D.G. Meuleman

Comparison of two experimental thrombosis models in rats effects of four glycosaminoglycans

Two experimental thrombosis models in rats have been compared with regard to the composition of the formed thrombi and the effects of various treatments on thrombus formation. In the first model thrombosis is induced in the vena cava by a combination of venous stasis and hypercoagulability; these thrombi consist merely of red cells and fibrin with only a few platelets. In the second model thrombosis is induced in an arterio-venous shunt in which the formed thrombi consist of red cells, fibrin and a large amount of platelet aggregates adhering to the foreign material. Antiplatelet serum and acetylsalicylic acid, which reduce blood platelet activity, inhibited thrombus formation only in the arteriovenous shunt model. Dicumoxane, an oral anticoagulant, was active in both models. The glycosaminoglycans heparin, Org 10172, Fragmin and the pentasaccharide, representing the AT-III binding sequence of heparin, were active in both models. However, there were qualitative and quantitative differences between the effects of the glycosaminoglycans suggesting differences in their modes of action.

A novel anti-thrombotic heparinoid (ORG 10172) devoid of bleeding inducing capacity: A survey of its pharmacological properties in experimental animal models

The pharmacological profile of Org 10172, a mixture of sulphated glycosaminoglycorunans derived from hog intestinal mucosa, has been assessed in experimental thrombosis and bleeding models in rats and compared with heparin USP. Org 10172 inhibited thrombus formation in arterio-venous shunts dose dependently, the dose required for 50% inhibition (ID50) of thrombus formation was 40 anti-Xa units/kg i.v. The ID50 for heparin USP was 70 anti-Xa units/kg i.v. Org 10172 hardly increased bleeding in doses upto 1600 anti-Xa units/kg i.v., whereas heparin USP dose dependently increased bleeding from 90 anti-Xa units/kg i.v. onwards. The benefit (anti-thrombotic)/risk (bleeding) ratio of Org 10172 was therefore considerably better than that of heparin USP. The improved profile of Org 10172 towards bleeding might be caused by differences in the interaction with blood platelets in comparison with heparin USP. Org 10172 had less effect on the platelet content in thrombi than heparin USP. Org 10172 did not inhibit collagen induced release of serotonin in contrast to heparin USP. Org 10172 inhibited factor Xa induced aggregation of rabbit platelets but only at anti-Xa levels which were fifteen times higher than for heparin USP. In contrast to heparin USP Org 10172 had only a very weak effect on the activated partial thromboplastin time (APTT) ex vivo.

From heparin to EP217609: The long way to a new pentasaccharide-based neutralisable anticoagulant with an unprecedented pharmacological profile

The elucidation of the structure of the antithrombin binding sequence in heparin has given a large impulse to the rational design of heparin related drugs. De novo chemical synthesis of the corresponding pentasaccharide as well as simplified analogues has provided very specific, antithrombin-mediated inhibitors of factor Xa with various pharmacokinetic profiles. Fondaparinux and idraparinux are examples of such compounds that have found clinical application as antithrombotics. Because of the very specific binding to antithrombin the pharmacokinetics of pentasaccharides can be predicted and transferred to other molecules covalently bound to them. The new chemical entities thus obtained display a wide array of antithrombotic activities, giving improved heparin molecules as well as new anticoagulants, devoid of the undesired side effects of heparin and with unprecedented pharmacological profiles. In this context, a direct thrombin inhibitor was covalently coupled to a pentasaccharide by an inert spacer. This compound, EP42675 exerts antithrombin mediated anti-factor Xa activity together with direct thrombin inhibiting capacity. It displays favourable pharmacokinetics as imposed by the pentasaccharide. EP42675 was further modified by the introduction of a biotin moiety in its structure. The new entity obtained, EP217609 exerts the same pharmacological profile as EP42675 and it can be instantaneously neutralised by injection of avidin. Due to this unprecedented mechanism of anticoagulant activity and its ability to be neutralised, EP217609 deserves to be investigated in clinical settings where direct thrombin inhibition is required.

Time courses of the antithrombotic effects, bleeding enhancing effects and interactions with factors Xa and thrombin after administration of iow molecular weight heparinoid Org 10172 or heparin to rats

Time response curves of the anti-thrombotic effects, bleeding enhancing effects, effects on APTT, anti-Xa activities, anti-thrombin activities and thrombin generation inhibitory activities of the low molecular weight heparinoid Org 10172 and heparin have been compared in rats. The time courses of these effects were similar for heparin but quite different for Org 10172. Org 10172 induced anti-thrombotic and anti-Xa effects which lasted approximately 3 times longer than those at the same anti-Xa doses of heparin, whereas the bleeding enhancing effects and effects on APTT of Org 10172 were of shorter duration than those of heparin. The half-life of the anti-thrombin effect after Org 10172 seemed somewhat longer than after heparin administration. Thrombin generation inhibition by Org 10172 showed a slightly longer duration than by heparin. The similarities between the time courses of the anti-thrombotic effect and the anti-Xa activity after Org 10172 administration suggest that the most appropriate parameter to monitor Org 10172 treatment is the plasma anti-Xa level.

Effects of intra-arterial cannulation on blood platelet consumption in rats

Experimental arterial thrombosis has been induced by an indwelling cannula in the abdominal aorta of rats. The involvement of blood platelets was studied by the determination of the blood platelet count and the survival of 51Cr labeled platelets. Cannulation transformed the platelet disappearance patterns from linear to curvi linear and exponential curves. The blood platelet consumption rate increased 2–3 fold immediately after cannulation and remained at that level for at least 8 days. The consumption rate was higher when longer cannulae were used. Initially the increased blood platelet consumption was not compensated by an increase in platelet production, this resulted in a decreasing platelet count. After 3 to 7 days a long lasting steady state thrombocytopenia was achieved, under these circumstances the platelet turnover in cannulated and normal rats was the same. Withdrawal of the cannula normalized the platelet consumption within a few hours, leading to a linear disappearance pattern of the residual labeled platelets. The platelet count was readily restored to normal values via a rebound overshoot.

PLACENTAL TRANSFER OF Org 10172, A LOW-MOLECULAR WEIGHT HEPARINOID, IN THE AWAKE LATE-PREGNANT GUINEA PIG

The placental transfer of Org 10172, a low-molecular weight heparinoid, was determined in 12 awake late-pregnant guinea pigs. Nine animals receiving placebo served as controls. After 5 days i.v. treatment with Org 10172 (2 x 300 anti-Xa U/kg/day), anti-Xa activity in fetal plasma amounted to 2.4% of the maternal concentration. The Org 10172 transfer across the placenta was also evaluated with 3H-labelled Org 10172. One hour after the administration of the latter compound, fetal Org 10172-bound 3H-activity had reached 1.5% of the maternal value. The associated extremely low placental Org 10172 transfer indicates that the Org 10172 transport across the placenta of the guinea pig is membrane-limited and that the placental permeability is negligibly low. Since the hemochorial placenta of the guinea pig closely resembles that of the human, it is likely that similar transplacental transfer properties of Org 10172 apply to man.

Inhibition of the early stages of the thrombin generation reaction by various glycosaminoglycans

Inhibition of thrombin generation was studied in strongly diluted human plasma by continuously measuring the splitting of the thrombin-specific chromogenic substrate S2288 as a function of the inhibitor concentration. To avoid the activation of clotting cascade proenzymes other than prothrombin, the thrombin generation reaction was initiated by a mixture of calcium, phospholipids and (bovine) factor Xa. Using this assay we have estimated the relative potency of the following glycosaminoglycans: heparin; a fraction of heparin with high affinity for antithrombin III (high affinity heparin); the low molecular weight heparin Fragmin; the low molecular weight heparinoid Org 10172; a fraction of Org 10172 with high affinity for antithrombin III (high affinity Org 10172) and the O-methyl derivative of the pentasaccharide, representing the minimal structure required for binding to antithrombin III. Based on concentrations expressed in amidolytic anti-Xa units, the descending order of potency observed is: Heparin approximately High Affinity Heparin greater than Fragmin greater than Org 10172 greater than High Affinity Org 10172 greater than O-methyl pentasaccharide. The more potent the glycosaminoglycan the stronger the concentration dependence of its inhibitory effect. These findings could be due to the different, additional anti-thrombin activities of these glycosaminoglycans and/or to their different anti-prothrombinase activities. With the pentasaccharide a striking saturation of the inhibition is observed, due to saturation of the antithrombin III.

Analysis of platelet survival curves in an arterial thrombosis model in rats.

Abstract A mathematical formula, which can objectively describe blood platelet disappearance patterns under normal, mild and severe thrombotic conditions, has been derived from experimental data. Platelet kinetics were studied in rats with an experimentally induced arterial thrombosis, in rats during the termination of the thrombotic stimulus and in normal rats. Normal populations and a cohort of young, radiolabeled platelets were used in the experiments. Our investigations strongly suggested that under normal and thrombotic conditions and under conditions in which the thrombotic stimulus is terminated, blood platelets ultimately disappeared from the circulation after the same finite life span. The obtained data could be best explained by assuming that two mechanisms can operate simultaneously: disappearance by ageing occurs only under non-thrombotic conditions, whilst both mechanisms operate together under thrombotic conditions. The derived mathematical formula, which describes both processes, offers a useful method to analyse data obtained from platelet survival studies.