Phase Separation of Intrinsically Disordered Protein Polymers Mechanically Stiffens Fibrin Clots

Abstract

Fibrin (Fb) networks self‐assemble through the coagulation cascade and serve as the structural foundation of blood clots. Following severe trauma or drug therapy, reduced integrity of Fb networks can lead to formation of clots with inadequate mechanical properties. A key feature of therapeutic interventions for hemostasis is therefore the ability to restore mechanical strength to clots formed under coagulopathic conditions. Here, an intrinsically disordered protein based on an elastin‐like polypeptide (ELP) sequence is described, which specifically binds Fb and modulates its mechanical properties. Hemostatic ELPs (hELPs) are designed containing N‐ and C‐terminal peptide tags that are selectivity recognized by human transglutaminase factor XIIIa and covalently linked into fibrin networks via the natural coagulation cascade. Phase separation of hELPs above their lower critical solution temperature leads to stiffening and rescue of clot biophysical properties under simulated conditions of dilutive coagulopathy. In addition to phase‐dependent stiffening, the resulting hELP‐Fb networks exhibit resistance to plasmin degradation, reduced pore sizes, and accelerated gelation rate following initiation of clotting. These results demonstrate the ability of protein‐based phase separation to modulate the physical and biochemical properties of blood clots and suggest protein phase separation as a new mechanism for achieving hemostasis in clinical settings.

Protein conformation and biomolecular condensates

“…Extracellular deposits of proteins are ubiquitous. These include but are not limited to collagen, blood clot forming fibrin, insect elastic matrix protein resilin, hinge ligament of bivalve molluscs abductin, spider and insect silks, matrix proteins of squid suckers, attachment fibers and adhesives of mussels, and bacterial biofilms ( Muiznieks et al., 2018 ; Urosev et al., 2020 ; Seviour et al., 2020 ). Only two representative extracellular protein condensates, elastin and silk, will be briefly described.…”

Protein-Based Biological Materials: Molecular Design and Artificial Production

“…The large body of literature on biomedical applications has been regularly reviewed before, including recently by Varanko et al Here we only highlight a few examples of recent progress in the use of genetically engineered ELPs for medical applications. Urosev et al demonstrated a hemostatic-ELP protein variant by introducing charge and polar residues at the N- and C-termini. The ELP could be used to selectively bind to blood clots and modulate their physicochemical properties, such as improved resistance to enzymatic degradation and mechanical stability.…”

Folding and self-assembly of short intrinsically disordered peptides and protein regions

“…These interactions took place through Gln- and Lys-residues on Fb γ-chains and α-chains, and AA cross-linked with hELP through its Gln- and Lys-blocks. 174 Hossain et al used intrinsically disordered peptide-polymers (IDPPs) for post-translational modifications (PTMs) adding a lipid chain to encode non-equilibrium phase behaviour transitions, an emergent frontier in biomacromolecular engineering. The IDR was based on a tropoelastin (Gly–X–Gly–Val–Pro) 80 domain (containing a mixture of Ala : Val 2 : 8 in the X position), while the lipids tested were a canonical PTM (M-IDPP) and an azide (–N 3 ) non-canonical PTM (ADA-IDPP).…”