Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation
Christoph Reinhardt, … , Steffen Massberg, Bernd Engelmann
Christoph Reinhardt, … , Steffen Massberg, Bernd Engelmann
Published February 14, 2008
Citation Information: J Clin Invest. 2008;118(3):1110-1122. https://doi.org/10.1172/JCI32376.
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Research Article

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

Abstract

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.

Authors

Christoph Reinhardt, Marie-Luise von Brühl, Davit Manukyan, Lenka Grahl, Michael Lorenz, Berid Altmann, Silke Dlugai, Sonja Hess, Ildiko Konrad, Lena Orschiedt, Nigel Mackman, Lloyd Ruddock, Steffen Massberg, Bernd Engelmann

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Figure 6

Constitutive glutathionylation of TF.

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TF disulfide exchange supports fibrin production in vivo. (A) Prevention...
(A) Basal glutathionylation of cell TF. Solubilized monocytes were immunoprecipitated with anti-GSH antibody or with isotype control antibody, as described in Methods. This was followed by western blotting with control antibody or anti-TF antibody. (B) Structure of C209 containing peptide STDSPVEC*MGQEK as identified by MS/MS fragmentation. The spectrum was generated by collision-induced dissociation (CID) of the doubly charged 858.33-m/z ion. CID of doubly charged precursor ions predominately forms singly charged product ions that are indicative of the sequence of a peptide. The product ions thus generated are b-ions from the N-terminal site and y-ions from the C-terminal site. Peptide fragments observed in the mass spectrometer are shown in bold. The mass difference between b7 and b6 ions shows that the cysteine contains a modification equal to a glutathionylation from the N-terminal site. This is confirmed by the same mass difference between the y5 and y6 ion from the C-terminal site. (C) Glutathionylation attenuates TF activation. Cell lysis increases procoagulant activity, relative to intact cells, which is completely inhibited by anti-TF antibody. Increasing concentrations of DTT suppress TF activity. Glutathionylation in lysed cells was induced by preincubation with DTT (0.1 mM), followed by incubation with GSH (0.1 mM plus 0.1 mM diamide; 15 minutes). This increased the density of the band representing glutathionylated TF in immunoblots of lysed monocytes by 5.5-fold. Procoagulant activity of the lysates was determined by coagulation factor concentrate. *P < 0.05 versus control. n = 3–9.