1. Iodinated Contrast Media and Blood Hemostasis Jean-Marc Idée

2. What is Hemostasis ? Highly regulated process
Maintains blood in a fluid, clot-free state in normal vessels
Induces the formation of a localized hemostatic plug at the site of vascular injury

3. Potential Risks of Thrombosis During PCI
Coagulation
Fibrin Formation
Primary Hemostasis
Platelet function
Fibrinolysis
Clot Dissolution
Balloon/stent
Injured Vessel Wall
Endothelium

4. 1. Interaction with the Endothelium
East TennesseeState Univ.

5. A Specific Vasomotor Response to Nonionic Contrast Media
QCA of the left coronary artery in patients with (n = 38) or without (n = 42) CAD
* Visipaque and Ultravist:
- vasoconstriction at the level of the stenosis
- vasodilation of the healthy segments
* dilation: still significant at 60 sec; constriction: peak at 60 sec, return to baseline at 180 sec
* Hexabrix: no effect on epicardial coronary dimensions
Limbruno U et al. Circulation 2000; 101:

6. Potential Contrast Media Interactions
Effects
Platelet Activation
Prothrombin
Thrombin
Aggregation Xa
TF-Vlla
Xa-Va X
Fibrinogen
Thrombin
In summary, we have shown that contrast agents do indeed impact every aspect of thrombus formation. Research on the effect of contrast on the endothelium is just beginning and as of yet, the results are mixed dependent on the type of experiment performed and the contrast agent class. In comparison, a wealth of in vitro data exists showing that fibrin formation, platelet activation and aggregation are effected by contrast agents. The ionic contrast agents have a much higher propensity to prevent thrombus formation than do the nonionic monomeric and dimeric agents.
Endothelium
Tissue Factor
Tissue Factor
Fibrin
Fibrin Formation
Potential effects on abrupt closure, thrombotic complications, vasospasm

7. 2. Interactions of Contrast Media
with Platelets

9. smallest element of flowing blood
WHAT ARE PLATELETS ?
white, discoid
smallest element of flowing blood
1 - 2 microns diameter
normal range x 10 9 / Liter blood
No nucleus

10. Normal Function of Platelets
Haemostasis
Preventing bleeding from wounds
Integrity and repair of the vessel wall
As slide

11. Hemostatic Role of Platelets in Health: How Do They Work?
Platelets circulate in a resting, inactive state
Must become activated
Must stick together = Aggregation
Platelets circulate in a resting, inactive state
Platelets are very small and in order to be effective, have to stick together!
They need to become activated
Stimulus can be damaged vessel wall -next slide

12. Platelet Morphology * Microtubules * Energy-producing apparatus
- mitochondria
- glycogen particles (GL)
* Dense Bodies
accumulate
- -serotonin (5-HT)
- adenosine-5'-diphosphate (ADP)
* Lysosomes
contain glycoprotein GP 53 (CD63)
* Alpha Granules (GR)
acumulate
- fibrinogen
- platelet factor 4 (PF4)
- beta-thromboglobulin (-TG)
- platelet-derived growth factor (PDGF)
- P-selectin (GMP-140)
* Dense tubular system (OCS)
* GPIIb/IIIa glycoprotein
* etc.

13. Haemostatic plug Blood Vessel Endothelium Subendothelium INJURY
Collagen
Tissue Factor
VWF
Vaso-
constriction
Platelet Adhesion
& Secretion
Coagulation Cascade
Thrombin
Platelet aggregation
Fibrin
Haemostatic plug
Dr Isobel Ford

14. Hemostasis: The Three Phases of Platelets Involvement
1. Adhesion. Binding of platelets to areas of
subendothelial injury via interaction of
GPIb receptors to vWF multimers.
2. Activation. Platelet surface changes.
Degranulation and release of procoagulant and
vasoactive factors.
3. Aggregation. Cross-linking of platelets
through the activated integrin GPIIb-IIIa
and fibrinogen.

15. Resting and Activated Platelets

17. Factors that Activate Platelets
ADP
Thrombin
Collagen
5-hydroxytryptamine (serotonin)
Thromboxane A2
Mechanical stimuli
Many stimuli
Several different receptors
Multiple signalling pathways

18. PLATELET ACTIVATION RESTING ACTIVATED ACTIVATION        
50,000
> 500 
Fibrinogen 
 granules
P-selectin 
GPIIb-IIIa 
GPIIb-IIIa
P-selectin
GPIV
GPIb/IX/V  
GPIb/IX/V
GPIV 
ACTIVATION 
25,000
ACTIVATION : - GPIb IX V : internalized
- GPIIbIIIa : 1) membrane expression increased
2) complex occupied by fibrinogen, v. Willebrand Factor ...
- P-selectin : translocated to the membrane

19. Flow Cytometry to Measure Platelet Activation
Detector &
Mechanical
Computer System
Fluidics
Schematic of 3 principle components of flow cytometer ie computer, detectors and flow cell; plus a picture of the flow cytometer used in BTS

20. Detection of activated platelets in circulation
Flow Cytometry
Principle: Labelling platelets with fluorescently-conjugated antibodies
USES:
Detection of activated platelets in circulation
Effects of treatment on platelet activation
CAN MEASURE:
Changes in platelet surface receptors
Expression of markers of activation: Fibrinogen binding, fibrinogen receptor, P-selectin
Calcium ion flux
Procoagulant changes in membrane phospholipid by binding of Annexin V
Adhesion to other blood cells

21. Interaction of CM with Resting Platelets: Two Possible Cases
Do CM activate them ?
Do CM interact with platelet activation by factors such as thrombin, ADP, etc.. ?

22. 1993: A Scoop !

23. No Platelet Degranulation with Hexabrix Unlike Omnipaque
Same pattern of platelet activation in blood of patients
anticoagulated with heparin and pretreated with aspirin
N Chronos et al. Circulation 1993; 88:

24. Li and Gabriel Acad Radiol 1997; 4: 108
Platelet Activation by Thrombin Inhibited by Hexabrix but not by Nonionic Contrast Media
Thrombin Control
Omnipaque
Fluorescence Spectroscopy
Visipaque
Late mortality was 20.1% in patients with chronic renal failure and increased to 43.3% in patients with baseline renal failure and a 25% rise in creatinine after contrast exposure. In patients who required dialysis after coronary intervention, mortality at one year was as high as 56.5%.
This associations may not indicate causality. That is, the development of contrast nephropathy may not have caused the increased mortality. Rather, more critically ill patients may have had a higher incidence of contrast nephropathy
Hexabrix 20 40 60 80
100
% Thrombin-Activated Platelets
Li and Gabriel Acad Radiol 1997; 4: 108

25. Clinically Relevant ? Prospective, randomized trial.
Hexabrix vs. Ultravist (20 pts each). Cardiac catheterization.
Before
30 minutes p
Serotonin
(nM)
Hexabrix
0.03
Ultravist
TAT
(ng/ml)
Lower platelet activation (/serotonin release) and lower thrombin generation (TAT) with Hexabrix.
Jung et al. Pathophysiol Haemost Thromb 2002;32:

26. Fibrinogen binding to Glycoprotein IIb-IIIa on activated platelets
Platelet Aggregation
Fibrinogen binding to Glycoprotein IIb-IIIa on activated platelets

27. Synergy Between Hexabrix and Plavix in Antithrombotic Effect in Rats
Carotid artery
Ultrasonic flow probe
Deposit of a filter paper soaked in FeCl3 applied to the ventral surface of carotid artery, distal to an ultrasonic flow probe
Labarthe B et al. Invest Radiol 2003; 38: 34-43

28. Synergy Between Hexabrix and Plavix in Antithrombotic Effect in Rats
Labarthe B et al. Investigative Radiology : 34-43

29. Unlike Visipaque, Hexabrix Boosts the Effects of ReoPro on Platelet-Dependent Thrombin Generation (Thrombogram)
"ioxaglate strongly boosts the effect of the GPIIb IIIa inhibitor (abciximab) whereas iodixanol does not."
R. Al Dieri et al. Journal of Thrombosis and Haemostasis 2003, 1:269:274

30. Isovue and Visipaque Potentiate ADP-induced Platelet Aggregation in Hirudinized Blood
Iodixanol
Iopamidol
control
Ioxaglate
time (min)
Heptinstall S et al. Brit J Haematol 1998; 103:

31. Systemic Anti-Platelet action of CM
N = 30 pts, diagnostic coronary angiography, randomized. ASA (75 mg) for all pts.
Arterial blood sampling before and after the last angiogram
Dalby MCD et al. Am Heart J 2002

32. What About Visipaque ?
Jones & Goodall, Thrombosis Research 2003; 112: 65-71

33. 3. Interactions of Contrast Media with the Blood Coagulation Cascade

34. 5. SEM of a clot, higher magnification
The clot is a jelly-like mass of fibrin and red blood cells, which is constantly being broken down and rebuilt. The longevity of the clot depends upon the balance between clot formation and fibrinolysis.

35. - - - Coagulation Cascade All Contrast Media Hexabrix
Al Dieri R, Béguin S, Hemker HC, J.Thromb. Haemost. 2003;1:

36. The First Law of Thrombosis
The more thrombin, the more thrombosis
The less thrombin, the less thrombosis
Prof. HC Hemker

37. Hexabrix Anticoagulant Effects
Hexabrix strongly inhibits thrombin generation 10 20 30 40 50 60 %
Saline
iodixanol
iopamidol
iomeprol
iopiperidol
diatrizoate
ioxaglate
Inhibition of thrombin generation by contrast agents. Error bars represent 1 standard deviation.
Valenti R et al. Acad Radiol 1999;6:

38. Binding site for Hexabrix on Human Thrombinl R2 R1 R3 R4
COOH
thrombin
Hexabrix
Anion-binding exosite II
Anion-binding exosite I
Catalytic site
Li and Gabriel Acad Radiol 1997; 4: 108
Al Dieri et al. J Thromb Haemosatsis 2003; 1:
Goodall

39. 4. Interaction of CM with Fibrinolysis
Cedenho FB et al. 1999

40. Interaction with Fibrinolysis
Changes in fibrin structure during
coronary angiography with the non-ionic agent Omnipaque
Thinner fibers formed in the presence of Omnipaque: more resistant to fibrinolysis
Melton LG et al Am J Cardiol 1998; 82:

41. Interaction with Fibrinolysis
Contrast media have profound effects on
fibrin assembly and clot structure.
Lysis Time
Normal Clot
1,000 sec
Clot with Iothalamate (ionic)
1,500 sec
Clot with Iohexol (nonionic)
72 hours
Late mortality was 20.1% in patients with chronic renal failure and increased to 43.3% in patients with baseline renal failure and a 25% rise in creatinine after contrast exposure. In patients who required dialysis after coronary intervention, mortality at one year was as high as 56.5%.
This associations may not indicate causality. That is, the development of contrast nephropathy may not have caused the increased mortality. Rather, more critically ill patients may have had a higher incidence of contrast nephropathy
In vitro, Iohexol produced very thin fibers that
were almost completely resistant to degradation.
Carr Haemostasis 1995; 25;172

42. Interaction with Fibrinolysis
Onset of Lysis with t-PA (in vitro samples)
600
Angiografin*
500
Omnipaque*
400
Time (sec)
300
*p<0.001
vs control
200
Late mortality was 20.1% in patients with chronic renal failure and increased to 43.3% in patients with baseline renal failure and a 25% rise in creatinine after contrast exposure. In patients who required dialysis after coronary intervention, mortality at one year was as high as 56.5%.
This associations may not indicate causality. That is, the development of contrast nephropathy may not have caused the increased mortality. Rather, more critically ill patients may have had a higher incidence of contrast nephropathy
Hexabrix
Control
100
In vivo samples, also show delays in
lysis by t-PA after angiography (Iohexol).
Dehmer JACC 95; 25:1069

43. No thrombi formed from blood incubated with Hexabrix (20 or 50% vol/vol)
Thrombi formed with Visipaque or Omnipaque weighed > x 10 times more than saline controls and were more resistant to fibrinolysis

44. Antithrombotic Effects of Contrast Media in Baboons
All CM: in situ infusion, 0.1 and 0.3 ml/min
Markou CP et al.Thromb Haemost 2001; 85:

45. Antithrombotic Effects of Contrast Media in Baboons
Markou CP et al. Thromb Haemost 2001; 85:

46. Antithrombotic Effects of Contrast Media in Baboons
Markou CP et al. Thromb Haemost 2001; 85:

47. Saline Iodixanol Ioxaglate Iohexol
Markou CP et al. Thromb Haemost 2001; 85:

48. Idée JM, Corot C, Fundam Clin Pharmacol 1999; 13: 613-623
To Summarize…
Idée JM, Corot C, Fundam Clin Pharmacol 1999; 13:

49. Take-Home Messages
All iodinated CM are anticoagulant, but ionic > nonionic.
Nonionic monomers activate resting platelets. No effects with Hexabrix.
Hexabrix inhibits platelet activation by endogenous factors. Visipaque and nonionic monomers have no inhibitory effect.
Hexabrix inhibits platelet aggregation, unlike nonionic agents.
Clinical data to support the in vitro antiplatelet potential of Hexabrix.