A Synthetic Blood Clot that Forms in Response to Numerous Specific Stimuli

Chan, Karen Y. T. (University of British Columbia)
Yeon, Ju Hun (University of British Columbia)
Wong, Ting-Chia (University of British Columbia)
Kastrup, Christian J. (University of British Columbia)


Creating smart materials that form in response to environmental cues will require strategies for detecting an onslaught of diverse molecules, materials, and cells, and responding to a specific subset. This has not been achieved in synthetic materials, but biological systems have evolved to utilize complex biochemical networks to do this. This responsiveness is often the result of complex biochemical and genetic networks that sense numerous, diverse regulators. For example, the formation of blood clots is carefully controlled by a multitude of co-operative on- and off-switches that regulate an intricate network of enzymatic reactions that polymerize and further cross-link fibrin. In contrast, current synthetic materials can typically only respond to a single signal with high specificity, or respond to diverse stimuli but cannot distinguish between similar signals. Materials have been developed that can be solidified by three or four distinct stimuli, but materials that form in response to multiple distinct stimuli from diverse classes of signals have not yet been produced. We asked the question: Can the blood coagulation network still form a material in response to its plethora of natural triggers when its natural product is replaced with a synthetic one? Here we show that the coagulation network’s output, the fibrin clot, can be replaced with a synthetic cross-linkable polymer, thereby creating a smart material that is controlled by the network’s same diversity of specific stimuli. During clotting, fibrin is stabilized when the inactive precursor factor XIII (FXIII) is converted to FXIIIa by thrombin, one of the network’s central proteases. FXIIIa is a transglutaminase that cross-links glutamine to lysine residues or other substrates containing primary amines. As a promiscuous enzyme, FXIIIa has many known substrates, including synthetic polymers that contain peptides. For example, a branched polyethylene glycol (PEG) macromer has been cross-linked by purified FXIIIa in buffered systems when conjugated to appropriate peptides. We used this well-characterized reaction, and the coagulation network’s ability to activate FXIIIa, to couple the polymerization of the PEG macromer to the coagulation network.

Materials and Methods

A peptide sequence derived from α2-antiplasmin, acetyl-NQEQVSPLTLLKKGC, was conjugated to malemide groups on eight arms of branched PEG. In a buffered solution, the macromer was polymerized in a reaction mixture containing human FXIIIa, spermidine, and CaCl2. To form the MCBN by polymerizing the macromer in a mixture containing blood plasma, the reaction mixture contained synthetic macromer, fibrinogen-deficient plasma, purified FXIII, spermidine, and CaCl2 in HEPES buffer. Thromboelastography (TEG) was used to determine the mechanical properties and shear elastic moduli of the materials.


To test whether the coagulation cascade enzymes could control the formation of a synthetic material that is polymerizable by FXIIIa, the PEG macromer was added to human plasma lacking fibrinogen, the soluble precursor of fibrin (Fig. 1a). When blood coagulation was triggered with silica, formation of the polymerized MCBN occurred and the shear elastic modulus increased over 40-fold compared to when no activator was added. The MCBN had mechanical properties that differed from fibrin. The MCBN had an elastic modulus over 10-fold that of a normal fibrin clot (Fig. 1b). Addition of serine protease inhibitors or a FXIIIa inhibitor prevented polymer formation, verifying that formation was coupled to the coagulation network. Using both conventional scanning electron microscopy (SEM) and cryo-SEM, the MCBN more resembled the fibrous and porous structure of fibrin (Fig. 1c). Coagulation is responsive to several distinct classes of stimuli. To test if the formation of the MCBN responds to diverse stimuli when coupled to the coagulation network, its formation time was measured after adding known activators and inhibitors of coagulation. The activators included two enzymes (coagulation factor Xa and thrombin), tissue factor, and inorganic silica nanoparticles. The inhibitors included two small molecules (rivaroxaban and DAPA), a small protein, hirudin, an enzyme, activated protein C, and a polysaccharide, fondaparinux. These 9 diverse and specific modulators of coagulation significantly altered the formation time of the MCBN (Fig. 2a, b). When the macromer was not coupled to the network, but was polymerized with purified FXIIIa alone, the stimuli did not significantly change formation time (Fig. 2c).

Discussion and Conclusion

This work shows that the formation of a material can be coupled to a functional biological network to achieve a level of precise control that is typically only seen in nature, and unprecedented in synthetic materials. The MCBN formed in response to a multitude of specific chemical and biological signals to polymerize via activation of its cross-linking enzyme. The MCBN formed in response to a distinct inorganic solid, polyphenol, polysaccharide, membrane protein, small molecules, phospholipids, enzymes, and specific cellular and viral phenotypes. Thus, smart materials can be engineered to utilize biochemical networks, replacing these networks’ outputs while preserving the ability to sense diverse signals with high selectivity.

Figure 1. a, A synthetic material coupled to the biochemical reaction network of blood coagulation that can indirectly sense stimuli of the network. b, Gel formation times (blue bars) and shear elastic modulus (red bars). c, Scanning electron micrographs (top) and cryo-SEM (bottom). Scale = 20 µm.

Figure 2. a-b, Formation of the MCBN can be controlled by diverse and specific modulators, including activators (a) and inhibitors (b) of coagulation. *p < 0.05. c, The formation times without the network, using purified FXIIIa, did not respond to the modulators (all p > 0.05).


This work was funded by Canadian Institutes of Health Research (MOP-119426 and MSH-130166), the Canadian Foundation for Innovation (31928), and the BC Knowledge Development Fund.

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