A critical role for extracellular protein disulfide isomerase during thrombus formation in mice
Jaehyung Cho, … , Shaun R. Coughlin, Bruce Furie
Jaehyung Cho, … , Shaun R. Coughlin, Bruce Furie
Published February 21, 2008
Citation Information: J Clin Invest. 2008;118(3):1123-1131. https://doi.org/10.1172/JCI34134.
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A critical role for extracellular protein disulfide isomerase during thrombus formation in mice

Abstract

Thiol isomerases, including protein disulfide isomerase (PDI), catalyze disulfide oxidation, reduction, and isomerization, thereby playing an important role in protein synthesis. To determine whether extracellular PDI mediates thrombus formation in an animal model, PDI expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice by intravital fluorescence microscopy following laser-induced arteriolar injury. A time-dependent increase in PDI was observed in murine thrombi following injury. Infusion of the PDI inhibitor bacitracin or a blocking monoclonal antibody against PDI inhibited platelet thrombus formation and fibrin generation. Fibrin deposition is normal in mice lacking the G protein–coupled platelet receptor Par4, although there is no stable accumulation of platelets. Infusion of monoclonal antibodies against PDI into the circulation of Par4–/– mice prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required in vivo in mice for both fibrin generation and platelet thrombus formation.

Authors

Jaehyung Cho, Barbara C. Furie, Shaun R. Coughlin, Bruce Furie

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Figure 5

Inhibition of fibrin formation and platelet accumulation in wild-type mice as a function of increasing concentrations of inhibitory RL90 antibody to PDI.

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Inhibition of fibrin formation and platelet accumulation in wild-type mi...
(A) Fibrin was labeled with a fibrin-specific antibody conjugated to Alexa Fluor 488, and platelets were labeled with Fab fragments of anti-CD41 antibodies conjugated to Alexa Fluor 647. The time course of appearance of fluorescence associated with fibrin (green) and platelets (red) is shown over 180 s following laser-induced vessel wall injury in wild-type mice within the context of the bright-field microvascular histology. Inhibitory monoclonal anti-PDI antibody RL90 was infused into the circulation 5 min prior to injury at 0, 0.3, 1.0, and 3.0 μg/g BW (columns 1–4, respectively). In all experiments when no inhibitory monoclonal anti-PDI antibody was infused, 3.0 μg/g BW of isotype-matched IgG2a was infused instead. (B and C) Median integrated platelet fluorescence (B) and median integrated fibrin fluorescence (C) for thrombi formed in the presence of increasing concentrations of the anti-PDI antibody RL90 — 0 μg (curve 1; 27 thrombi, 3 mice), 0.3 μg (curve 2; 28 thrombi, 3 mice), 1.0 μg (curve 3; 23 thrombi, 3 mice), and 3.0 μg (curve 4; 21 thrombi, 3 mice) — are presented versus time after vessel wall injury. A representative binarized image is shown in A, and the complete data sets of this and multiple identical experiments are presented in B and C, which plot the integrated fluorescence intensity of all pixels in the image, regardless of their intensity, as a function of time.