Although unfractionated heparin (UFH) remains a vital agent for rapid anticoagulation, its reliance on animal-derived sources results in batch-to-batch variability and contamination risks, and its clinical application is further restricted by the possibility of heparin-induced thrombocytopenia. Dendritic polyglycerol sulfates were investigated as heparin analogues in 2004 due to their ability to mimic its charge, which is essential for its acting mechanism, and revealed an anticoagulant activity of 15-35% compared to UFH. In the current study, we found that the anticoagulant effect of polyglycerol sulfates further increases with their flexibility, resulting in a comparable activity of linear polyglycerol sulfate to UFH. Furthermore, we comprehensively analyzed the mechanism of action and discovered an antithrombin-independent, thrombin-selective mechanism. Moreover, we confirmed that FDA-approved protamine sulfate is a viable reversal agent for polyglycerol sulfate.