Heparin Modulates SARS-CoV-2 Spike−Platelet Factor 4 Interactions through Concentration-Dependent Interface Remodeling

Abstract

Heparin, a highly sulfated glycosaminoglycan, interacts with numerous proteins through electrostatic and multivalent binding mechanisms. In the context of SARS-CoV-2 infection, platelet factor 4 (PF4) has been proposed to form complexes with the viral spike protein (S protein), potentially contributing to immune-mediated thrombotic complications. However, the molecular mechanisms by which heparin modulates PF4-S protein interactions remain poorly understood. Here, we investigate how unfractionated heparin (UFH) influences the formation and stability of PF4-S protein complexes using a combination of ensemble binding assays, single-molecule force spectroscopy, and molecular dynamics simulations. ELISA measurements reveal concentration-dependent and partner-specific effects of UFH on S protein interactions. While low concentrations of UFH enhance ACE2-S protein binding, higher concentrations produce a modest reduction, indicating biphasic modulation of receptor engagement. In contrast, UFH inhibits PF4-S protein binding at low to intermediate concentrations, with partial restoration at higher levels. Single-molecule force spectroscopy confirms that UFH decreases the mechanical stability of the PF4-S protein complex, as reflected by reduced unbinding forces that reach saturation at concentrations ≥5 IU mL−1. Molecular dynamics simulations using the dp5 heparin fragment demonstrate energetically favorable binding to both PF4 and the S1 subunit of the S protein. In ternary S1-PF4-dp5 assemblies, heparin alters interfacial contacts and reshapes the energetic landscape of the protein-protein interaction, suggesting modulation through electrostatic reorganization and interfacial remodeling. These findings provide mechanistic insight into glycosaminoglycan-mediated regulation of PF4-S protein complexes and highlight how heparin can modulate biomacromolecular interactions at viral-host protein interfaces.