Keratin hydrogel hemostasis–cellular and molecular mechanisms
Abstract number: PP-WE-703
Orebaugh1 C., Bahawdory2 M., Stahle1 M.C., Rouse3 J., Burnett3 L., Van Dyke3 M., Hantgan1 R.R.
11Biochemistry 22Molecular Medicine and Translational Sciences 33Wake Forest Institute of Regenerative Medicine, Wake Forest University School of Medicine, Winston Salem, USA
How-to-cite Orebaugh C, Bahawdory M, Stahle MC, Rouse J, Burnett L, Van Dyke M, Hantgan RR. Keratin hydrogel hemostasis–cellular and molecular mechanisms. Journal of Thrombosis and Haemostasis 2009; Volume 7, Supplement 2: Abstract PP-WE-703
Keratin hydrogels display unprecedented efficacy in preventing blood loss in a porcine model of severe arterial hemorrhage. This study tested the hypothesis that keratin: integrin interactions avidly link activated platelets to vascular injury sites, forming an emergency seal quickly reinforced by tissue factor-mediated coagulation.
Methods: Keratin hydrogels, formulated from proteins extracted by reductive denaturation of human hair, were characterized by SDS-PAGE, UV-vis absorbance, protein and thiol assays, and turbidity measurements, as well as scanning electron microscopy, to determine their composition and structure. Platelet adhesion to keratin, fibrin, collagen hydrogels was measured under static conditions by a colorimetric microtiter plate assay. Time-dependent turbidity measurements examined the effects of keratin hydrogels on the kinetics of tissue factor-catalyzed coagulation in plasma (+/- platelets).
Results: Protein analyses demonstrated that concentrated keratin extracts, mixtures of a-keratin monomers and multimers, as well as g-keratins, formed stable hydrogels following 18 h incubation at 37°C; turbidity data indicated fibers ∼150 keratin subunits/cross section. Adhesion assays demonstrated that gel filtered platelets adhered to keratin hydrogels nearly as avidly as to fibrin and more effectively than to collagen. Inhibitory monoclonal antibodies specific for the A2, A5 and B1 integrin subunits reduced adhesion to near-background levels measured with formaldehyde-inactivated platelets. SEM revealed activated platelets adherent to a porous keratin gel network. These observations suggest that keratin's integrin-targeting sequences, presented on a coiled-coil fibrous scaffold, enable it to bind to A2B1 or A5B1 integrins. Coagulation kinetic data indicated platelet adhesion to keratin hydrogels yielded a modest acceleration of the extrinsic coagulation pathway.
Conclusions: Our mechanistic observations suggest that keratin hydrogels function so effectively in animal models of traumatic arterial injury because they trigger sustained and localized hemostasis by filling the wound with an exogenous matrix protein (EXM) that avidly recruits platelets and accelerates coagulation.
Disclosure of interest: Mark Van Dyke, KeraNetics, Shareholder, Grant Research Support.