RF9 powerfully stimulates gonadotrophin secretion in the ewe: evidence for a seasonal threshold of sensitivity.

GPR147 and its endogenous ligands, RFRPs, are emerging as important actors in hypothalamic-pituitary axis control. The role of this system would be to inhibit gonadotrophin secretion. However, data on the subject are contradictory. The discovery of RF9 (adamantanecarbonyl-RF-2-NH(2)), a GPR147 antagonist, prompted us to use this new tool to further investigate this system in the ewe. Accordingly, we tested the effect of i.c.v. administration of RF9 on gonadotrophin secretion in the ewe during anoestrous and the breeding season. Intracerebroventricular injections of RF9 (from 50-450 nmol) caused a clear elevation in peripheral blood plasma luteinising hormone (LH) concentrations. The effect of RF9 on LH was more pronounced during the anoestrous season. Furthermore, peripheral administration of RF9 as a bolus (2.1, 6.2 and 12.4 µmol per ewe) or as a constant i.v. infusion (2.1, 6.2, 12.4 and 18.6 µmol/h per ewe) to anoestrous acyclic ewes induced a sustained increase in LH plasma concentrations. A pharmacokinetic study showed that RF9 (12.4 µmol bolus i.v.) has an effective half life of 5.5 h in the plasma. Conversely, RF9 is not detectable in the cerebrospinal fluid, suggesting that it does not cross the blood-brain barrier. The increase in LH plasma concentrations induced by RF9 was blocked by previous administration of 1.3 µmol per ewe of gondotrophin-releasing hormone (GnRH) antagonist Teverelix. This suggests that GnRH is involved in the stimulatory effect of RF9 on gonadotrophin secretion. Finally, no variation in LH plasma concentrations could be detected in ovariectomised ewes injected either i.c.v. or i.v. with RFRP3 (VPNLPQRF-NH(2)). The lack of effect of RFRP3 in our experimental setting suggests that the mechanisms involved in RF9 action are probably more complex than previously assumed. Our results indicate that delivery of RF9 in the ewe greatly increases gondadotrophin secretion in both the oestrus and anoestrus season, suggesting a potential new way of controlling reproduction in mammals.

Phospholipid composition of in vitro endothelial microparticles and their in vivo thrombogenic properties.

INTRODUCTION: Microparticles from activated endothelial cells (EMP) are well known to expose tissue factor (TF) and initiate coagulation in vitro. TF coagulant activity is critically dependent on the presence of aminophospholipids, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), but it is unknown whether or not TF-exposing EMP are enriched in such aminophospholipids. Furthermore, despite the fact that EMP have been reported in several pathological conditions, direct evidence for their (putative) coagulant properties in vivo is still lacking. We investigated the phospholipid composition of endothelial MP (EMP) and their thrombogenic properties in vivo. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC; n=3) were incubated with or without interleukin (IL)-1alpha (5 ng/mL; 0-72 h). Phospholipid composition of EMP was determined by high-performance thin layer chromatography. The association between EMP, TF antigen and activity was confirmed in vitro (ELISA, Western blot and thrombin generation). Thrombogenic activity of EMP in vivo was determined in a rat venous stasis model. RESULTS: Levels of TF antigen increased 3-fold in culture medium of IL-1alpha-treated cells (P<0.0001). This TF antigen was associated with EMP and appeared as a 45-47 kDa protein on Western blot. In addition, EMP from activated cells were enriched in both PS (P<0.0001) and PE (P<0.0001), and triggered TF-dependent thrombin formation in vitro and thrombus formation in vivo. In contrast, EMP from control cells neither initiated coagulation in vitro nor thrombus formation in vivo. CONCLUSIONS: EMP from activated endothelial cells expose coagulant tissue factor and are enriched in its cofactors PS and PE.

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

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.

Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner.

BACKGROUND: Circulating microparticles of various cell types are present in healthy individuals and, in varying numbers and antigenic composition, in various disease states. To what extent these microparticles contribute to coagulation in vivo is unknown. OBJECTIVES: To examine the in vivo thrombogenicity of human microparticles. METHODS: Microparticles were isolated from pericardial blood of cardiac surgery patients and venous blood of healthy individuals. Their numbers, cellular source, and tissue factor (TF) exposure were determined using flow cytometry. Their in vitro procoagulant properties were studied in a fibrin generation test, and their in vivo thrombogenicity in a rat model. RESULTS: The total number of microparticles did not differ between pericardial samples and samples from healthy individuals (P = 0.786). In both groups, microparticles from platelets, erythrocytes, and granulocytes exposed TF. Microparticle-exposed TF antigen levels were higher in pericardial compared with healthy individual samples (P = 0.036). Pericardial microparticles were strongly procoagulant in vitro and highly thrombogenic in a venous stasis thrombosis model in rats, whereas microparticles from healthy individuals were not [thrombus weights 24.8 (12.2-41.3) mg vs. 0 (0-24.3) mg median and range; P < 0.001]. Preincubation of pericardial microparticles with an inhibitory antibody against human TF abolished their thrombogenicity [0 (0-4.4) mg; P < 0.01], while a control antibody had no effect [19.6 (12.6-53.7) mg; P > 0.05]. The thrombogenicity of the microparticles correlated strongly with their TF exposure (r = 0.9524, P = 0.001). CONCLUSIONS: Human cell-derived microparticles promote thrombus formation in vivo in a TF-dependent manner. They might be the direct cause of an increased thromboembolic tendency in various patient groups.

Pre-clinical pharmacological profile of the novel glycoconjugate Org 36764 with both factor Xa and thrombin (IIa) inhibitory activities.

Org 36764, is an antithrombin III (AT) and thrombin binding carbohydrate, which accelerates the inactivation of both factor Xa and thrombin by AT. It displays in buffer an anti-Xa and anti-thrombin activity of 415 and 2 U/mg, respectively, compared to 172 and 114 U/mg, respectively, for unfractionated heparin (UFH), Org 36764 does not cross-react with HIT (heparin induced thrombocytopenia) antibodies and is not neutralised by PF4. In experimental models in rats, on a molar basis. Org 36764 was more active than the pentasaccharide SanOrg 34006 (= AT binding domain of Org 36764) in arterial thrombosis, but both were equally active in venous thrombosis. In arterial thrombosis following endothelial damage by ferric chloride, Org 36764 was more active than the LMW heparin enoxaparin and SanOrg 34006 and similar active to UFH. At AT saturating doses the bleeding enhancement was not more than 3.5 times the control value. Org 36764 was more active in suppressing in vivo thrombus formation on stents than UFH. SanOrg 34006 or a combination of ticlopidine and aspirin. The results indicate that the novel drug Org 36764 is a drug with antithrombotic potential against venous and arterial thrombosis.

Two new closely related rat models with relevance to arterial thrombosis--efficacies of different antithrombotic drugs.

Two thrombosis models in rats are described in which mixed type thrombi are formed at arterial and venous flow rates. The models, containing a silk thread in the aorta and vena cava, respectively, were characterised for the activity of three platelet inhibitors, three thrombin active site inhibitors and five glycosaminoglycans (GAGs). In the two models a similar highly platelet-dependent thrombus developed both in size and composition during the first 10 min after insertion of the silk thread. The thrombotic processes were self-limiting, thus maintaining blood flow, but persisted twice as long in the vena cava model. In both models the thrombus consisted for more than 65% of platelets. Thrombus development under arterial as well as under venous flow conditions was inhibited dose dependently by all tested compounds including aspirin and the synthetic alpha-methyl glycoside copy of the ATIII binding pentasaccharide within heparin, Org31540/SR90107A. Simultaneous fibrin deposition and platelet activation, which represents an essential element of arterial thrombosis, initially dominated-in both models. The gradual thrombus outgrowth, in the cava model, was more sensitive to factor Xa selective anti-coagulants, as is venous thrombosis.

Biochemical and pharmacological properties of SANORG 32701. Comparison with the "synthetic pentasaccharide' (SR 90107/ORG 31540) and standard heparin.

SANORG 32701 is a new sulfated pentasaccharide obtained by total chemical synthesis. It is analogue of the "synthetic pentasaccharide" (SR 90107/ORG 31540), which represents the antithrombin III (AT-III) binding site of heparin. Like SR 90107, it shows high affinity for human AT-III (Kd = 3.7 +/- 0.7 nmol/L) and is a potent catalyst of its inhibitory effect with regard to factor Xa (1100 +/- 31 versus 850 +/- 27 anti-Xa U/mg for SR 90107). SANORG 32701 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathways in vitro. After intravenous or subcutaneous administration to rabbits or rats, SANORG 32701 displayed prolonged anti-factor Xa activity and inhibition of thrombin generation ex vivo. SANORG 32701 was slowly eliminated, showing elimination half-lives between 2.8 and 4.9 hours with different doses. SANORG 32701 displayed antithrombotic activity by virtue of its potentiation of the anti-factor Xa activity of AT-III. It strongly inhibited thrombus formation in an experimental model of thromboplastin-induced venous thrombosis in rats (intravenously) and rabbits (subcutaneously) (ED50 values were 25.5 +/- 4.1 and 91 +/- 12.7 nmol/kg, respectively). SANORG 32701 inhibited the accretion of fibrinogen I 125 to a preformed thrombus in the rabbit jugular vein and significantly reduced thrombus growth occurring after electrical stimulation of the rabbit carotid artery. In the rabbit, intravenous injection of SANORG 32701 enhanced tissue plasminogen activator (TPA)-induced thrombolysis, suggesting that concomitant use of SANORG 32701 during TPA therapy may be helpful in preventing thrombus accretion, thus facilitating clot lysis. In the rat, SANORG 32701 potently inhibited thrombus formation induced on a silk thread in an arteriovenous shunt and in the vena cava. Compared with standard heparin, SANORG 32701 (1000 nmol/kg IV) caused only minimal bleeding enhancement and exhibited a favorable antithrombotic activity/ bleeding risk ratio, therefore showing that it might be considered as a promising compound in the treatment and prevention of various thrombotic diseases.

Synthetic analogues of the antithrombin III-binding pentasaccharide sequence of heparin. Prediction of in vivo residence times.

The synthetic pentasaccharide Org 31540/SR 90107A represents the antithrombin III (ATIII) binding region of heparin and accelerates the ATIII-mediated inhibition of coagulation factor Xa. This compound and 15 structural analogues with ATIII binding constants (Kd) ranging from 2.7 to 2600 nmol/L were compared for their plasma elimination in rats as measured from their factor Xa inhibiting activity. After administration of a low dose (100 nmol/kg body wt IV), each pentasaccharide showed a characteristic plasma half-life varying from a minimum of 0.3 hour for pentasaccharides with low affinity for ATIII to 10.9 hours for pentasaccharides with high affinity for the protein. The latter value was close to the half-life measured for radioiodinated rat ATIII (11.8 hours). We hypothesized that the elimination half-life of pentasaccharides is markedly extended by ATIII binding, of which the extent is governed by the Kd of the complex. The following observations support this hypothesis. The low-dose, low-affinity pentasaccharides were almost fully recovered in the urine without having lost anti-factor Xa activity, whereas compounds with high ATIII binding affinity were only partly recovered in the urine. With a high dose (500 nmol/kg body wt), a rapid plasma clearance of pentasaccharide was observed until a concentration similar to that of endogenous ATIII was reached, in accordance with their expected 1:1 stoichiometric interaction. The elimination of half-life was similar to that of the low dose. The relation between Kd values and plasma half-lives could be explained by assuming rapid clearance of free and coclearance of ATIII-bound pentasaccharide with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentasaccharide and Orgaran arrest, whereas heparin delays thrombus formation in a rat arteriovenous shunt.

The mode of action of glycosaminoglycans (GAGs) towards thrombus formation in a rat arteriovenous shunt was studied by simultaneous examination of thrombus weight, platelet consumption and thrombin generation during 45 min of blood circulation. A comparison was made between the effects of heparin, the heparinoid Org 10172 (Orgaran), and the chemically synthesized methoxy derivative of the antithrombin III binding pentasaccharide fragment of heparin (Org 31540). All three compounds inhibited thrombus growth by 30% at a dose of 80 anti-Xa U/kg i. v. when assessed after 15 min of circulation through the shunt. In addition, a systemic decrease of 27% of platelet numbers in the placebo group was inhibited by heparin and Orgaran with 63% and by pentasaccharide with 48%. At a later stage, after 45 min of circulation, at comparable plasma anti-Xa levels, thrombi which had formed in the presence of Orgaran or pentasaccharide, but not in the presence of heparin, became less or non thrombogenic. This non-thrombogenicity was reflected by i) an inhibition of the local deposition of [51Cr]platelets of 75% with Orgaran and of 57% with pentasaccharide, and ii) an inhibition of ex-vivo thrombus-induced thrombin generation in pooled rat plasma of 67% with Orgaran and of 52% with pentasaccharide (inhibition compared to placebo). Although the mechanism of inducing non-thrombogenicity of a (developing) thrombus by Orgaran and pentasaccharide requires further investigation, the suppression of the local thrombin generation potency, measured by thrombus-induced thrombin generation in pooled plasma, is much more correlated with thrombus growth than systemic anticoagulant activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antifactor Xa activity and antithrombotic activity in rats of structural analogues of the minimum antithrombin III binding sequence: discovery of compounds with a longer duration of action than of the natural pentasaccharide.

The total chemical synthesis of a series of structural analogues of the so-called natural AT III binding pentasaccharide together with the natural pentasaccharide itself has been accomplished. The structural analogues all contain an extra 3-O-sulfate group on glucosamine residue H of the pentasaccharide, some carry additional 3 or 4-O-sulfate groups on glucosamine residue D. All these structural analogues elicit a higher specific anti-Factor Xa activity than the natural pentasaccharide (700 anti-Factor Xa U/kg). The structural analogue carrying only an additional 3-O-sulfate on glucosamine unit H (Org 31550) has the highest specific activity (1230 anti-Factor Xa U/kg). The increased specific activity is presumably attributed to the stronger binding to AT III. All structural analogues have a prolonged duration of action of the plasma anti-Factor Xa activity. (T1/2, approximately 9 hours) compared with that of the natural pentasaccharide (T1/2, approximately 5 hours) after single intravenous administration of 600 anti-Factor Xa U/kg. All structural analogues exert dose-dependent antithrombotic activity in a rat stasis thrombosis model after intravenous administration. On an anti-Factor Xa basis, the compounds have the same potency as the natural pentasaccharide (ED50s are 35 to 55 anti-factor Xa U/kg). Of two structural analogues (Org 31550 and Org 31706), the time course of antithrombotic activity was assessed in the same model after subcutaneous administration of 600 anti-Factor Xa U/kg. The duration of antithrombotic activity of these compounds was four to five times longer than that of the natural pentasaccharide.

Pharmacological profile of the chemically synthesized antithrombin III binding fragment of heparin (pentasaccharide) in rats.

The antithrombotic and haemostatic effects of a pentasaccharide, the chemically synthesized antithrombin III (AT-III) binding fragment of heparin (PENTA), were investigated in rats in comparison with heparin. PENTA showed a dose-dependent antithrombotic effect in three thrombosis models in which thrombus formation was induced by different triggers. PENTA was consistently less potent than heparin in these models if doses were expressed in anti-Xa U/kg but PENTA showed more or less the same potency as heparin if doses were expressed in mg/kg. The antithrombotic effect of PENTA was strongly related to its anti-Xa activity as judged from its antithrombotic potency in the various models and from the time courses of both activities. PENTA caused a dose-dependent increase in blood loss in a bleeding model but the dose response curve was rather flat; the effect of PENTA on blood loss was small compared to that of heparin. The duration of action of PENTA as measured by the plasma anti-Xa levels was long compared to that of heparin and the duration of the antithrombotic effect was that expected on the basis of the plasma anti-Xa levels. Finally, PENTA showed comparable antithrombotic activity after s.c. and i.v. administration, as expected because of the approximately 100% bioavailability of the anti-Xa activity after s.c. administration.

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.

Time courses of the antithrombotic effects, bleeding enhancing effects and interactions with factors Xa and thrombin after administration of low 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.

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.