Experimental Thrombosis on a Collagen Coated Arterioarterial Shunt in Rats: A Pharmacological Model to Study Antithrombotic Agents inhibiting Thrombin Formation and Platelet Deposition

Abstract

SummaryA rat thrombosis model was developed to assess the efficacity of antithrombotic drugs. It had the following characteristics: controlled hemodynamic and rheological conditions corresponding to arterial flow, a collagen coated surface as a relevant thrombogenic stimulus, a method of measurement allowing dynamic monitoring of thrombus formation and the possibility to assess the thrombus structure. A shunt composed of polyethylene and silicone catheters, including in the middle of the shunt a collagen coated glass capillary, was inserted between the two primitive carotids of the rat. The duration of patency of the shunt was recorded using a thermic probe fixed on its central part. In this model, the patency of the shunt was 539 ± 55 s. Platelet and fibrinogen-fibrin accumulation in successive one centimeter segments along the shunt were measured using 111In labeled platelets and 125I labeled fibrinogen. Platelet accumulation occurred on the collagen coated surface and at the junctions between the different components of the shunt, where flow was disturbed. The effects of four antithrombotic agents were measured: aspirin, clopidogrel, heparin and r-hirudin. Clopidogrel, heparin and hirudin significantly prolonged patency duration of the shunt, whereas aspirin was inactive. Aspirin did not reduce platelet or fibrinogen-fibrin accumulation on the collagen coated surface. Platelet accumulation on the collagen surface was significantly lower in the clopidogrel group (50 mg/kg) than in the group treated with heparin (500 U/kg), demonstrating the direct antiplatelet effect of clopidogrel. Hirudin at doses giving similar values of APTT as heparin (500 U/kg) prolonged the occlusion time to over 2 h while the heparin occlusion time was only 20 min. We conclude that this experimental arterioarterial thrombosis model is sensitive to both antiplatelet and anticoagulant agents, allowing discrimination between antiplatelet and anticoagulant mechanisms of thrombosis prevention. The technique is easily performed in a small animal, fast and reproducible, and thus comparable to primate models for use in the screening of new antithrombotic drugs.

Anti‐thrombotic and haemorrhagic effects of active site‐inhibited factor VIIa in rats

“…After the bolus administration of 4 mg/kg, FFR-rFVIIa disappeared with a mean half-life of 86 min and the occlusion time returned to its basal value in less than 5 h. FFR-rFVIIa was devoid of any anti-thrombotic effect in the uncoated shunts whatever the dose delivered. Thus, the anti-thrombotic effect of FFR-rFVIIa has a completely different pattern to that of heparin or hirudin tested in this model (Freund et al, 1993;Leger et al, 1999). These anticoagulants prolong the occlusion time independent of the thrombogenic surface, while FFR-rFVIIa normalizes the TF-induced shortened occlusion time.…”

“…Description of the thrombosis model The thrombosis model was an arterioarterial shunt inserted between the two carotids of the animal. The shunt was constructed as described previously (Freund et al , 1993). The extremities inserted in the rat carotid arteries consisted of 5 cm‐long polyethylene catheters (Merck Clevenot, ref 3403, i.d.…”

“…Similarly, knowing the plasma fibrinogen concentration, the number of cpm emitted by the radiolabelled fibrinogen in the 100 μl blood sample and the capillary, the haematocrit and the volume of the capillary, the amount of fibrinogen–fibrin deposited was calculated. The methods used for platelet and fibrinogen labelling have been reported previously (Freund et al , 1993).…”

A revised model of platelet aggregation

“…Blood was collected from healthy donors or individuals with Type III von Willebrand's disease (vWD; < 1% plasma and platelet vWf) or Glanzmann's thrombasthenia (GT; < 1% α IIb β 3 ). Washed platelets were prepared from human donors (11) or Wistar rats as reported previously (12). Flow-based assays were performed according to a modified method of Cooke et al (13).…”

“…Preincubated platelets were injected in the jugular vein, and their interaction with pre-formed arterial and venous thrombi were viewed using fluorescence microscopy. In vivo rat platelet survival upon pretreatment with the RPM antibodies was examined as described previously (12,17). Shear rates in mesenteric vessels were determined by intravenous injection of fluorescent beads (0.25 µm diameter; Molecular Probes) through a jugular catheter (18).…”