Evaluation of thrombin-antithrombin complexes and fibrin fragment D in carbohydrate-induced acute laminitis.

Carbohydrate-induced laminitis has been associated with decreased platelet survival, decreased blood flow to the hoof wall and with the deposition of platelets and microthrombi within venules in the dermal laminae. To evaluate further the systemic prothrombotic events occurring in the prodromal stages of laminitis, plasma samples from control and laminitis-affected ponies and horses were tested for the presence of thrombin-antithrombin (TAT) complexes and fibrin fragment D (D-dimer). No statistically significant differences between the control and laminitis-affected animals were observed for either the D-dimer or the TAT complexes. Few of the values for individual animals exceeded the reference ranges for control animals. These data indicate that the prothrombotic events observed in carbohydrate-induced laminitis may not be associated with systemic activation of the coagulation or fibrinolytic systems.

Comparison of the effects of low-molecular-weight and unfractioned heparin in horses.

Thirty healthy male horses were allotted to 3 groups and treated blindly during 4 days. Group-1 horses received unfractioned calcium heparin (100 IU/kg of body weight, SC, q 12 h). Group-2 horses received a single dose of a low-molecular-weight heparin (50 anti-Xa IU/kg, SC) every morning, and a similar volume of saline solution every evening. Group-3 horses received the vehicle (saline solution), SC, every 12 hours. Citrated and EDTA-anticoagulated blood samples were collected before starting the medication (T-0) and once daily 3 hours after each morning injection (T-3, T-27, T-51, and T-75). The PCV, hemoglobin concentration, RBC and platelet counts, and clotting times (activated partial thromboplastin time and thrombin time) were determined, and a microscopic examination to detect hemagglutination was performed. Plasma concentration of heparin was measured by use of the antifactor Xa, activity assay. Bleeding time was determined on the first and fourth days, using a double-template method. The horses given unfractioned heparin had marked agglutination of erythrocytes after the first injection that became more pronounced as treatment progressed. Also, significant decrease in PCV, hemoglobin concentration, and RBC count was observed during treatment. Platelet count was significantly decreased after the first day, and clotting times were significantly prolonged. In contrast to the horses given unfractioned heparin, those given low-molecular-weight heparin did not have any agglutination of erythrocytes during the 4 days of treatment, and there were no significant changes in PCV, hemoglobin concentration, or RBC and platelet counts. Activated partial thromboplastin time increased slightly in the horses given low-molecular weight heparin, although the values remained within reference range. Both groups of horses achieved adequate concentrations of heparin in plasma for prophylactic purposes, but those given low-molecular-weight heparin achieved those values after the first injection. Bleeding times were not significantly different between heparin-treated horses and horses given saline solution during treatment. We conclude that low-molecular-weight heparin may be used more safely and conveniently in horses, because it does not affect equine erythrocytes, platelets, or clotting and bleeding times.

Effects of two different doses of hirudin on APTT, determined with eight different reagents.

The APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study. Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica-based reagents. In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica-containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

Effects of recombinant hirudin on D dimer levels before and after experimental venous thrombosis.

In a randomized, blind study, the effect of saline, a low-molecular-weight heparin (Fragmin), and a recombinant hirudin (CGP 39393) on thrombin-antithrombin (TAT) complexes and D dimer (DD) levels were studied in 60 rabbits. The drugs were always injected subcutaneously 2 h before the induction of thrombosis in the jugular vein by a combination of endothelial damage and flow reduction. A sample of blood was obtained just before surgery, and a further sample was obtained 10 min after thrombus formation. No significant differences were found in TAT plasma levels between the different study agents, either before or after thrombus development. However, 2 h after drug administration DD values were significantly lower in hirudin-treated animals (292 +/- 69 vs. 372 +/- 138 ng/ml; p < 0.05). Then, after thrombus formation, DD levels significantly increased in all three groups of animals, as compared to baseline levels. But the increase was significantly higher in hirudin-treated rabbits; DD levels after thrombus development were significantly higher in this group as compared to placebo (779 +/- 188 vs. 664 +/- 152 ng/ml; p < 0.05). There are no previous reports in the literature about the effect of hirudin on DD levels. Our hypothesis is that the effect of hirudin on DD may be the result of its ability to inhibit the thrombin-induced thrombus growth. If the thrombus is not allowed to grow then it will lyse more quickly, producing more DD and the process will not be impaired by the constant deposition of fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

[Normal values for hematological and hemostatic parameters in the rabbit. Determination of new parameters for experimental models of thrombosis and hemostasis]. / Valores normales de los parámetros hematológicos y hemostáticos en el conejo. Determinación de nuevos parámetros para modelos experimentales de trombosis y hemostasia.

PURPOSES: Assessment of normal haematological values in the rabbit using a semi-automatic analyser. Evaluation of new haemostatic parameters necessary for experimental models in thrombosis and haemostasis. MATERIAL AND METHODS: 20 New Zealand male rabbits of 2-2.5 kg were anaesthetized and blood was collected from the jugular vein. The following haematological determinations were carried out using a semi-automatic analyser: red blood cells count, haemoglobin, haematocrit, indexes, white blood cells count, platelet count and mean platelet volume. The parameters used to assess the physiological haemostasis in rabbit were: bleeding time, fibrinogen, prothrombin time, activated partial thromboplastin time, thrombin time; and others not yet determined in the rabbit: antithrombin III, thrombin-antithrombin complexes and F1+2 fragments to measure the basal thrombin-formation activity, and fibrin D-dimer and fibrinogen degradation products for fibrinolysis activity. RESULTS: The haematological results obtained were similar than with other techniques, but with no stress influence. Normal values for haemostatic parameters studied are shown, including the parameters for the determination of physiological thrombin-formation and fibrinolysis activity. F1+2 fragments and fibrinogen degradation products could not be evaluated. CONCLUSIONS: The haematological results obtained were completely normal. All the haemostatic parameters studied were found useful to determine the physiological activity of coagulation and fibrinolysis in the rabbit, and specially for experimental models of hypercoagulability and hyperfibrinolisys states.

Comparative study on the antithrombotic efficacy of hirudin, heparin and a low-molecular weight heparin in preventing experimentally induced venous thrombosis.

In a random, blind study, we compared the antithrombotic potential of unfractioned heparin, a low-molecular weight heparin (LMWH) and a recombinant hirudin (r-hirudin) given subcutaneously in a jugular vein thrombosis model in rabbits. All drugs and placebo were injected subcutaneously 2 h before inducing experimental thrombosis. A good thromboprophylactic effect was obtained with either LMWH and r-hirudin as compared with placebo. By contrast, no significant differences were found between groups in bleeding time. We also found a small (non-significant) prolongation of the activated partial thromboplastin time in the r-hirudin-treated animals. As for thrombin time, r-hirudin-treated rabbits exhibited too much sensitivity, with noncoagulable end-points. In our study, r-hirudin at the dose used seemed to be as effective as LMWH is in the prophylaxis of venous thrombosis.

Comparative study on the antithrombotic efficacy of four low-molecular-weight heparins in three different models of experimental venous thrombosis.

In a randomized, blind study, both the antithrombotic efficacy (reduction of thrombus weight) and potency (anti-Xa activity) of several commercially available low-molecular-weight heparins (LMWHs) were compared with those of unfractioned heparin (UFH) and placebo. Three different thrombogenic challenges were used: venous thrombosis was induced by direct endothelial damage in 60 New Zealand rabbits (group I), intracarotid injection of bovine thrombin in an additional series of 60 rabbits (group II), or after inferior-vena-cava ligature in 60 Wistar rats (group III). The drugs were administered subcutaneously 2 h before surgery in a blind fashion. The doses recommended for clinical practice were used (adjusted by body weight), except in group II animals, in whom doses were doubled. No differences were found between UFH and most LMWHs in terms of reduction of thrombus weight in group I animals. But UFH showed a weaker antithrombotic efficacy in the other two models. Similarly, one of the LMWHs used (Clexane) proved to be not as effective as the remainder. However, only clinical studies will provide enough information to verify these differences. Additionally, our findings confirm that the antithrombotic efficacy of a given drug differs according to the stimulus used to induce venous thrombosis.

Heparin-related osteoporosis in rats. A comparative study between unfractioned heparin and a low-molecular-weight heparin.

In an animal model, the effect of a high dose of conventional heparin (2 IU/g s.c. twice a day) and a low-molecular-weight heparin (LMWH; Fragmin, 1 anti-Xa U/g once a day) was compared with that of placebo on the mineral bone mass in the femur of rats. After 33 days of treatment no differences were found in the weight of the femur. But heparin-treated rats exhibited a lower density (1,249 +/- 0.046 g/ml as compared with that in control rats (p = 0.00007) and also in LMWH-treated rats (p = 0.001). Similarly, statistically significant differences have been found in ash contents. They were higher in control rats than in heparin-treated rats (p = 0.0002), and also slightly higher than in LMWH-treated rats (p = 0.01). Our findings suggest that LMWH may have a lower osteopenic effect than that of conventional heparin.