In Situ Crosslinking of Bioorthogonal Nanoparticles to Restore Clot Stability in Coagulopathic Blood

Intravenous hemostats have shown significant promise in prolonging survival for severe noncompressible and internal injuries in preclinical animal models. Existing approaches include the use of liposomes with or without procoagulant enzymes, as well as polymer nanoparticles or soluble biopolymers. While these methods predominantly target or mimic tissue components that are present during coagulation, such as activated platelets and collagen, they may not account for the loss of fibrinogen, which is not only key to clot formation but also the first protein to fall below critical levels in dilutional coagulopathy. This protocol describes the synthesis and in vitro or ex vivo characterization of a crosslinkable nanoparticle system that seeks to address dilutional coagulopathy by leveraging the critical gelation concentration and bioorthogonal click chemistry. The system was shown to only gel at high nanoparticle and crosslinker concentrations, increase the rate of platelet recruitment, and decrease the rate of clot degradation in a low-fibrinogen environment, providing a platform for treating severe hemorrhage in a coagulopathic environment. Ultimately, the contents of this protocol may assist researchers in the in vitro characterization and screening of other crosslinkable nanoparticle systems or hemostats, with potential expansions to other categories of coagulation dysfunction, such as embolism treatment. Key features • A protocol for the synthesis of nanoparticles with activated-platelet-binding moieties to mimic fibrin. • In vitro and ex vivo assays assessing complement activation, accumulated platelet recruitment, platelet recruitment under hemodilution, coagulation potential, and clot lysis. • The inclusion of hemodiluted and plasminolytic conditions creates a more physiologically relevant environment for screening of hemostatic agents. • The use of a two-component system helps reduce complement activation in intravenous therapies.