Extração, caracterização, atividade antitrombótica e o efeito na síntese de Heparan Sulfato Endotelial de Dermatan sulfato de diferentes origens.

Abstract

Dermatan sulfate (DS) is an anionic, linear, and structurally complex polysaccharide belonging to the glycosaminoglycans (GAG) class, found in a wide variety of tissues in many species attached to a core protein as proteoglycans (PG). DSPGs have been implicated in various cellular processes and cell-matrix interactions. DS chains may also influence the coagulation process, binding to serum proteins and inhibiting thrombin (interacting with HCII). This study aimed to extract GAGs from different tissues, and investigate DS effects upon coagulation hemostasis. GAGs were extracted by proteolysis, precipitated, desalted, lyophilized, submitted to agarose gel electrophoresis and chemical dosages (hexosamine, uronic acid). GAGs from porcine skin (PS) and bovine cornea (BC) were, further, purified by anion exchange chromatography and characterized by agarose gel electrophoresis, molecular mass, chemically (hexosamine, uronic acid, and sulfate contents), and spectroscopically (IR, RAMAN, and RMN). The purified polysaccharides were evaluated in experimental animal models of thrombosis and the stimulation of heparan sulfate proteoglycan synthesis by endothelial cells in culture. Different GAGs could be extracted from various tissues with high yields. The purified polysaccharides from CB and PS have a distinct polymeric structure. BC-DS showed the highest molecular mass and lower sulfate: hexosamine ratio (DS-BC=0,802, DS-PS=1,189), also it seems to have the highest content of IdoA when compared with PS-DS (GlcAC/HEX: DS-BC=0,619, DSPS=1,109). Although the spectroscopical analysis of the purified carbohydrates showed similar spectra, it is possible to find some subtle differences that might be influenced by the chemical environment on each of their polymeric structure. No other contaminants were identified on the samples. DS-BC and DS-PS showed similar effects on coagulation, with BC-DS ceasing thrombi formation with a lower dose (50µg/g) compared to DS-PS (75µg/g). Both GAGs were capable of stimulating the synthesis of endothelial HSPG secreted to the medium. We highlight the findings on antithrombotic properties of DS from PS and BC, and presented a new result regarding its properties on the endothelial cell wall, stimulating the synthesis of HS, which have been found to promote angiogenic responses of endothelial cells beyond its antithrombotic properties.