Differential effects of the iodinated contrast agents Ioxaglate, Iohexol and Iodixanol on thrombus formation and fibrinolysis

Abstract

Introduction: Contrast media (CM) possess both pro-thrombotic and anticoagulant properties. Here we investigate the effect of three classes of CM; Iohexol, Iodixanol and Ioxaglate, on thrombus formation and fibrinolysis in vitro and evaluated the contribution of platelets to this process. Materials and methods: Non-anticoagulated blood was mixed with CM or saline (50% or 20% (v/v)) for 1 min then citrated. Thrombi were produced in vitro under flow in a Chandler loop. Thrombus structure was visualized by immunohistochemistry using FITC-fibrin/ogen antibodies, and propidium iodide to identify nucleated blood cells. To measure fibrino/thrombolysis release of FITC-labeled fibrinogen, added prior to thrombus formation, was measured over 24 h. Platelet degranulation was analyzed by whole blood flow-cytometry. Results: No thrombi formed from blood incubated with Ioxaglate. Thrombi formed with Iohexol or Iodixanol weighed ≥10× more than saline controls (116±52 and 230±128 mg vs. 11±3 mg, respectively; p<0.0005), and were more resistant to thrombolysis as evidenced by the release of FITC over 24 h (19.1±8.9 and 31.9±17.2 U vs. 65.1±19.1 U, respectively; p<0.02). Thrombi formed with Iodixanol and Iohexol had larger, more diffuse platelet-rich head areas and tail regions composed of a more open fibrinogen/fibrin meshwork enclosing denser RBC rich areas compared to controls. Iodixanol and Ioxaglate did not increase platelet degranulation, but Iohexol caused a significant increase compared to the saline control (percentage of platelets expressing P-selectin 68.9±23.43% vs. 4.93±1.44%, respectively; p=0.001). Conclusion: Thus thrombi formed with Iodixanol or Iohexol will be larger and more resistant to thrombolysis, whereas Ioxaglate may reduce the risk of thrombus formation.

Introduction

Since their introduction, iodinated, angiographic contrast agents have been widely used in interventional procedures for coronary artery disease. The early, high osmolar agents have, since the late 1970s, been largely superseded by low/iso osmolar contrast media (CM). There are currently four general classes of contrast agents, which can be ionic or non-ionic, and either monomeric or dimeric.

Low osmolar CM are generally found to be anticoagulant in vitro, with ionic CM having a greater anticoagulant effect than the non-ionic agents [1], [2], [3], [4], [5], [6]. Despite these anticoagulant effects in vitro, a body of evidence has accumulated that suggests that non-ionic CM are associated with increased thrombus formation in animal models [7], [8], [9], [10], [11], and thrombi have been reported in syringes containing non-ionic CM contaminated with blood [12]. This does not appear to be the case with the dimeric ionic CM, Ioxaglate, which has been shown to reduce thrombus occlusion in a canine arterial balloon injury model [8], reduce time to formation of occlusive thrombi at clinically relevant concentrations in a rat carotid artery injury model [13], reduce thrombus formation on catheters [14] and to inhibit platelet and fibrin deposition on stents in a baboon femoral shunt model [15]. Similarly, ionic CM inhibited platelet adhesion and aggregation more than non-ionic CM in a parallel plate flow model [16] and Ioxaglate has been shown to reduce platelet aggregation in response to collagen [17].

Part of the effects of the different CM on haemostasis may stem from their differential effects on platelets. Early reports suggested CM inhibited platelet aggregation [18]. However, more recently it has been recognized that low osmolar CM cause platelet degranulation, as evidenced by expression of α-granule membrane P-selectin (CD62P) and lysosomal membrane antigen CD63 (GP53), and by the release of platelet α-granule (βTg, PF4) and dense granule (5-HT) contents [19], [20], [21]. This effect does not equate to platelet activation as it is seen without the activation of platelet GPIIbIIIa [19]. Comparison of the different types of CM has demonstrated that degranulation is greater with non-ionic than with ionic CM, whilst the ionic dimer, Ioxaglate, causes little or no platelet degranulation or activation [19], [20], [21]. Ioxaglate also has a marked inhibitory effect on thrombin-induced platelet activation [19], [22], which appears to be mediated though a direct interaction with thombin [22] and is not seen when platelets are stimulated directly through the PAR-1 thrombin receptor [18], [21]. This is supported by recent studies which have demonstrated that Ioxaglate inhibits thrombin generation in platelet poor and platelet rich plasma through interference of the activation of FV and FVIII by thrombin [4], [23].

In addition, CM have an effect on fibrinolysis/thrombolysis. This may, in part, be a consequence of enhanced platelet degranulation, since platelet α-granules contain large amounts of PAI-1, FXIII, and TAFI which become incorporated into the growing thrombus, and are responsible for resistance to fibrinolytic agents. As well as this CM have a negative effect on fibrinolysis, by giving rise, to thinner fibrin filaments, that are more resistant to fibrinolysis/thrombolysis [24], [25], [26], [27]. This has been reported both in vitro and in vivo [24], [25], [26], [27], [28], [29], and in those studies where comparisons have been made between contrast agents, the ionic dimer Ioxaglate has less antifibrinolytic effect than non-ionic CM [29].

The literature on the effects of CM is contradictory and difficult to interpret due to the array of different CM used and because components of the haemostatic system have been investigated individually and out of context of changes in the rest of the system. Therefore there is a need for a more encompassing approach to understand the clinically relevant effect of CM on thrombus formation and fibrinolysis. Here we utilized a model to create whole blood thrombi in vitro under conditions of flow (Chandler loop [30]). Thrombi formed in this way have been shown to closely resemble arterial thrombi with a distinct platelet rich head and fibrin tail [31]. Adding CM to the blood prior to thrombus formation gives a physiologically relevant model to evaluate the effects of CM on thrombus formation and fibrinolysis.

In this study, we set out to compare the effect of three different classes of contrast agents non-ionic monomer Iohexol (Omnipaque), non-ionic dimer Iodixanol (Visipaque), and ionic dimer Ioxaglate (Hexabrix) on thrombus formation, fibrinolysis and tPA mediated thrombolysis, and to evaluate the role of platelets in this process.