Venous thrombosis model

The compounds potently inhibit factor Xa in vitro with reversible binding kinetics and are able to inhibit not only free but also prothrombinase-bound factor Xa (Ki 41 nM, 0.1 I nM, and 0,5 nM, respectively) (58-60), In contrast, no direct effect on platelet aggregation has been described (60-62), Antithrombotic activity in arterial and venous thrombosis models has been demonstrated and it has a reduced effect on hemorrhage in comparison to standard therapy (58,60,63). Factor Xa inhibitors are able to reduce the endogenous thrombin potential in platelet-poor as well as in platelet-rich plasma (64,65). Thus, thrombin generation seems to be a suitable biomarker for clinical evaluation and has been evaluated in phase I studies (66,67). 

FIBROLASE, AN ACTIVE THROMBOLYTIC ENZYME IN ARTERIAL AND VENOUS THROMBOSIS MODEL SYSTEMS... 

The present report outlines studies with fibrolase in several different animal thrombosis model systems. Infusion of fibrolase proximal to an occlusive thrombus produces rapid and effective thrombolysis in carotid (22) and renal arterial, and iliac venous thrombosis model systems (23). Recently the recombinant form of fibrolase has been purified from a yeast expression system (24). It appears to be identical in all respects to the natural enzyme and has been used successfully in the carotid arterial thrombosis model system (22). Both natural and recombinant forms of the enzyme have effective thrombolytic activity in the different animal models employed there are no observable side effects nor toxicity, and minimal or no observable hemorrhaging. 

Figure 2. Thrombolytic effectiveness of natural fibrolase in a subacute iliac venous thrombosis model in the rabbit. Occluding spring coils were introduced retrograde from the jugular vein into both iliac veins. After 48 hours cutdowns on each iliac vein were performed below the coils and a catheter was introduced. Standard heparin infusion was initiated via the ear vein. A control angiogram was taken. Infusion of venom enzyme was then begun and repeat venograms were taken to document the extent of lysis. A, venogram at 0 time B, following 1 hour of fibrolase infusion C, following 2 hours of enzyme infusion.

Rabbit Subacute Iliac Venous Thrombosis Model... 

In the iliac venous thrombosis model system six animals were studied with good lysis observed venographically in five (23). Figure 2 presents representative venograms illustrating the effectiveness of thrombolysis induced by selective application of natural fibrolase. No toxicity, little or no hemorrhaging, and no evidence of other side effects were observed in the animals studied. These studies again demonstrate the favorable therapeutic potential of native fibrolase. 

The present results demonstrate that natural fibrolase exhibits effective in vivo thrombolytic activity in several animal thrombosis model systems. Infusion of the enzyme proximal to an occlusive thrombus induced rapid and specific thrombolysis in rabbit renal arterial and iliac venous thrombosis model systems. No evidence of hemorrhaging or alterations to the hemostatic system were observed in these studies. Additionally, no toxicity was observed and no angiographic, histologic, or functional evidence of side effects were obtained. The enzyme rapidly lysed 48 hr aged thrombi in the venous thrombosis model. This suggests that one of the primary mechanisms of thrombus resistance to PA-based agents. 

Thrombosis Models of arterial or venous thrombosis Shebuski et al.116... 

Table 1 Factor Xa Inhibitors (TAP, Antistasin) in Experimental Models of Thrombosis Rat and rabbit models of venous thrombosis [25]...

As low levels of protein C activation peptide are found in healthy individuals, it is suggested that protein C is constantly activated to a small degree (124). Protein C administration has been shown to inhibit both arterial and venous thrombosis in animal models (125). Heterozygous protein C deficiency or activated protein C resistance due to factor V mutation is thought to explain 60% to 70% of the cases of familial thrombophilia (I 16). 

Elevated TAFI levels have been found in men with symptomatic coronary artery disease (142). TAFI is also reported to be a risk factor for deep venous thrombosis, A recent report on the high levels of TAFI in the acute phase of ischemic stroke revealed not only elevated levels but also an incremental increase in TAFI with the degree of neurologic deterioration (143). Therefore, the observation by Boffa et al. on the acute phase nature of this protein requires further validation, In addition, Juhan-Vague et al, stated that there is a correlation between TAFI levels and cardiovascular risk factors (144). Animal models may be needed to truly validate studies on TAFI upregulation and its relation to thrombosis. 

R. A, G. Smith, R J. Dupe, P, D, English, and J. Green. Acyl-cnzymes as thrombolytic agents in a rabbit model of venous thrombosis. Thromb. Uaemost. 47 269 (1982). 

It was found that rivaroxaban competitively inhibits human FXa and prothrombinase activity (/C50 = 2.1 nM). It inhibits endogenous FXa more potently in human and rabbit plasma (/C50 = 21 nM) than in rat plasma (/C50 = 290 nM). Rivaroxaban has demonstrated anticoagulant effects in human plasma, doubling the prothrombin time (PT) and activated partial thromboplastin time (APTT) at 0.23 and 0.69 pM, respectively. In vivo, rivaroxaban reduced venous thrombosis dose dependency (EDS0 = 0.1 mg/kg i.v.) in a rat venous stasis model. The pharmacological actions of rivaroxaban have been described in more detail by Perzbom et al.12... 

Stenosis- and Mechanical Injury-Induced Arterial and Venous Thrombosis Harbauer-Model... 

Harbauer (1984) first described a venous model of thrombosis induced by mechanical injury and stenosis of the jugular vein. In a modification, both arterial and venous thrombosis is produced in rabbits by stenosis of the carotid artery and the jugular vein with simultaneous mechanical damage of the endothelium. This activates platelets and the coagulation system and leads to changes in the bloodstream pattern. As a consequence, occluding thrombi are formed as detected by blood flow measurement. The dominant role of platelets in this model is shown by the inhibitory effect of an antiplatelet serum in both types of vessels (Just 1986). The test is used to evaluate the antithrombotic capacity of compounds in an in vivo model of arterial and venous thrombosis where thrombus formation is highly dependent on platelet activation. 

Venous thrombosis is produced in rats by insertion of a stainless steel wire coil into the inferior caval vein. Platelets as well as plasmatic coagulation are activated on the wire coil. Thrombus formation onto the wire is quantitated by measuring the protein content of the thrombotic material isolated. The kinetics of thrombus formation show an increase in weight and protein content within the first 30 min followed by a steady state between thrombus formation and endogenous thrombolysis leading to a constant protein content of thrombi between 1 and up to 48 h following implantation of the wire coil. Thrombosis incidence in untreated control animals in this model is 100%. The test is used to evaluate antithrombotic and thrombolytic properties of compounds in an in vivo-model of venous thrombosis in rats. 

Kumada T, Ishihara M, Ogawa H, Abiko Y (1980) Experimental model of venous thrombosis in rats and effect of some agents. Thrombosis Res 18 189-203 Mellot MJ, Stranieri MT, Sitko GR et al. (1993) Enhancement of recombinant tissue plasminogen activator-induced reper-... 

Compared to the arterial system, it seems to be more difficult to develop a thrombosis model in venous... 

Hollenbach SJ, Wong AG, Ku P et al. (1995) Efficacy of FXa inhibitors in a rabbit model of venous thrombosis. Circulation 92 1486-1487... 

De ite differmces in their mechanisms of action and in vitro activities, pentasaccharide, DX-9065a and TAP have been shown to be effective antithrombotic agents in experimental models of venous thrombosis, coronary artery occlusion, arterial thrombolysis and acute reocclusion, restenosis after angioplasty, dialysis, and DIG. Pentasaccharide has also demonstrated measurable antithrombotic effects in human trials. Both TAP and DX-9065a produce measurable in vitro anticoagulant effects. In contrast, pentasaccharide does not produce an anticoagulant effect by the typical clot based assays. Thus, with fector Xa inhibitors there is not necessarily a correlation between current lab assays and antithrombotic efficacy as there is with heparin. 

The thrombolytic effect in vivo was studied in rabbits with a radioactive thrombus produced in the inferior vena cava. The radioactivity over the occluded venous segment was measured by a scintillation probe fitted with a collimator adapted to the anatomical conditions. Comparison of equivalent doses of urokinase and benzoyl-urokinase in the model of venous thrombosis showed that the acylated form of the enzyme was more active than the free enzyme [38). 

The history of utilization of the earthworm as a therapeutic drug source for various diseases extends back for several thousand years in China and other parts of the Far East. However, practical pharmacological studies have not been performed except on lumbrofebrin as an antifebrile. The lyophilized powder of the earthworm L. rubellus is administered orally to rats and dogs and its fibrinolytic and antithrombotic effects are investigated [1], Experiments in an arteriole-venous shunt model of rats show that the thrombus weight decreased significantly [74]. These results support that earthworm powder is valuable for prevention and/or treatment of thrombosis conditions. 

Compounds (27.4) and (27.5) were evaluated in an in vivo irterio-venous shunt thrombosis model in baboons, ind both compounds at 1 p-g min were found to be iffective in blocking platelet deposition, as monitored in eal time by accumulation of Indium oxine-labelled laboon platelets on a dacron graft. Encouraged by these esults, compounds (27.4) and (27.5) were further evaluated... 

Toomey J, Abboud M, Valocik R, Roster P, Bums-Kurtis C, Pillarisetti K, Danoff T, Erhardt J. A comparison of the (3-D-xyloside, odiparcil, to warfarin in a rat model of venous thrombosis. J. Thromb. Haemost. 2006 4 1989-1996. 

The present investigations, although limited to the local application of fibrolase, demonstrate that under these conditions the enzyme lyses venous or arterial thrombi rapidly and with no observable systemic or hematologic side effects. In these thrombosis model systems the enzyme, either alone or in combination with antiplatelet therapy, offers a unique, safe, and specific mechanism for clot dissolution and may prove useful as a clinically effective alternative to, or for use in synergistic combination with, presently used thrombolytic agents. 

Santos, J. C, Mesquita, J. M. F., Belmiro, C. L. R., Carolina da Silveira, . M., Viskov, C., Mourier, P. A., and Pavao, M. S. G. (2007). Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (Chordata-Tunicata). Distinct effects on venous and arterial models of thrombosis. Thromb. Res. 121,213-222. 

PURPOSE AND RATIONALE A classical method to produce thrombosis is based on the insertion of wire coils into the lumen of blood vessels. The model was first described by Stone and Lord (1951) in an aorta of a dog and was further modified to be used in arterial coronary vessels of opened-chest dogs. The use in venous vessels was described by Kumada et al. (1980). 

In order to compare the effects of 7E3F(ab )2 on arterial versus venous thrombolysis, a canine model was employed in which thrombosis was electrically induced simultaneously in the carotid artery and jugular vein [159], Whilst 7E3F(ab )2 (0.8mg/kg i.v.) prevented reocclusion in the carotid artery after thrombolysis, no effect was seen on rethrombosis in the jugular vein, reflecting the different thrombotic mechanisms in the arterial and venous circulation. 

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