The vitronectin-binding domain of plasminogen activator inhibitor-1 plays an important functional role in lipopolysaccharide-induced lethality in mice


Victoria A. Ploplis, W. M. Keck Center for Transgene Research, University of Notre Dame, 230 Raclin-Carmichael Hall, Notre Dame, IN 46556, USA.
Tel: +1 574 631 4017; fax: +1 574 631 4414.


Narasaki R, Xu Z, Liang Z, Fung LCW, Donahue D, Castellino FJ, Ploplis VA. The vitronectin-binding domain of plasminogen activator inhibitor-1 plays an important functional role in lipopolysaccharide-induced lethality in mice. J Thromb Haemost 2012; 10: 2678–21.

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor superfamily, and is the main physiologic inhibitor of urokinase plasminogen activator (u-PA) and tissue-type plasminogen activator [1]. Studies using PAI-1-deficient (PAI-1−/−) mice have demonstrated that PAI-1 not only regulates the fibrinolytic system, but also modulates other physiologic and pathophysiologic processes, including inflammation, angiogenesis, tumor growth, and cardiovascular disease [2–6]. Regions of human PAI-1 that are critical for inhibition of plasminogen activation, binding to vitronectin (VN) and binding to LDL receptor-related protein have been identified [7–9]. Our laboratory has demonstrated that these functional sites are conserved in the murine system [10]. In order to define functional roles for domains within PAI-1, we generated mice that express PAI-1 with altered VN-binding capacity. Binding of VN to PAI-1 stabilizes the biological activity of PAI-1 and prolongs its half-life in plasma [11–13]. Moreover, the binding of VN by PAI-1 modulates cell adhesion and cell migration by limiting the binding of VN to its integrin receptor and u-PA receptor (uPAR) [13–17].

It has been demonstrated that a Q123K mutation in PAI-1 results in a significant reduction in the ability of PAI-1 to bind to VN [18], and transgenic mice overexpressing this PAI-1 variant have been generated and characterized [19]. Previous studies have shown that a recombinant murine PAI-1 variant that has the double mutation R101A/Q123K has significantly diminished ability to bind to VN, even more so than that with the single mutation at amino acid 123 [10]. On the basis of these findings, genetic mutations that translate into these alterations (R101A and Q123K) of PAI-1 were targeted into the PAI-1 gene in the mouse genome. A 2806-bp PCR genomic fragment containing PAI-1 exon 2 and exon 3 was subcloned into the pCR.21-TOPO vector as the 5′-flank for the targeting vector (TV), and nucleotide substitutions were introduced by site-directed mutagenesis to generate the R101A and Q123K changes in exon 3. A 3674-bp PCR genomic fragment containing PAI-1 exons 4 and 5 was subcloned into the pCR.21-TOPO vector as the 3′-flank for the TV. The 5′-flank and 3′-flank were cloned into the multicloning site of a pre-made TV backbone in which the neomycin resistance gene (Neo) cassette (NEO) was flanked by two lox P (Lox) sites and two flippase recombination target (FRT) sites to yield the final TV for PAI-1 VN with the R101A and Q123K mutations (Fig. 1A).

Figure 1.

 (A) The final targeting vector (TV) for plasminogen activator inhibitor-1 (PAI-1)R1010A/Q123K. PAI-1 exons are black rectangles, and the red circle shows the location of the R101A and Q123K mutations in exon 3.The red vertical bar at the edge of the neomycin resistance gene (NEO) site is flippase recombination target (FRT) site. The triangles represent the Lox P site. The cytosine deaminase cassette (CDA) was inserted for negative selection, which was achieved with 5-fluorocytosine. Positive selection of embryonic stem (ES) cells containing the NEO gene was achieved via G418 resistance. (B) Wild-type (WT), homologously recombined and final alleles, with arrows representing the location of primers for genotyping. The gel on the left shows PCR amplicons confirming homologous recombination of the TV with the WT allele and incorporation of the Lox–FRT–NEO–FRT–Lox cassette. The right gel shows PCR amplicons of WT and final mutated alleles (PAI-1R1010A/Q123K). The Lox–FRT–NEO–FRT–Lox cassette was inserted between exon 3 and exon 4. This cassette was removed by crossing with transgenic mice expressing flippase. The forward primer was 5′-GCTCAACATGAGCCTAATGGATC-3′, and the reverse primer was 5′-CATTCATGAGTTCCTGGCTCCAG-3′. These primers generate a 484-bp amplicon in the WT and a 646-bp amplicon for PAI-1R101A/Q123K. (C) Characterization of WT (white bar) and PAI-1 R101A/Q123K (black bar) mice 8 h after injection of lipopolysaccharide (LPS) (2 μg g−1 body weight) for induction of PAI-1. (a) PAI-1 antigen levels (F7 mice, n = 3 for WT mice and n = 5 for PAI-1R101A/Q123K mice). (b) Plasma PAI-1 inhibitory activity against tissue-type plasminogen activator (t-PA) relative to WT activity (F1 mice, n = 3 for WT mice and n = 4 for PAI-1R101A/Q123K mice). (c) PAI-1 mRNA levels in liver tissue relative to WT mice (F7 mice, n = 3 for WT mice and n = 5 for PAI-1R101A/Q123K mice). The detection of PAI-1 was performed with a murine PAI-1 total antigen assay by ELISA. For mRNA analyses, the values indicate the fold difference relative to ribosomal protien L19 and converted to fold differences relative to WT mice. Values are expressed as the mean ± standard error of the mean. (D) Survival of WT, PAI-1−/− and PAI-1R101A/Q123K mice after LPS challenge. LPS was injected intraperitoneally (10 μg g−1 body weight). The data are presented as Kaplan–Meier survival curves. WT mice, n = 33; PAI-1 mice, n = 25; and PAI-1R101A/Q123K mice, n = 36 (F7). Survival rate differences were compared by use of the log-rank test. P-values: WT vs. PAI-1−/−, 0.03; WT vs PAI-1R101A/Q123K, 0.011. VN, vitronectin.

The TV was electroporated into C57BL/6/129 embryonic stem (ES) cells. The ES cells surviving negative selection with 5′-fluorocytosine for the cytosine deaminase cassette gene and positive selection with G418 for the NEO gene were screened by southern blot analysis, and the mutations were confirmed by PCR (data not shown). A PCR strategy was also employed to confirm homologous recombination (Fig. 1B). Recombined ES cells were injected into blastocysts, and chimeric males were identified. The F1 offspring resulting from crossing chimeric male mice with C57BL/6 female mice were tested for proper germline transmission by PCR and sequence analysis (Fig. 1B). F1 mice were then bred with transgenic mice expressing flippase, Tg-CAG_FLPe37, to remove the NEO gene (Fig. 1B). The PCR forward primer 5′-GCTCAACATGAGCCTAATGGATC-3′ and reverse primer 5′-CATTCATGAGTTCCTGGCTCCAG-3′ were used to detect the removal of the NEO gene. A PAI-1 genomic fragment from PAI-1R101A/Q123K mice was cloned and sequenced, and it was found to contain the mutations for R101A and Q123K, and the FLPe/FRT recombination sequence. Blood counts and blood analyses were performed, and body weights and litter sizes were determined, for wild-type (WT), PAI-1−/− and PAI-1R101A/Q123K mice, and all values were within the normal range.

Lipopolysaccharide (LPS) is derived from the outer membrane of Gram-negative bacteria, and is a known inducer of PAI-1 gene expression [20]. In order to determine whether this response is equivalent between WT and PAI-1R101A/Q123K mice, LPS (2 μg g−1 body weight, E.C. 0111:B4; Sigma-Aldrich Co. LLC, St. Louis, Mo) was injected intraperitoneally into 8–12-week-old male WT and PAI-1R101A/Q123K mice. After 8 h, plasma PAI-1 levels and PAI-1 inhibitory activity were determined. Plasma levels of PAI-1 in WT and PAI-1R101A/Q123K mice were equivalent (Fig. 1Ca), as was plasma PAI-1 inhibitory activity (Fig. 1Cb). In addition, the mRNA levels of PAI-1 in liver were also the same for the two genotypes (Fig. 1C-c). These results indicated that PAI-1R101A/Q123K mice have the same ability to produce PAI-1 as WT mice, and that the PAI-1 from PAI-1R101A/Q123K mice maintained plasminogen activator inhibitory activity. LPS is the major causative agent in Gram-negative endotoxemia. Hallmark features of this disease are a systemic inflammatory response and hypercoagulablility. The systemic microthrombosis that develops leads to disseminated intravascular coagulation, and subsequently to hypoxic organ failure [21–23]. Indeed, our laboratory has previously shown that a coagulation factor VII deficiency protects against lethal endotoxemia [24]. Thus, a balance between the fibrinolytic system and the coagulation system plays an important role in regulating the downstream effects of endotoxemia. As PAI-1 is a target following LPS exposure, it was determined whether a lack or mutation of PAI-1 would affect survival after exposure to a lethal dose of LPS. For this study, 8–12-week-old male WT, PAI-1−/− and PAI-1R101A/Q123K mice were injected intraperitoneally with LPS (10 μg g−1 body weight), and survival was monitored every 3 h. PAI-1−/− and PAI-1R101A/Q123K mice were shown to have a significant (P = 0.03 and P = 0.011, respectively) survival advantage relative to WT mice (Fig. 1D). Studies have demonstrated that VN levels in lungs following intratracheal administration of LPS are significantly increased [25]. With this model, VN null mice were protected against LPS-induced acute lung injury [25]. Additionally, other studies have demonstrated that there is an increased occurrence of neutrophil apoptosis in LPS-treated VN null mice [26], and apoptotic cells can protect mice from LPS toxicity [27]. In the current study, PAI-1 played a critical role in LPS-induced lethality, and the VN-binding capacity of PAI-1 is important for this function, potentially through the enhanced effect of VN on PAI-1 inhibition of the anti-coagulant, anti-inflammatory protein, protein C [28].

Disclosure of conflict of interests

This study was funded, in part, by a grant from NIH (NHLBI) HL63682 (V. A. Ploplis). The other authors state that they have no conflict of interest.