Precision chemoenzymatic tailoring of bioengineered low-molecular-weight heparins for protamine-reversible anticoagulation.

Animal-derived low-molecular-weight heparins (LMWHs) are essential anticoagulants but are associated with supply chain vulnerabilities and contamination risks, alongside incomplete protamine reversibility in clinical practice. Herein, we report the precision chemoenzymatic tailoring of bioengineered LMWHs from Escherichia coli K5 capsular polysaccharide (heparosan) to achieve protamine-reversible anticoagulation. Through the integration of chemical N-deacetylation/N-sulfation, precisely regulated C5-epimerization/2-O-sulfation, controlled β-eliminative depolymerization, and sequential enzymatic 6-O/3-O-sulfation, the molecular weight distribution and specific sulfation patterns were rationally modulated. The representative bioengineered product, L3S-2, exhibited potent anti-factor Xa activity, an optimized anti-factor Xa/anti-factor IIa ratio, and pharmacokinetic properties comparable to enoxaparin following subcutaneous administration. Crucially, L3S-2 demonstrated significantly improved protamine reversibility both in vitro and in vivo compared to commercial enoxaparin. In rat thrombosis models, L3S-2 potently inhibited venous and arterial thrombus formation, demonstrating efficacy comparable to enoxaparin. These findings establish a rational design framework for bioengineered anticoagulants, demonstrating that optimizing multivalent electrostatic interactions with protamine via tailored sulfation and molecular weight yields highly reversible agents with optimized therapeutic indices.