Christian Doutremepuich

Heparin and a low molecular weight fraction enhances thrombolysis and by this pathway exercises a protective effect against thrombosis

In human volunteers unfractionated heparin and a low molecular weight fraction of heparin (LMWH) caused an increase in plasma plasminogen activator (PA) which peaked at 3 hours after subcutaneous injection. Using a perfused isolated rabbit ear model the enhancement of PA activity was confirmed and was related to the anti-Xa activity of both products infused. Using a modified rabbit Wessler model for thrombus formation it was found that, when using doses of heparin and LMWH sufficient to give a 100% antithrombotic effect, antifibrinolytic drugs (eg. epsilon-ACA and aprotinin), negated this protective effect. It is concluded that the effect of heparin and LMWH on haemostasis is mediated in part through the enhancement which these drugs have on fibrinolysis, the latter being arguably a major defence against fibrin formation during thrombosis.

Experimental thrombosis model induced by free radicals. Application to aspirin and other different substances

A large number of experimental studies suggests that oxygen free radicals play a major role in the pathogenesis of the myocardial lesions observed during the sequence ischemia-reperfusion. The purpose of this study was to determine whether oxygen free radicals can induce thrombosis. In so doing we have developed a new experimental thrombosis model. Reproducible focal thrombosis has been achieved by irradiating mesenteric arterioles of rat for variable time with green filtered light issuing from a mercury lamp after systemic injection of different rose bengal doses. The number of emboli that remove in the blood (N), the duration of total occlusion (T) and the number of emboli per minute were then measured. As control, no rose bengal administration was done and the vessels were exposed to the filtered light. In comparison with this control, results clearly showed that free radicals always induced thrombosis and the induced thrombus was mainly composed of platelets. In this new thrombosis model induced by free radicals antithrombotic drugs (aspirin, 200 mg/Kg, heparin, 2 mg/Kg) and antioxidants (vitamin C, 10 and 20 mg/Kg, allopurinol, 200 and 300 mg/Kg, vitamin E, 500 and 1000 mg/Kg) have been tested. Results have shown that only heparin and vitamin E had an antithrombotic effect on thrombus formation induced by free radicals. This model should be useful in studying the effects of different drugs and could lead to new treatment modalities for ischemic accident and other cardiovascular diseases.

Thromboembolic Complications Several Days After a Single-Dose Administration of Aspirin

The antithrombotic properties of acetyl salicylic acid (ASA) used at current doses are largely demonstrated. However, our previous study showed unexpected thrombotic potencies associated with the use of this drug. In this study we investigate the effect of aspirin on an experimental thrombosis induced by laser beams, according to its in vivo plasma concentration. Experiments were done on nine groups of seven Wistar male rats. The groups are defined by the delay between aspirin administration time and the laser-induced thrombosis time. Results from this study showed an enhancement of thromboembolic complications when thrombosis was induced 8 or 10 days after aspirin administration; the number of emboli and the duration of embolization are increased, compared to the control group. The prothrombotic properties of ASA demonstrated in this study, might limit its therapeutic benefit and might explain thromboembolic complications observed in some ASA-treated patients. These results also suggest a biological monitoring several days after aspirin administration to patients.

Thrombogenic properties of ultra-low-dose of acetylsalicylic acid in a vessel model of laser-induced thrombus formation

Various studies have demonstrated the antithrombotic effect of high dose acetylsalicylic acid (ASA) and more reports show the similar efficiency of the much better tolerated low dose ASA. Recently, in vitro studies hinted the potent thrombotic properties of ultra-low-dose ASA (ULD-ASA, < 1 mg/day) showing a significant decrease in bleeding time( 1,2). This effect was, at least in part, related to the inhibition of endothelial cyclooxygenase -and not to platelet oneleading to a decrease of prostacyclm release (3). To investigate the effect of ASA in vivo, we used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of the microvasculature. Thrombus formation was induced by Argon-laser shot. The instrumental test set-up was completed with a video-system. The kinetic feature of thrombus formation was estimated in terms of number of laser shots required to induce endothelium injury leading to thrombosis, number of emboli removed by blood stream and duration of embolization. The reliability and reproductibility of our laser-induced thrombus model have been previously tested in rat mesenteric arterioles and veinules and we showed that ASA used at current doses (50-200 mgkg) decreased the number of emboli and the duration of embolization (4). The aim of this preliminary study was the investigation of the effects of ULD-ASA in vivo. In so doing, we evaluated ULD-ASA in our experimental laser-induced thrombus model. This might represent a further step in the understanding of the action of ASA according to the range of concentration used.

Reverse Effect of Aspirin: Is the Prothrombotic Effect after Aspirin Discontinuation Mediated by Cyclooxygenase 2 Inhibition?

Background: While aspirin is the drug most often used to prevent cardiovascular complications, its discontinuation induces an increased risk of acute coronary syndrome and ischemic stroke in some patients. Objectives: We hypothesized that infinitesimal concentrations of aspirin could persist in plasma after its discontinuation, thereby inducing a prothrombotic effect that could be due to a modification in the mechanism of action of aspirin via the cyclooxygenase 1 (COX-1) and COX-2 pathways. Methods and Results: We studied the effects of ultra-low-dose aspirin (ULDA) as well as those of sc-560 and ns-398, specific COX-1 and COX-2 inhibitors, on induced hemorrhagic time and in a model of laser-induced thrombosis in rats. In the laser-induced thrombosis model, ULDA treatment increased the number of emboli and the duration of embolization, thereby confirming its prothrombotic effect described in previous publications. This effect was also observed in rats pretreated with sc-560 but not in those pretreated with ns-398. Conclusions: We demonstrated that ULDA induced a prothrombotic effect in the rats studied. This strongly suggests that a very small amount of aspirin could remain in the patient’s blood after aspirin therapy, leading to cardiovascular complications. This effect may be mediated by the COX-2 pathway.

Combination of two doses of acetyl salicylic acid: experimental study of arterial thrombosis.

The antithrombotic effect of high dose acetylsalicylic acid is well known, and recently, in vitro studies hinted the potent thrombotic effect of ultra-low dose of acetylsalicylic acid (<1mg/day) showing a significant decrease in bleeding time. In this study, we investigated the effect of a combination between a high and an ultra-low dosage (100 mg/kg+ 10(-30) mg/kg) on an arterial thrombosis induced by a laser beam. We used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of microvasculature. Thrombus formation was induced by argon-laser shot. The instrumental test setup was completed with a video system, to select mesenteric arterioles with the same diameter (between 15 and 25 microm). The changes in platelet aggregability were determined by Cardinal and Flower method, and the concentration of acetylsalicylic acid in the plasma was measured by high pressure liquid chromatography. Antithrombotic effect of high dose (100 mg/kg) acetylsalicylic acid was confirmed in all results obtained. Asa injected at ultra-low dose (10(-30) mg/kg) had a potent thrombotic properties and decreased significantly the bleeding time. The subcutaneous administration of the combination of the two doses permitted to come back to the control values, and the bleeding time was shortened compared to control group.

Time Related Neutralization of Two Doses Acetyl Salicylic Acid

Aspirin has a well established role in the prevention of arterial thrombosis. Discussion on the efficacy and safety of aspirin in the treatment and prophylaxis of thrombosis has become an important issue. In fact, hemorrhage complications are often associated with its use. On the other hand, previous studies showed unexpected thrombotic potencies associated with the presence of this drug at ultra low doses (ULD) in the circulation. In this study, we aimed to evaluate the effect of aspirin at ULD, injected 1, 2, or 3 hours after the administration of aspirin at 100 mg/kg, on hemostasis and bleeding in rats. We used an experimental model of thrombosis induced by laser beams to evaluate these effects. Platelet aggregation was determined by Cardinal and Flower method. Results from this investigation demonstrate that the neutralizing effect of aspirin at ULD did not operate significantly 1 hour after the injection of aspirin at 100 mg/kg. This effect was observed 2 and 3 hours after. The use of aspirin at ULD to neutralize the side effects of aspirin at high doses will reduce the hemorrhagic risk during extra corporeal circulation. The therapeutic benefit and safety of aspirin therapy in the treatment of cardiovascular diseases can be obtained.

IMPORTANCE OF PHOTO ACTIVATION OF ROSE BENGAL FOR PLATELET ACTIVATION IN EXPERIMENTAL MODELS OF PHOTOCHEMICALLY INDUCED THROMBOSIS

Generation of oxygen free radicals in the lumen of a vessel leads to NO inactivation, modification of lipid components of platelets and endothelial cell membranes, and platelet activation. On this basis, many experimental models of thrombosis have been developed, where the formation of a platelet rich thrombus follows the illumination of a vessel with an appropriately filtered light after intravenous administration of Rose Bengal or another sensitizing dye. However the detailed mechanism of thrombus formation remains poorly known. This work appreciates the contribution of platelet activation directly induced by oxygen free radicals in formation of the platelet rich thrombus, from a study of human platelet aggregation in presence of photo-activated Rose Bengal. The results demonstrate that direct activation of platelets by free radicals generated by Rose Bengal is of low or no importance in formation of the thrombus. Therefore, the main trigger of platelet aggregation and thrombosis in these models is primary endothelial cells injury.

Prothrombotic and Hemorrhagic Effects of Aspirin

Aspirin remains the most widely used drug for prevention of vascular events. Recent observational epidemiological evidence has raised the concern that aspirin withdrawal for treatment noncompliance, surgery, or side effects can carry an increased thrombotic risk. The delay to the thrombotic event was between 7 to 30 days in most reports and most frequently 7 to 10 days. The mechanism underlying this effect remains poorly understood. Using an in vivo model of laser-induced thrombosis, aspirin injected in 1 single dose of 100 mg/kg body weight has also shown a prothrombotic activity in the rat 8 to 10 days after injection in the normal rat. The hypothesis was made that minimal concentrations of aspirin or ultra-low dose aspirin (ULDA) could induce this effect. ULDA showed prothrombotic properties in the same model of induced thrombosis that were very similar to those described after aspirin withdrawal, but the effect was observed only 1 hour after aspirin administration. This prothrombotic effect of ULDA is very similar to the effect observed after COX 2 selective inhibition with NS 398. The administration of both the selective COX 2 inhibitor and ULDA did not produce further changes. In conclusion, the prothrombotic effects described in recent observational studies are likely produced by a direct effect of aspirin, whose putative mechanism involving COX 2 inhibition remains poorly understood.

Platelet Aggregation in Portal Hypertension and Its Modification by Ultra-Low Doses of Aspirin

Aspirin (ASA) is widely accepted as antithrombotic drug, but several reports point out that its use in ultra-low doses (ULD) has prothrombotic properties. In this study, we evaluate the effect of portal hypertension in rats on platelet aggregation in an in vivo arterial thrombosis model induced by a laser beam. Portal hypertension was produced by calibrated stenosis of the portal vein. ASA in ULD was injected to both control and portal hypertensive groups. Platelet aggregation induced by ADP, prothrombin time, activated partial thromboplastin time, fibrinogen and induced hemorrhagic time test were also performed. Portal hypertensive rats showed a diminished number of emboli and duration of embolization in the laser procedure and an increase in induced hemorrhagic time. These changes were reverted by one injection of ASA at ULD. This observation could be of importance for primary prevention or the treatment of recurrence in upper digestive tract hemorrhage in portal hypertensive patients.