Immunogenic risk assessment of porcine, bovine, and ovine heparins for diversifying the global heparin supply.

BACKGROUND

The global heparin supply relies heavily on porcine mucosa, prompting renewed interest in ruminant-derived alternatives. However, concerns regarding heparin-induced thrombocytopenia remain a major barrier to their clinical reintroduction.

OBJECTIVES

To systematically evaluate the platelet factor 4 (PF4)-dependent immunogenic potential of porcine mucosal heparin, bovine lung heparin, and ovine mucosal heparin, as well as their low-molecular-weight heparin (LMWH) derivatives, and to delineate the structural features governing these interactions.

METHODS

Size-exclusion chromatography and a competitive ELISA established in this study were used to monitor PF4-heparin complex stoichiometry and antibody recognition. Flow cytometry quantified functional heparin-induced platelet activation (CD62P/CD63 expression). PF4 affinity chromatography-hydrophilic interaction liquid chromatography-mass spectrometry and molecular docking characterized high-affinity glycan features and evaluated sulfation patterns in stabilizing PF4-heparin interactions.

RESULTS

Under equipotent dosing conditions, PF4 complex formation, antibody recognition, and platelet activation followed the order bovine lung heparin > ovine mucosal heparin > porcine mucosal heparin. Differences among heparins were largely explained by molecular weight-dependent stoichiometry. In contrast, source-dependent differences among LMWHs persisted after potency normalization, with bovine LMWH forming significantly more PF4 complexes and triggering the most robust platelet activation. Affinity profiling identified degree of polymerization 6-degree of polymerization 8 (dp6-dp8) as minimal PF4-binding units and revealed that increased sulfation and reduced N-acetylation enhance PF4 affinity. Molecular docking confirmed that N-sulfation stabilizes PF4-heparin interactions.

CONCLUSION

Heparins derived from different animal sources exhibit distinct PF4-binding properties and immunogenic profiles. These findings provide mechanistic insight into species-dependent heparin-induced thrombocytopenia risk-driven by stoichiometry in heparin and fine structural features in LMWH-and support diversification of the global heparin supply.