The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis
Junsong Zhou, Yi Wu, Lu Wang, Lubica Rauova, Vincent M. Hayes, Mortimer Poncz, David W. Essex
Junsong Zhou, Yi Wu, Lu Wang, Lubica Rauova, Vincent M. Hayes, Mortimer Poncz, David W. Essex
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Research Article Hematology

The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis

Abstract

Protein disulfide isomerase (PDI) has two distinct CGHC redox-active sites; however, the contribution of these sites during different physiologic reactions, including thrombosis, is unknown. Here, we evaluated the role of PDI and redox-active sites of PDI in thrombosis by generating mice with blood cells and vessel wall cells lacking PDI (Mx1-Cre Pdifl/fl mice) and transgenic mice harboring PDI that lacks a functional C-terminal CGHC motif [PDI(ss-oo) mice]. Both mouse models showed decreased fibrin deposition and platelet accumulation in laser-induced cremaster arteriole injury, and PDI(ss-oo) mice had attenuated platelet accumulation in FeCl3-induced mesenteric arterial injury. These defects were rescued by infusion of recombinant PDI containing only a functional C-terminal CGHC motif [PDI(oo-ss)]. PDI infusion restored fibrin formation, but not platelet accumulation, in eptifibatide-treated wild-type mice, suggesting a direct role of PDI in coagulation. In vitro aggregation of platelets from PDI(ss-oo) mice and PDI-null platelets was reduced; however, this defect was rescued by recombinant PDI(oo-ss). In human platelets, recombinant PDI(ss-oo) inhibited aggregation, while recombinant PDI(oo-ss) potentiated aggregation. Platelet secretion assays demonstrated that the C-terminal CGHC motif of PDI is important for P-selectin expression and ATP secretion through a non-αIIbβ3 substrate. In summary, our results indicate that the C-terminal CGHC motif of PDI is important for platelet function and coagulation.

Authors

Junsong Zhou, Yi Wu, Lu Wang, Lubica Rauova, Vincent M. Hayes, Mortimer Poncz, David W. Essex

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Figure 9

Intravascular PDI is required for fibrin deposition and platelet accumulation in vivo.

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Intravascular PDI is required for fibrin deposition and platelet accumul...
(A–C) Cremaster arteriole injury was induced in Pf4-Cre Pdifl/fl mice or Cre– Pdifl/fl littermate control mice. Platelets and fibrin were detected using anti-CD41 F(ab)2 fragments (which bind to platelet β3) conjugated to Alexa Fluor 488 and anti-fibrin antibody conjugated to Alexa Fluor 647. (A) Representative intravital microscopy fluorescence images show platelet accumulation (green) and fibrin deposition (red) at selected time points up to 180 seconds after vascular injury. Original magnification, ×64. Scale bar: 25 μm. The median integrated FIs of (B) anti-CD41 (platelet) and (C) anti-fibrin (fibrin) antibodies over 270 seconds. (D–F) Cremaster arteriole injury was induced in Mx1-Cre Pdifl/fl mice or Cre– Pdifl/fl littermate control mice. Mx1-Cre Pdifl/fl mice were infused with rPDI(oo-ss) (200 μg per mouse). (D) Representative intravital microscopy fluorescence images show platelet accumulation (green) and fibrin deposition (red). Original magnification, ×64. Scale bar: 25 μm. The median integrated FIs of (E) anti-CD41 (platelet) and (F) anti-fibrin (fibrin) antibodies. Fluorescence signal was not observed using fluorescently labeled control IgG (data not shown). The data were analyzed by area under curve, with a Mann-Whitney rank-sum test (37). Only significant differences are shown; *P < 0.05; ***P < 0.001. The data were obtained from 30 thrombi in 3 mice for each experimental condition.