Bleeding Time in Rats: A Comparison of Different Experimental Conditions

Four low-molecular-weight-heparin (LMWH) preparations have recently become available for clinical usage. Some ten preparations are currently under preclinical development. The safety/efficacy profile of each of these LMWHs is now known to be quite distinct from one and other. In order to minimize the variations in the pharmacologic responses and to cross-reference different products on a common scale, the WHO has recently introduced a LMWH standard. This standard is a nitrous-acid-depolymerized product provided by KabiVitrum (Stockholm, Sweden) with a specific physiochemical and biological profile. Although the in vitro characteristics of this standard are comparable to other LMWH, the in vivo behavior of this reference and the parent product, Fragmin®, are quite different in comparison to other products. In a well-defined uniform biochemical and pharmacologic screening, seven LMWH were compared at WHO reference-adjusted potency using pharmacologically valid dose-response curves. Cross-referencing of various LMWHs against the WHO standard in terms of anti-Xa activity resulted in a marked elevation of the anti-IIa component of some of the LMWHs. Establishment of this WHO standard stipulated that the clinical performance of cross-referenced LMWHs would be equivalent to one another and variation in potency could be minimized. Our data suggest that utilizing the WHO, standard individual potency (anti-Xa)-adjusted LMWHs exhibit distinct safety/efficacy profiles which were not related in their observed in vitro potency. Each LMWH has distinct multiparametric molecular and biochemical characteristics which determine the pharmacologic activities of individual agents. Thus cross-referencing of LMWHs by one in vitro assay is of questionable value as it may result in serious dosimetric errors, resulting in serious thrombotic and bleeding complications.

Section: Discussionmentioning

confidence: 99%

Validity of the Newly Established Low-Molecular-Weight Heparin Standard in Cross-Referencing Low-Molecular-Weight Heparins

Fareed

Walenga²,

Racanelli³

et al. 1988

“…Visible veins were avoided. The tail was then placed vertically in a 37° C saline with magnetic rotation and the visible bleeding measured for a maximum of 20 min [12], Simulta neously a 2-mm cut was made in the glandular part of the stomach mucosa with a pair of microscissors. The bleeding from the cut was monitored as described by Whittle et al [13] during perfusion of the chamber with 37°C saline at a rate of 2 ml/min.…”

Section: Experimental Modeimentioning

confidence: 99%

The Haemorrhagic Effect of Low Molecular Weight Heparins, Dermatan Sulphate and Hirudin

Matthíasson

Lindblad²,

Stjernquist³

et al. 1995

In a randomized, blind study the primary effect on haemostasis after intravenous administration of dermatan sulphate (DS), recombinant hirudin (r-hirudin) and four commercial low molecular weight heparins (LMWHs) (nadroparine, enoxaparin, dalteparin and tinzaparin) was investigated in rats and compared with saline (control). The tail bleeding time, the bleeding from the gastric mucosa [the mucosal bleeding time (min) and the mucosal bleeding (μ1)] as well as changes in activated partial thromboplastin time, antifactor IIa and Xa activities were investigated. DS and r-hirudin were investigated in a dose potentially suitable in thomboprophylaxis and the LMWHs in doses recommended by the manufacturers for thromboprophylaxis, adjusted to body weight. All substances significantly prolonged the mucosal bleeding time. Dalteparin, tinzaparin, DS and r-hirudin increased the mucosal bleeding when compared with controls whereas nadroparine and enoxaparin did not. The effect of r-hirudin was also significantly more pronounced compared with other treatments. Moreover, r-hirudin prolonged the tail bleeding time significantly whereas the other substances did not. The antifactor Xa activity in plasma correlated well with the given dose of the LMWHs (r_s = 0.7). However, the monitored bleeding parameters in the LMWH groups did not correlate with the plasma activities of antifactor IIa or Xa. The results indicate that the tested LMWHs are not equipotent in their effect on haemostasis in this model and that antifactor Ha or Xa activities do not directly correlate with their effect on haemostasis although increased haemorrhage was observed in the LMWHs with lower antifactor Xa/antifactor IIa ratios. Our findings indicate that the different commercial LMWHs may differ in their haemorrhagic effect when given in recommended clinical doses adjusted to body weight. DS (3.0 mg/kg) did not cause more bleeding than the tested LMWHs but r-hirudin (2.0 mg/kg) caused considerable bleeding.

“…An experimental model of IHT [16] was performed 10 min before laser-induced thrombosis. The tail of the rat was immersed in a bain-marie for 5 min at 37 ° C and sectioned 6 mm from the extremity.…”

Section: Ex Vivo Studymentioning

confidence: 99%

Effects of External Electrical Stimulation on Laser-Beam-Induced Experimental Thrombosis

Aguejouf¹,

Doutremepuich²,

Doutremepuich³

2006

External electrical stimulation (EES) has demonstrated venous antithrombotic properties. The aim of this investigation was to study its antithrombotic properties using unfractionated heparin (UFH) as a reference in arterial and venous mesenteric microvessels. For this purpose a rat model of laser-beam-induced thrombosis was used. Four groups of rats were investigated (n = 7): control, UFH (5 mg /kg), UFH (2.5 mg/kg) and electrical stimulation [e-vascular®, E = 10, alternate polarity, in two protocols: (i) 30 min before and during thrombosis induction and (ii) only 30 min before thrombosis induction as preventive stimulation]. This effect was tested in arterial and venous microvessels in the four groups using the continuous protocol and only in arterial microvessels using the preventive protocol. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli and the duration of embolization. Induced hemorrhagic time (IHT), platelet aggregation induced by ADP and coagulation tests (activated partial thromboplastin time, aPTT, prothrombin time, pTT, and fibrinogen level) were also determined. Continuous electrical stimulation decreased the number of emboli and the duration of embolization both in arterioles and in venules. It also significantly reduced the amplitude and velocity of the ex vivo platelet aggregation induced by ADP. Contrary to UFH, neither aPTT nor pTT were affected in platelet-poor plasma collected after the induction of thrombosis by laser beam, nor did it modify IHT. Preventive electrical stimulation produced similar results with fewer effects on induced venous thrombosis. These data suggest that EES has a potent antithrombotic effect on arterial and venous thrombosis without any hemorrhagic adverse effects, making it a very promising tool in the prevention and treatment of venous and arterial thromboembolic complications.