Abstract

Chondroitin sulfate (CS) polysaccharides exhibit intrinsic structural heterogeneity, limiting precise studies of their bioactivities and therapeutic applications. To obtain homogeneous CS oligosaccharides and elucidate structure-activity relationships, we developed a controlled enzymatic method for preparing well-defined CSA oligosaccharides and investigated their chain length-dependent anticoagulant mechanisms. Herein, hyaluronidase was used to depolymerize chondroitin sulfate A and subsequently purified by sequential ultrafiltration and anion-exchange chromatography, yielding CSA oligosaccharide fractions (CSA4, CS6, CS8, CS10 and CSA12). Their structures were confirmed by NMR spectroscopy and mass spectrometry. In vitro anticoagulation assays showed significant chain-length dependence: longer oligomers (CSA8, CSA10 and CSA12) profoundly prolonged activated partial thromboplastin time (APTT) and thrombin time (TT), suggesting potentiation of endogenous serine protease inhibitors. However, shorter oligomers (CSA4 and CSA6) just selectively prolonged prothrombin time (PT), indicating extrinsic pathway modulation. This work provides a reliable method for preparing CSA oligosaccharides and establishes a molecular foundation for developing safer, predictable CS-based anticoagulants.