Dirk G. Meuleman

Experimental Study of Thrombogenicity and Foreign Body Reaction Induced by Heparin-Coated Coronary Stents

BACKGROUND Results of recent randomized clinical trials have revealed a significant reduction in angiographic restenosis rate when adjunctive stenting was performed after conventional coronary balloon angioplasty. The thrombogenicity of metal stents, however, remains a concern. In the present study, we compare the thrombogenicity of heparin-coated coronary stents with that of bare metallic coronary stents. METHODS AND RESULTS Thrombogenicity of metallic coronary stents (four heparin-coated and eight bare stents) was studied in a rat arteriovenous shunt model with the use of 125I-labeled fibrinogen and 51Cr-labeled platelets. Total clot weight after 30-minute follow-up was significantly lower in the heparin-coated stents compared with the bare stents (8.1 +/- 3.7 versus 25.8 +/- 4.6 mg; P < .001). Relative 125I and 51Cr activities in the stents were significantly higher in the bare stents than in the heparin-coated stents (125I, 1.03 +/- 0.43 versus 0.18 +/- 0.04, P = .003; 51Cr, 17.5 +/- 6.8 versus 4.4 +/- 1.0, P = .004). Subsequently, heparin-coated and bare stents were randomly implanted in the right coronary artery of 20 domestic pigs. Angiographic parameters were similar between both groups at baseline and after 6-week follow-up. Morphometry also did not show a significant difference in lumen area (bare, 1.03 +/- 0.83 mm2; heparin-coated, 1.12 +/- 0.73 mm2; P = NS) or neointimal hyperplasia (bare, 1.01 +/- 0.81 mm2; heparin-coated, 1.21 +/- 0.57 mm2; P = NS). CONCLUSIONS Heparin coating of metallic coronary stents decreases their thrombogenicity but does not improve late vessel patency and neointimal hyperplasia at follow-up in a porcine coronary model.

Tibolone Prevents Atherosclerotic Lesion Formation in Cholesterol-Fed, Ovariectomized Rabbits

Tibolone (Org OD14), a synthetic steroid with estrogenic and progestogenic/androgenic properties, is clinically effective for the treatment of climacteric symptoms and the prevention and treatment of osteoporosis in postmenopausal women. The effect on atherogenesis, however, is not known. In the current study, we investigated the effect of tibolone in comparison with that of estradiol and norethisterone acetate on atherogenesis in 140 ovariectomized New Zealand White rabbits that had been induced by an atherogenic diet (0.4% cholesterol, 20 weeks). Tibolone at 18, 6, or 2 mg/d orally completely prevented cholesterol accumulation and fatty streak formation in the aorta; the impairment of endothelium-dependent smooth muscle relaxation of the aorta; and complex lesion formation after endothelial denudation in the carotid artery. Tibolone also reduced the increased postovariectomy plasma lipid concentrations. Analysis of the results, however, indicated that a substantial part of the strong, beneficial effects were plasma lipid independent. Compared with subcutaneous estradiol decanoate (150 microgram once weekly) and oral 17beta-estradiol (4 mg/d), the effects of tibolone were more pronounced at equipotent uterotropic activity. Norethisterone acetate (1 mg/d) did not affect atherosclerotic lesion formation. There are no indications that the progestogenic/androgenic properties of tibolone counteracted its atheroprotective effect on the vessel wall. Therefore, tibolone has the intrinsic potential to be a compound that protects the arterial vessel wall against atherosclerotic processes.

Syntheses of heparin-like pentamers containing opened uronic acid moieties

Abstract The syntheses of four analogues of pentasaccharide Ia , which corresponds to the minimal AT III binding region of heparin, are presented and the biological activities of these analogues will be discussed. Three of these analogues (i.e. compounds II , III and IV ) contain an R -glyceric acid oxymethylene residue ( i.e. B in fig.3 ) instead of α-L-iduronic acid and in the other analogue ( i.e. compound V ) the β-D-glucuronic acid unit has been replaced by an s -glyceric acid oxymethylene residue ( i.e. A in fig3 ). The R and S -glyceric acid oxymethylene residues represent an “opened” iduronic acid unit and an “opened” glucuronic acid unit, respectively, containing the essential carboxylate function in the appropriate configuration. The crucial step in the syntheses of these “opened” uronic acid pentamer analogues, was the preparation of the required glyceric acid oxymethylene residues 8a , 8b and 8c . Analogues II and III , containing an “opened” iduronic acid moiety, display a significant AT III mediated αXa activity. Compound III contains two extra sulphate groups at unit 2 . Removal of the contributing O-sulphate groups at position 3 and 6 of unit 6 of compound II ( i.e. compound IV ) results in a seven-fold drop in αXa activity. Replacement of the β-D-glucuronic acid unit by an S -glyceric acid oxymethylene residue ( i.e. compound V ) leads to almost a complete loss of αXa activity, notwithstanding the fact that all the essential and contributing charged groups are present in the molecule.

Comparison of the antiatherosclerotic effect of tibolone with that of estradiol and ethinyl estradiol in cholesterol-fed, ovariectomized rabbits.

Objective Tibolone is a synthetic steroid with tissue-specific estrogenic, progestogenic, and androgenic properties. The drug relieves climacteric symptoms and prevents osteoporosis but does not stimulate the endometrium. We have previously shown that in laboratory animals tibolone inhibits the atherogenesis induced by a high-cholesterol diet. Therefore, we compared the antiatherosclerotic effect of oral tibolone at different dose levels with that of oral 17&bgr;-estradiol (E2) and ethinyl estradiol (EE). Design Atherosclerotic lesion formation (increase in vessel wall cholesterol deposition and fatty streak formation) was measured in ovariectomized rabbits after 20 weeks on an atherogenic diet (fed daily 80 g of a rabbit chow containing 0.4% cholesterol, 3.75% peanut oil, and 3.75% coconut oil) in eight groups: group 1, placebo (n = 35); group 2, control (n = 34) received normal rabbit chow; group 3, E2 group (E2 4 mg, n = 12); group 4, EE group (EE 60 &mgr;g, n = 10); and groups 5–8, tibolone (6 mg, n = 12; 2 mg, n = 13; 0.6 mg, n = 25; and 0.15 mg, n = 11, respectively). During the study, blood samples were obtained for the evaluation of plasma triglycerides, cholesterol, lipoproteins, and glutamate pyruvate transaminase. After 20 weeks, the animals were killed, and cholesterol concentration and the formation of fatty streaks in the wall of the aortic arch were evaluated. Results In the placebo group, the atherogenic diet induced a mean increase in total plasma cholesterol concentration from 1.1 ± 0.1 mmol/L (control group) to 34.1 ± 1.8 mmol/L (mean ± SE). This resulted in an accumulation of cholesterol in the aortic arch from 48 ± 4 (control group) to 608 ± 44 nmol/mg protein and in the formation of fatty streaks (41.8 ± 3.2% of the surface of the aortic arch was covered with fatty streaks). Tibolone had strong dose-dependent antiatherosclerotic effects. It reduced the accumulation of cholesterol in the aortic arch at doses of 6 to 0.15 mg by 99, 97, 87, and 57% and the formation of fatty streaks by 98, 97, 81, and 38%, respectively. E2 had only a marginal antiatherosclerotic effect, whereas EE showed an effect comparable to that of tibolone at doses of 2 to 0.6 mg. With EE, the accumulation of cholesterol in the vessel wall was reduced by 93% and the formation of fatty streaks by 73%. Mean plasma cholesterol concentrations were also reduced by tibolone (64, 70, 61, and 47%) and EE (57%). This reduction was mainly mediated via a reduction in &bgr;-very-low-density lipoprotein cholesterol. Analysis, however, indicated that the observed antiatherosclerotic effects of tibolone and EE, at least partly, are due to a direct effect on the vessel wall and independent of the changes in plasma cholesterol. At equipotent antiatherosclerotic doses, EE showed a stronger uterotropic effect (measured as the increase in uterine weight) than tibolone. EE increased uterine weight from 0.57 g/kg body weight (BW) (control group) to 3.5 g/kg BW; tibolone at doses of 6, 2, 0.6, and 0.15 mg increased uterine weight to 2.5, 2.8, 2.2, and 1.3 g/kg BW, respectively. Conclusion Tibolone can protect the arterial vessel wall against atherosclerotic lesions induced by a hypercholesterolemic diet. However, it has much less estrogenic effects on the uterus compared with EE at equipotent doses, indicating tissue selectivity for tibolone. The clinical implications of these findings require investigation.

Antithrombotic properties of a direct thrombin inhibitor with a prolonged half‐life and AT‐mediated factor Xa inhibitory activity

Summary.  Rebound thrombin generation after successful thrombolysis might be related to (i) too short‐term anticoagulant therapy and to (ii) the inability of heparin derivatives to inhibit clot‐bound thrombin. To meet these shortcomings, a compound was synthesized, which consists of a pentasaccharide conjugated to a direct thrombin inhibitor. This compound (Org 42675) has a 10 times longer half‐life compared with the original half‐life of the direct thrombin inhibitor, while the thrombin inhibitory activity is maintained. An extra advantage of this product is the inhibitory activity on thrombin generation via antithrombin III (AT)‐mediated factor (F)Xa inhibition. Org 42675 inhibited in vitro clot‐bound thrombin with similar activity to the direct thrombin inhibitor argatroban. In experimental models in rats, Org 42675 showed on a molar base similar antithrombotic activity to unfractionated heparin, was more active than argatroban and was more active than fondaparinux sodium (AT‐mediated FXa inhibitor) in arterial thrombosis. Finally, Org 42675 was far more active than the three reference compounds in an experimental thrombolysis model in rabbits. These properties of Org 42675, with its FXa and (clot‐bound) thrombin inhibitory activity in combination with its long half‐life, make this compound a powerful drug that is likely to be effective in the prevention of re‐occlusion after successful thrombolysis in man.

Glycosaminoglycans: Synthetic fragments and their interaction with serine protease inhibitors

Glycosaminoglycans such as e.g. heparin, heparan sulphate and dermatan sulphate display a broad variety of biological activities. Unique, Well-defined domains in some glycosaminoglycans have been characterized that are responsible for the biological activity. For instance, a unique pentasaccharide domain in heparin could be identified which binds and activates the serine protease inhibitor (serpin) anti-thrombin I11 (AT 111). The structure-activity relationships of various synthetic counter-parts of the heparin pentasaccharide fragment reveal the highly specific nature of the pentasaccharide mediated activation of AT 111. With the aid of molecular modelling and the availability of crystal structures of serpins and their target proteases, the activation process of AT I11 by heparin becomes understood at the molecular level. Some attention will also be paid to well-defined domains in heparan sulphate and dermatan sulphate.

The action of serpins and heparin as leads in drug discovery

Abstract The natural product heparin is an important lead in research on antithrombotics. By molecular modification of a unique heparin fragment new derivatives were discovered. One of them, the synthetic counterpart of the antithrombin III binding pentasaccharide 1 , is now being tested clinically. Recently, the structure elucidation of a number of blood coagulation proteins and serpins initiated a new breakthrough in the anticoagulant research. Molecular modelling of the crucial antithrombin III — heparin — thrombin complex inspired the design and synthesis of a novel class of anticoagulants. Their anticoagulant properties can be adjusted in a rational way leading to compounds with unprecedented characteristics.