Jean-Claude Lormeau

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. Breakthroughs in oligosaccharide chemistry made possible the total synthesis of the pentasaccharide antithrombin-binding site of heparin,. This pentasaccharide represents a new family of potential antithrombotic drugs, devoid of thrombin inhibitory properties, and free of undesired interactions with blood and vessel components. To obtain more potent and well-tolerated antithrombotic drugs, we wished to synthesize heparin mimetics able to inhibit thrombin, that is, longer oligosaccharides. Like thrombin inhibition, undesired interactions are directly correlated to the charge and the size of the molecules, so we had to design structures that were able to discriminate between thrombin and other proteins, particularly PF4. Here we describe the use of multistep converging synthesis to obtain sulphated oligosaccharides that meet these requirements.

Chemical synthesis of glycosaminoglycans : new approaches to antithrombotic drugs

Science, Optics and Music in Medieval and Early Modern Thought.By A. C. Crombie. The Hambledon Press: 1990. Pp. 474. £37.50,

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

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Glycosaminoglycans enhance megakaryocytopoiesis by modifying the activities of hematopoietic growth regulators

The binding of [125I]‐factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7°C, [125I]‐factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 ± 0.8 nM and a binding site density of 57,460 ± 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo‐first order and gave association and dissociation rate constant values of 0.15 × 106 M‐1 s‐1 and 4.0 × 10‐4 s‐1, respectively. [125I]‐factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin‐III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor‐1 (EPR‐1), a well‐known receptor of factor Xa on various cell types. [125I]‐factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide‐antithrombin‐III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC‐bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR‐1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall. J. Cell. Physiol. 172:36–43, 1997.

Studies on the structural requirements of heparin for the catalysis of thrombin inhibition by heparin cofactor II

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), dermantan 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. We also show that heparin significantly potentiates the megakaryocytopoietic activity of C‐Mpl ligand and interleukin (IL)‐6 but not IL3, GM‐CSF, SCF, and Epo. In addition, the GAGs significantly neutralize the inhibitory action of platelet factor 4 (PF4) and transforming growth factor β1 (TGFβ1) on megakaryocyte colony growth. These results demonstrate a stimulating activity of GAGs on megakaryocytopoiesis by modifying the activity of several growth‐regulating factors.

Selectively O-acylated glycosaminoglycan derivatives

The structural requirements of heparin for the catalysis of thrombin inhibition by heparin cofactor II (HC II) were investigated. A series of well characterized heparin derivatives were prepared and their activities were measured using human thrombin in the presence of an excess of purified human HC II and, for comparison, antithrombin III (AT III). The 50% inhibitory concentrations of each derivative were calculated and compared with those of unmodified heparin. Heparin activity was strongly dependent on molecular weight (Mr) in a manner grossly comparable for the two inhibitors. High-Mr fractions were the most active. Below 10 kDa, the activity dropped rapidly. A minimum size of 26 residues appeared to be required for HC II activation (against 16-18 for AT III). Below 5 kDa, a residual activity two orders of magnitude lower than that of high-Mr species remained with HC II (but not with AT III). Heparin was selectively desulfated or oversulfated in the O- and/or N-position. When an N-acetyl group was substituted for the original N-sulfate in the glucosamine and the derivative was oversulfated in the O-position, a strong activity with HC activities with both inhibitors decreased when the overall sulfate content (i.e., the charge density) was reduced, and vice-versa. Carboxyl-reduced heparin was also inactive but activity could be restored after O-sulfation. Our results thus suggest that, unlike the case of AT III, no functional group in heparin is critical for optimal thrombin inhibition by HC II. Sulfate and carboxylate are important in as much as they contribute to the global charge of the molecule.

O-Acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase α-smooth muscle actin expression in cultured arterial smooth muscle cells

Abstract Glycosaminoglycans, particularly heparin and heparin fragments, were specifically O -acetylated by use of tetrabutylammonium or tributylammonium salts of the anionic polysaccharides, carboxylic acid anhydrides, and 4-dimethylaminopyridine in an homogeneous way in N,N -dimethylformamide. The number of acyl chains introduced on the carbohydrate backbone was determined either after transesterification and quantitative analysis of the butyl esters thus obtained by GLC or by 1 H NMR spectroscopy.

The first total synthesis of the antithrombin III binding site of porcine mucosa heparin

Selectively O-acylated derivatives of various glycosaminoglycans were prepared and tested in vitro for their anticoagulant activity and their antiproliferative effect on rat and rabbit smooth muscle cells. When O-acylation (butyrylation or hexanoylation) had been performed on periodate-depolymerized heparin fragments having very low anticoagulant activity, the antiproliferative potency was markedly increased (IC50 = 2 and 1 micrograms/ml respectively, versus 31 micrograms/ml for starting compound) without an increase in anticoagulant activity. The antiproliferative activity was related to the degree of acylation. The O-acylated derivatives of heparin fragments were also very active in reversing the de-differentiation of smooth muscle cell in culture, as estimated by the increase in the expression of alpha-smooth muscle actin and alpha-smooth muscle actin mRNA.

Synthesis and biological activity of a new anti-factor Xa pentasaccharide with a C-interglycosidic bond

Abstract The synthesis of a pentasaccharide containing a N-acetyl-glucosamine unit and corresponding to the major natural sequence required in heparin for binding to antithrombin III is reported for the first time. This compound elicits anti-factor Xa activity and high affinity for antithrombin III.

Structural Determinants of the Capacity of Heparin to Inhibit the Proliferation of Vascular Smooth Muscle Cells. II. Evidence for a Pentasaccharide Sequence That Contains a 3-O-Sulfate Group

Abstract A mixed synthetic C, O-pentasaccharide 17 has been synthesized and shown to display an anti-factor Xa activity similar to that of the corresponding O-pentasaccharide 18. 17 represents the first example of a synthetic C-oligosaccharidic mimetic eliciting a significant biological response.