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Background <p>Hereditary plasminogen (PLG) deficiency represents an extremely rare autosomal recessive disorder characterized by impaired fibrinolytic capacity resulting from diminished PLG enzymatic activity. In this study, we identify and characterize a novel compound heterozygous PLG mutation clinically associated with cerebral infarction. Our findings demonstrate that structural conformational alterations in the mutation PLG protein disrupt normal fibrinolytic pathway function.</p> Methods <p>In this study, the proband presented at the First Affiliated Hospital of Wenzhou Medical University with a chief complaint of “left-sided limb weakness lasting for two days.” PLG activity (PLG: A) and PLG antigen (PLG: Ag) levels were measured in the proband and eight family members spanning three generations using the chromogenic substrate assay(CSA) and enzyme-linked immunosorbent assay (ELISA), respectively. The genetic mutation site was identified through direct DNA sequencing. Bioinformatics software was employed to analyze the conservation and potential pathogenicity of the mutation site. A mutation protein model was constructed to investigate structural alterations in the protein before and after the mutation. Furthermore, a recombinant plasmid expression vector was developed, and the in vitro expression of the recombinant PLG protein was evaluated using real-time quantitative PCR (RT-qPCR), ELISA, and Western blot(WB) analysis.</p> Results <p>The propositus had a significantly reduced PLG: A to 27%, but the PLG: Ag level was normal at 103%, and was diagnosed with type II plasminogen deficiency (dysplasminogenemia). Sanger sequencing identified compound heterozygous missense mutations in the PLG gene: c.1702G &gt; A (p.Gly568Arg) in exon 14 and c.1858G &gt; A (p.Ala620Thr) in exon 15. Both mutation sites are highly conserved across species, and four independent bioinformatics tools consistently predicted pathogenic effects. Molecular modeling demonstrated that the p.Gly568Arg substitution causes steric hindrance through side chain elongation and establishes a new hydrogen bond with Leu686; p.Ala620Thr destabilized the catalytic triad (His603-Asp646-Ser741). In vitro functional assays, including RT-qPCR, ELISA, and WB analysis, confirmed that neither mutation significantly altered the expression level of mRNA、protein biosynthesis and secretion. However, the PLG: A/PLG: Ag ratio in the media was markedly reduced compared to wild-type controls.</p> Conclusions <p>We report the first case worldwide of a novel p.Gly568Arg mutation in the PLG gene, which coexists with the p.Ala620Thr mutation in a compound heterozygous form. Structural and functional analyses indicate that although the transcription, protein synthesis and secretion of the PLG gene are not affected, these two mutations specifically impair the catalytic activity of the PLG protein through different conformational changes. This expands the mutation spectrum and clarifies a novel thrombosis mechanism in type II PLG deficiency.</p>

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Novel compound heterozygous mutations in plasminogen (p.Gly568Arg/p.Ala620Thr) impair protein structure and function in type II deficiency: mechanistic insights into a hereditary thrombogenic disorder

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  • 发表日期：

    2025年12月15日

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  • Yifan Lu,
  • Fengjiao Wang,
  • Dandan Yu,
  • Haixiao Xie,
  • Yanhui Jin,
  • Mingshan Wang,
  • Lihong Yang

摘要

Background

Hereditary plasminogen (PLG) deficiency represents an extremely rare autosomal recessive disorder characterized by impaired fibrinolytic capacity resulting from diminished PLG enzymatic activity. In this study, we identify and characterize a novel compound heterozygous PLG mutation clinically associated with cerebral infarction. Our findings demonstrate that structural conformational alterations in the mutation PLG protein disrupt normal fibrinolytic pathway function.

Methods

In this study, the proband presented at the First Affiliated Hospital of Wenzhou Medical University with a chief complaint of “left-sided limb weakness lasting for two days.” PLG activity (PLG: A) and PLG antigen (PLG: Ag) levels were measured in the proband and eight family members spanning three generations using the chromogenic substrate assay(CSA) and enzyme-linked immunosorbent assay (ELISA), respectively. The genetic mutation site was identified through direct DNA sequencing. Bioinformatics software was employed to analyze the conservation and potential pathogenicity of the mutation site. A mutation protein model was constructed to investigate structural alterations in the protein before and after the mutation. Furthermore, a recombinant plasmid expression vector was developed, and the in vitro expression of the recombinant PLG protein was evaluated using real-time quantitative PCR (RT-qPCR), ELISA, and Western blot(WB) analysis.

Results

The propositus had a significantly reduced PLG: A to 27%, but the PLG: Ag level was normal at 103%, and was diagnosed with type II plasminogen deficiency (dysplasminogenemia). Sanger sequencing identified compound heterozygous missense mutations in the PLG gene: c.1702G > A (p.Gly568Arg) in exon 14 and c.1858G > A (p.Ala620Thr) in exon 15. Both mutation sites are highly conserved across species, and four independent bioinformatics tools consistently predicted pathogenic effects. Molecular modeling demonstrated that the p.Gly568Arg substitution causes steric hindrance through side chain elongation and establishes a new hydrogen bond with Leu686; p.Ala620Thr destabilized the catalytic triad (His603-Asp646-Ser741). In vitro functional assays, including RT-qPCR, ELISA, and WB analysis, confirmed that neither mutation significantly altered the expression level of mRNA、protein biosynthesis and secretion. However, the PLG: A/PLG: Ag ratio in the media was markedly reduced compared to wild-type controls.

Conclusions

We report the first case worldwide of a novel p.Gly568Arg mutation in the PLG gene, which coexists with the p.Ala620Thr mutation in a compound heterozygous form. Structural and functional analyses indicate that although the transcription, protein synthesis and secretion of the PLG gene are not affected, these two mutations specifically impair the catalytic activity of the PLG protein through different conformational changes. This expands the mutation spectrum and clarifies a novel thrombosis mechanism in type II PLG deficiency.