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 December 1, 2015; First 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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Figure 1

Deletion of Pdi in platelets.

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Deletion of Pdi in platelets. (A) Scheme of the targeting strategy. Homo...
(A) Scheme of the targeting strategy. Homologous recombination of the Pdi conditional targeting vector into the Pdi gene in embryonic stem cells introduced 2 loxP sites (triangles) upstream of exon 1 and the ATG start codon and downstream of exon 2 and 2 FRT sites (surrounding the Neo cassette that was used for selection). The targeted neo cassette allele (recombined locus) was removed by Flp-mediated recombination of FRT sites (to form the conditional allele, fl). Pf4-Cre recombinase (Cre) expressed with the Pdi conditional allele deletes exons 1–2, generating the Pdi-deleted allele. (B) Southern blot analysis of heterozygous Flp-excised Pdi-floxed mice. The PciI-digested mouse genomic DNAs (digestion sites indicated in A) were analyzed by hybridization with the external 5′ probe (ext 5′ probe, A). The wild-type allele with the Pdi endogenous locus (WT) and the Flp-mediated excised allele (Flp exc) gave rise to 5.1-kb and 8.1-kb hybridization bands, respectively. (C–F) Characterization of platelets of Pf4-Cre Pdifl/fl mice. (C) Platelet mRNA levels were evaluated by RT-PCR to demonstrate the absence of Pdi mRNA. (D) Western blots of lysates using a polyclonal rabbit anti-PDI antibody and antibodies against ERp57, ERp5, and ERp72. The PLCγ2 loading controls for PDI are shown. Separate loading controls were run for ERp57, ERp5, and ERp72 with similar amounts of protein found in each sample (data not shown). Blots in C and D are representative of 3 separate experiments. (E) Comparison of platelet counts between Pdifl/fl and Pf4-Cre Pdifl/fl mice; mean ± SEM, n = 10, t test. (F) Glycoprotein (αIIbβ3, GPIb, or GPVI) expression on platelets from Pf4-Cre Pdifl/fl and Pdifl/fl littermate controls was analyzed by flow cytometry; mean ± SEM, n = 10, t test. MFI, mean FI.