Teasing apart active site contributions

Thrombus formation at sites of vascular injury is essential for the healing process. Protein disulfide isomerase (PDI) is a secreted enzyme that catalyzes disulfide bond formation and has been shown to both inhibit and promote coagulation. There are 2 redox-active catalytic sites, at the N- and C-terminus, respectively, within PDI; however, the roles of these sites in platelet function during coagulation are unclear. Junsong Zhou, Yi Wu, and colleagues at Soochow University and Temple University School of Medicine developed several mouse models to evaluate PDI in thrombosis and the contribution of the N- and C-terminal CGHC redox-active sites. Mice specifically lacking PDI within platelets had increased tail bleeding times and impaired platelet function but no defect in fibrin deposition, while animals lacking PDI within platelets and vessel walls had substantial defects both in platelet function and fibrin deposition at the site of injury. Platelet accumulation was attenuated in PDI-deficient animals with transgenic expression of PDI lacking the C-terminal CGHC motif (PDI(ss-oo)). In PDI-deficient mice and those expressing PDI(ss-oo), transfusion of PDI with a functional C-terminal CGHC motif, but lacking the N-terminal active site (PDI_ss-oo) rescued these defects. In human platelets, addition of PDI(ss-oo) inhibited aggregation, while PDI(oo-ss) enhanced aggregation. Moreover, the PDI C-terminal CGHC motif mediated P-selectin secretion from platelets in an αIIbβ3-independent manner. Together, these results demonstrate that the C-terminal redox-active site of PDI is essential for platelet function and coagulation. The accompanying movie shows that leukocytes are not involved in thrombus formation in the model used in this study (Videos 1 and 2) and that recombinant PDI(ss-oo) accumulates at the site of injury in both WT (Video 3) and PDI-deficient mice (Video 4).

Published November 3, 2015, by Corinne Wiliams

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The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis
Junsong Zhou, … , Mortimer Poncz, David W. Essex
Junsong Zhou, … , Mortimer Poncz, David W. Essex
Published November 3, 2015
Citation Information: J Clin Invest. 2015;125(12):4391-4406. https://doi.org/10.1172/JCI80319.
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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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