasc. Diagn. 28 (1993), KAPLAN K. & OWEN J.: Plasma levels of thromboglobulin and platelet factor 4 as indices of platelet activation in viva Blood 57 (1981), LEVORSTAD K., VATNE K., BRODAHL U., AAKHUS T., B- MONSEN S. & VIK H.: Cerebral thromboembolic cornplications associated with the use of non-ionic contrast medium in coronary angiography. Acta Radiol. 36 (1995), RVIM U., BARSTAD R. M., VLASUK G.F! & SAKARIASSEN K. S.: Effect of selective factor Xa inhibition on arterial thrombus formation triggered by tissue factodfactor VIIa or collagen in an ex vivo model of shear-dependent human thrombogenesis. Arterioscler. Thromb. Vasc. Biol. 15 (1995), ORVIM U., ROALD H. E., STEPHENS R. W., RWs N. & SAKA- RIASSEN K. S.: Tissue factor-induced coagulation triggers platelet thrombus formation as efficiently as fibrillar collagen at arterial blood flow conditions. Arterioscler. Thromb. 14 (1994), ROALD H. E., BARSTAD R. M., ENGEN A,, KIERULF I?, SKJBRTEN E & SAKARIASSEN K. S.: HN a novel thromboxane A2 receptor antagonist with antithrombotic activity in humans at arterial blood flow conditions. Thromb. Haemost. 71 (1994), ROALD H. E., BARSTAD R. M., KIERULF? et al.: Clopidogrel - a platelet inhibitor which inhibits thrombogenesis in human non-anticoagulated blood independently of the blood flow conditions. Thromb. Haemost. 71 (1994), ROALD H. E., ORVIM U., BAKKEN I. J., BARSTAD R. M., KIERUL F! & SAKARIASSEN K. S.: Modulation of thrombotic responses in moderately stenosed arteries by cigarette smoking and aspirin ingestion. Arterioscler. Thromb. 14 (1994), ROSENBERG M. C. & HOLMSEN H.: Adenine nucleotide metabolism of gel filtered blood platelets. I\! Platelet aggregation responses to ATP and AD! Biochem. Biophys. Acta 157 (1968), ROTH G. J. & MAJERUS F! W.: The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein. J. Clin. Invest. 56 (1975), SAKARIASSEN K. S., BARSTAD R. M., HAMERS M. J. A. G. & STORMORKEN H.: Iohexol, platelet activation and thrombosis. I. Iohexol-induced platelet secretion does not affect thrombus formation in native blood. Acta Radiol. 39 (1998), SAKARIASSEN K. S., Joss R., MUGGLI R. et al.: Collagen type I11 induced ex vivo thrombogenesis in humans. Role of platelets and leukocytes in deposition of fibrin. Arteriosclerosis 19 (1990), SAKARIASSEN K. S., MUGGLI R. & BAUMGARTNER H. R.: Measurements of platelet interaction with components of the vessel wall in flowing blood. In: Methods in enzy- 360

8 IOHEXOL, PLATELET ACTIVA TION AND THROMBOSIS - I1 mology, vol. 169, p. 37. Edited by J. Hawiger. Academic Press Inc., New York SKALPE I. 0.: Complications in cerebral-angiography with iohexol (Omnipaque) and meglumine metrizoate (Isopaque cerebral). Neuroradiology 30 (1988), STORMORKEN H. & SAKARIASSEN K. S.: Contrast media effects on hemostatic and thrombotic parameters. Consequences for practial techniques and prophylactic measures. Acta Radio]. (1999, Suppl. 399, p STORMORKEN H., SKALPE I. 0. & TESTART M. C.: Effect of various contrast media on coagulation, fibrinolysis, and platelet function. An in vitro and in vivo study. Invest. Radio]. 21 (1986), TURITTO V. T. & BAUMGARTNER H. R.: Initial deposition of platelets and fibrin on vascular surfaces in flowing blood. In: Hemostasis and thrombosis. Basic principles and clinical practice, vol. 39, p Edited by R. W. Colman et al. J. B. Lippincott Co., Philadelphia VASSBOTN E S., HAVNEN 0. K., HELDIN C.-H. & HOLMSEN H.: Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha receptor. J. Biol. Chem. 269 (1994),