[New antithrombotic oligosaccharides].

In the early eighties, following breakthroughs in oligosaccharide chemistry, the total chemical synthesis of pentasaccharides has been achieved, representing the antithrombin binding domain of heparin (the active site). The selective inhibitors of coagulation factor Xa thus obtained represent a new type of antithrombotic drugs. In a further step, based on the knowledge of the mechanism of antithrombin activation by heparin, oligosaccharides (pentadeca- to eicosasaccharides), comprising an antithrombin binding domain prolonged by a thrombin binding domain, were designed and synthesised in the Sanofi group.

Synthetic oligosaccharides having various functional domains: potent and potentially safe heparin mimetics.

A synthetic heptadecasaccharide, comprising an antithrombin III binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained through a convergent synthesis. This compound displayed in vitro anticoagulant properties similar to that of standard heparin but, in contrast with heparin, escaped neutralization by platelet factor 4, a protein released by activated platelets.

New synthetic heparin mimetics able to inhibit thrombin and factor Xa.

Synthetic pentadeca-, heptadeca- and nonadecasaccharides, comprising an antithrombin III (AT III) binding pentasaccharide prolonged at the non-reducing end by a thrombin binding domain have been obtained. The pentadecasaccharide is the shortest oligosaccharide able to catalyse thrombin inhibition by AT III. The nonadecasaccharide is a more potent thrombin inhibitor than standard heparin.

Synthesis of thrombin-inhibiting heparin mimetics without side effects.

Unwanted side effects of pharmacologically active compounds can usually be eliminated by structural modifications. But the complex heterogeneous structure of the polysaccharide heparin has limited this approach to fragmentation, leading to slightly better-tolerated heparin preparations of low molecular mass. Despite this improvement, heparin-induced thrombocytopaenia (HIT), related to an interaction with platelet factor 4 (PF4) and, to a lesser extent, haemorrhages, remain significant side effects of heparinotherapy.

Contribution of erythrocytes to thrombin generation in whole blood.

Thrombin generation (TG) initiated by diluted tissue-factor was investigated in whole human blood, in platelet-rich plasma (PRP), in platelet-poor plasma (PPP), and in PPP supplemented with red blood cells (RBCs). TG was characterized by the lag time preceding the thrombin burst and by the endogenous thrombin potential (ETP). RBCs at normal haematocrit were found to influence the lag time to the same extent as platelets.

First Synthetic Carbohydrates with the Full Anticoagulant Properties of Heparin.

A single disaccharide building block is required to obtain synthetic carbohydrates that reproduce the anticoagulant activity of heparin and inhibit thrombin (n>6) and/or factor Xa (n≥2; see reaction scheme). Thus, there is evidence that heparin fragments with at least 15 saccharide units are required for thrombin inhibition. Lev=levulinoyl.

Introducing a C-interglycosidic bond in a biologically active pentasaccharide hardly affects its biological properties.

We describe here the synthesis and the biological activity of a 'C-pentasaccharide', a new analogue of the antithrombin III (AT III) binding region of heparin containing a methylene bridge in place of an interglycosidic oxygen atom. The affinity for AT III and the anti-factor Xa activity of this compound have been compared with that of the corresponding selected 'O-pentasaccharide'. Such a structural modification slightly decreased the affinity of this compound for AT III as well as its anti-factor Xa activity (880 +/- 40 anti-Xa units versus 1180 +/- 30 anti-Xa units for the C-pentasaccharide and the O-pentasaccharide, respectively).

Biochemical and pharmacological properties of SANORG 34006, a potent and long-acting synthetic pentasaccharide.

SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the "synthetic pentasaccharide" (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.

Comparative effects of two direct and indirect factor Xa inhibitors on free and clot-bound prothrombinase.

Factor Xa, as with thrombin, binds to the clot and contributes to the propensity of thrombi to activate the coagulation system. The aim of this work was to compare the extent of prothrombinase inhibition produced by two factor Xa inhibitors: the antithrombin III-dependent synthetic pentasaccharide (SR 90107/Org 31540) and DX-9065A, a direct factor Xa inhibitor. When incubated together with prothrombin, factor Xa, phospholipids, antithrombin III and calcium, clots formed from human plasma exhibited a prothrombinase activity as measured through fragment 1-2 (F1+2) generation.

Human umbilical vein endothelial cells express high affinity receptors for factor Xa.

The binding of [125I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7 degrees C, [125I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 +/- 0.

Synthesis and pharmacological properties of a close analogue of an antithrombotic pentasaccharide (SR 90107A/ORG 31540).

The synthetic pentasaccharide (1) corresponding to the heparin sequence which binds to, and activates, antithrombin III (AT III) is a potent antithrombotic compound in several animal models of venous thrombosis. We describe here the preparation and the pharmacological properties of 34, an analogue of oligosaccharide 1 with the latter's N-sulfates being replaced by sulfate esters and hydroxyl groups being methylated. These structural modifications allow a simpler and more efficient synthesis of such anionic oligosaccharides.

Long-term persistence of low-molecular-weight material with anti-factor Xa activity contributes to anticoagulant and antithrombotic effects after subcutaneous injection of CY 216 in the rabbit.

This study was done to document further the mechanism of the antithrombotic effect of CY 216 after subcutaneous injection in the rabbit. We first measured the circulating anti-factor Xa and anti-thrombin activities expressed in either International Unit or Standard Independent Unit and from these activities we calculated the above critical length material (ACLM) and below critical length material (BCLM) levels. The clearance of the BCLM was half that of the ACLM.

Pharmacokinetic study of three synthetic AT-binding pentasaccharides in various animal species-extrapolation to humans.

The 'synthetic pentasaccharide', SR 90107A/Org 31540 (SP) representing the minimal AT-binding sequence of heparin is a catalyst of factor Xa inhibition. Affinity of SP, Sanorg 32701 (32701) and SR 80027 (80027), two close analogues of SP for rat, rabbit, baboon and human AT has been evaluated. The dissociation constants (Kd) for AT of the three species were in the range of 41-132, 1.

Absence of cross-reactivity of SR90107A/ORG31540 pentasaccharide with antibodies to heparin-PF4 complexes developed in heparin-induced thrombocytopenia.

New carbohydrate-based anticoagulants devoid of the side effects of unfractionated heparin are currently under development and show a major potential for patients with heparin-induced thrombocytopenia (HIT) who still require efficient antithrombotic therapy. As HIT is usually associated with antibodies to heparin-platelet factor 4 (H-PF4) complexes, cross-reactivity of the heparin pentasaccharide SR90107A/ORG31540 was tested in the presence of PF4 with the plasma from 49 patients with HIT. No cross-reactivity was observed whatever the pentasaccharide concentrations.

Determination of the anti-factor Xa activity of the synthetic pentasaccharide SR 90107A/ORG 31540 and of two structural analogues.

The anti-factor Xa activity of the synthetic pentasaccharide SR 90107A/ORG 31540 was assayed by a chromogenic method at pH 8.4 and pH 7.35, comparatively to the 4th International Heparin Standard (IHS) or to the Ist International Low Molecular Weight Heparin Standard (LMWHS).

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.

Antithrombin-mediated inhibition of factor VIIa-tissue factor complex by the synthetic pentasaccharide representing the heparin binding site to antithrombin.

We examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor VIIa bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor VIIa, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor VIIa complex.

Glycosaminoglycans enhance megakaryocytopoiesis by modifying the activities of hematopoietic growth regulators.

We have previously reported that heparin is capable of stimulating in vitro and in vivo megakaryocytopoiesis in mice and has a thrombopoietic effect when given in chronic immune thrombocytopenic purpura and that heparin and several other glycosaminoglycans (GAGs) promote the growth of human megakaryoblastic cell lines in the presence of serum. We show here that GAGs, including heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA), also stimulate in vitro growth of murine megakaryocyte progenitors and augment the diameter of individual megakaryocytes in the presence of serum. However, in a serum-free agar system, the GAGs alone had no effect on megakaryocyte colony formation, suggesting that GAGs cooperate with some serum factor(s) to exert their activity.

DX 9065A a novel, synthetic, selective and orally active inhibitor of factor Xa: in vitro and in vivo studies.

DX 9065A is the first member of a newly developed series of synthetic and selective inhibitors of factor Xa. DX 9065A inhibited in a dose-dependent manner human factor Xa with K iota value of 3.1 +/- 0.

Heparin reverses the procoagulant properties of stimulated endothelial cells.

We examined the ability of unfractionated heparin to modulate the procoagulant activities of stimulated endothelial cells (EC). Confluent human venous umbilical EC were incubated with heparin before or after stimulation, then rinsed extensively to eliminate any heparin in the solution. EC, stimulated for 4 h with endotoxin and interleukin 1 beta, expressed tissue factor and prothrombinase activities.

The effect of the synthetic pentasaccharide SR 90107/ORG 31540 on thrombin generation ex vivo is uniquely due to ATIII-mediated neutralization of factor Xa.

The inhibition of thrombin generation (TG) was studied in plasma from human volunteers after single subcutaneous administrations of 4000, 8000 or 12,000 anti-Xa units (i.e., 6, 12 or 18 mg) of the synthetic pentasaccharide (SR 90107/ORG 31540) (SP).

Molecular profiling and weight determination of heparins and depolymerized heparins.

The recently proposed calibrant LHN-1 (lot F537; henceforth designated F537), for the molecular weight (MW) determination by high-performance size-exclusion chromatography of heparins, is shown here to have a range too narrow to allow for the accurate MW determination of all low molecular weight heparins (LMWHs). We have recently demonstrated, by this same methodology, that a chemically degraded benzyl ester of unfractionated heparin, heparin mass calibrator (HMC), is a better calibrant. Weight-average MW, number-average MW, peak MW, and dispersity values were calculated with F537, HMC, and by a reference narrow-range-calibration method for various LMWHs and unfractionated heparins.

Comparison of a low-molecular-weight heparin (nadroparin calcium) and unfractionated heparin as adjunct to coronary thrombolysis with alteplase and aspirin in dogs.

Background: Low-molecular-weight heparins may have a higher benefit to risk ratio than unfractionated heparin in preventing perioperative thrombosis. The antithrombotic effects of low-molecular-weight heparins, given as adjunctive therapy to alteplase and aspirin, have not previously been compared with those of unfractionated heparin in experimental models of coronary artery thrombosis.

Methods: Occlusive coronary thrombosis was induced in 5 groups of 10 dogs by placing a copper coil into the left anterior descending coronary artery.

Antithrombin III affinity dependence on the anticoagulant, antiprotease, and tissue factor pathway inhibitor actions of heparins.

To investigate AT-III affinity dependence on heparin's actions, heparin (UH) was fractionated on an AT-III-Sepharose column into a high (HAH) and a low affinity (LAH) fraction. Molecular profiling revealed a molecular weight of 11.8 kDa for (UH), 12.

Heparin and low molecular weight heparins inhibit prothrombinase formation but not its activity in plasma.

Factor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma.