A synthetic heparin oligosaccharide decouples anticoagulation from heparin-induced thrombocytopenia risk

Low–molecular weight heparin (LMWH) remains clinically indispensable for the prevention and treatment of thrombosis in cardiovascular surgery, hemodialysis, and other critical care settings. However, LMWH is constrained by heparin-induced thrombocytopenia (HIT) and supply chain vulnerabilities of animal-derived sources. This study used chemoenzymatic synthesis to engineer homogeneous dodecasaccharide 12G-2, decoupling anticoagulant efficacy from HIT risk via dual structural modulation. Substitution with 2- O -sulfated glucuronic acid conferred conformational rigidity, and engineered sulfation attenuated platelet factor 4 (PF4) binding, whereas the antithrombin-binding domain remained intact. 12G-2 exhibited nanomolar anti–Factor Xa (FXa) activity in vitro, enoxaparin-equivalent venous antithrombotic efficacy, and superior arterial antithrombotic activity in murine thrombosis models, and it showed diminished PF4 binding, attenuated pathological β sheet formation, and minimal recognition by HIT-related antibodies in vitro. Both in vitro and in a murine tail-bleeding model, 12G-2 demonstrated high protamine reversibility, exceeding standard LMWH enoxaparin neutralization efficiency. In washed human platelets, 12G-2–PF4 complexes with the HIT-mimicking antibody KKO failed to induce integrin αIIbβ3 activation or P-selectin release and elicited minimal platelet spreading comparable to resting controls. Safety assessments in mice showed no hepatotoxicity at tested doses and low immunogenicity in a murine PF4-heparin immunization model. In PF4-heparin antibody–positive mice, 12G-2 attenuated platelet activation while exerting stronger FXa inhibition than enoxaparin. These findings established 12G-2 as an alternative to LMWH.