A fibrin biofilm covers blood clots and protects from microbial invasion
Fraser L. Macrae, … , Heiko Herwald, Robert A.S. Ariëns
Fraser L. Macrae, … , Heiko Herwald, Robert A.S. Ariëns
Published May 3, 2018
Citation Information: J Clin Invest. 2018;128(8):3356-3368. https://doi.org/10.1172/JCI98734.
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Research Article Hematology Vascular biology

A fibrin biofilm covers blood clots and protects from microbial invasion

Abstract

Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.

Authors

Fraser L. Macrae, Cédric Duval, Praveen Papareddy, Stephen R. Baker, Nadira Yuldasheva, Katherine J. Kearney, Helen R. McPherson, Nathan Asquith, Joke Konings, Alessandro Casini, Jay L. Degen, Simon D. Connell, Helen Philippou, Alisa S. Wolberg, Heiko Herwald, Robert A.S. Ariëns

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Figure 2

Film only requires fibrin for formation.

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Film only requires fibrin for formation. (A) The film was peeled from cl...
(A) The film was peeled from clots using a needle. (B) Films removed from clots produced with or without T101 were run alongside FXIII, BSA, purified fibrin, and purified fibrinogen (Fgn) on reducing SDS-PAGE. MW, molecular weight marker. (C) SEM of the film in a clot produced from purified fibrinogen. A tear in the film exposes the underlying clot, as was often observed in SEM. Scale bar: 10 μm. (D) LSCM of the film at the air-liquid interface of a clot produced from purified fibrinogen, without breaks in fully hydrated conditions. Fibrinogen fluorescently labeled with Alexa Fluor 488. Scale bar: 50 μm. (E) SEM image of areas of film transitioning into fibers. Scale bar: 1 μm. (F) LSCM of tethering fibers connected to the film. Scale bar: 50 μm. Images in A–F are representative of n = 3 experiments. (G–Q) Mean sheet fluorescence (MSF) measurements of the film comparing different conditions: (G) thrombin concentration, n = 4 experiments; (H) CaCl2 concentration, n = 4 experiments; (I) fibrinogen concentration, n = 6 experiments; (J) normal pool (NP; n = 3 patients) versus dysfibrinogenemia (Dysfib, n = 2 patients) and afibrinogenemia (Afib, n = 3 patients); (K) thrombin versus reptilase, n = 4 experiments; (L) temperature, n = 4 experiments; (M) FXIII, n = 4 experiments; (N) PPP versus PRP, n = 6 individuals; (O) γA/γA fibrinogen versus γA/γ′ fibrinogen, n = 6 experiments; (P) fibrinogen triple γ-chain crosslinking mutant (3xγ), n = 6 experiments; (Q) fibrinogen α-chain deletion mutants (α390 and α220), n = 6 experiments. Asterisks indicate difference from first column; crosses, difference between other columns. *P < 0.05; **P < 0.01, ††P < 0.01; †††P < 0.001; ****P < 0.0001, ††††P < 0.0001. Mean ± SD. Unpaired t test (2 groups); ANOVA (multiple groups).See Supplemental Table 1 for estimated corresponding film thicknesses.