Efficient Synthesis of Heparinoid Bioconjugates for Tailoring FGF2 Activity at the Stem Cell-Matrix Interface.

Heparan sulfate glycosaminoglycans (HS GAGs) attached to proteoglycans harbor high affinity binding sites for various growth factors (GFs) and direct their organization and activity across the cell-matrix interface. Here, we describe a mild and efficient method for generating HS-protein conjugates. The two-step process utilizes a "copper-free click" coupling between differentially sulfated heparinoids primed at their reducing end with an azide handle and a bovine serum albumin protein modified with complementary cyclooctyne functionality. When adsorbed on tissue culture substrates, the glycoconjugates served as extracellular matrix proteoglycan models with the ability to sequester FGF2 and influence mesenchymal stem cell proliferation based on the structure of their HS GAG component.

Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/Rf ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments.

Comparative study on the in vitro and in vivo activities of heparinoids derivative investigated on the animal model.

In this study we compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulfate (Na-PPS, Ca-PPS), unfractionated heparin (UFH), and low-molecular-weight heparin (Fraxiparin). The antithrombotic effects of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules with a diameter of 20-30 microm were injured by well-defined argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities [activated partial thromboplastin time (aPTT), Heptest] of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses >10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS, an antithrombotic effect was not observed. Oral application of Ca-PPS in doses >20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose of 10 mg/kg, the aPTT increased threefold and the Heptest 2.5-fold compared with controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS, no effect on the coagulation test could be measured. Intravenous injection of UFH prolonged the Heptest after 1 min and the aPTT after 30 min. In ex vivo studies of aPTT and Heptest performed in rat plasma between 2 and 24 h after s.c. injection of 0.2 mg/kg Fraxiparin, no inhibition of any coagulation test was measured. The antithrombotic effect of 0.2 mg/kg Fraxiparin after s.c. injection was significant. Intravenous injection of 20 U/kg UFH significantly inhibited thrombus formation. The smallest antithrombotic effect was after i.v. injection of UFH.

Heparinoids: structure, biological activities and therapeutic applications.

Heparin is an important polyanionic drug having a wide variety of different biological activities. Substantial research effort has focused on the preparation of improved heparins and heparin analogues that might exhibit higher specificity and decreased side effects. These heparin analogues or heparinoids include sulfated polysaccharides from plant and animal origin, synthetic derivatives of polysaccharides, and acidic oligosaccharides and their small synthetic analogues. The structure, biological activities and therapeutic potential of these heparinoids are discussed.