The generation and characterization of mice expressing a plasmin-inactivating active site mutation
Article first published online: 24 JUL 2010
© 2010 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 8, Issue 10, pages 2341–2344, October 2010
How to Cite
IWAKI, T., MALINVERNO, C., SMITH, D., XU, Z., LIANG, Z., PLOPLIS, V. A. and CASTELLINO, F. J. (2010), The generation and characterization of mice expressing a plasmin-inactivating active site mutation. Journal of Thrombosis and Haemostasis, 8: 2341–2344. doi: 10.1111/j.1538-7836.2010.03995.x
- Issue published online: 24 JUL 2010
- Article first published online: 24 JUL 2010
- Accepted manuscript online: 24 JUL 2010 12:00AM EST
- Received 28 April 2010, accepted 20 July 2010
Human plasminogen (Plg) is a 791 amino acid single-chain protein zymogen that is activated to the two-chain serine protease, plasmin (Plm), by mammalian activators, for example tissue-type Plg activator and urokinase-type Plg activator (u-PA), as well as bacterial activators, for example streptokinase, staphylokinase , and a surface protease from Yersinia pestis . Activation occurs as a result of cleavage of a single peptide bond at Arg561–Val562, yielding a heavy chain linked by two disulfide bonds to a light chain. The former region possesses five kringle domains that are responsible for binding of Plg/Plm to effector molecules, and the latter region contains the serine protease catalytic triad, His603, Asp646, and Ser741 (human Plg numbering). Plm is the primary fibrinolytic enzyme, functioning in maintaining vascular patency through degradation of fibrin-rich thrombi, and in clearing extravascular fibrin, for example as occurs in ligneous conjunctivitis in humans and in mice [3,4]. Plm directly degrades fibrin and matrix proteins, but also catalyzes the activation of other zymogens, for example pro-matrix metalloproteases [5,6], latent forms of growth factors [7,8], and complement proenzymes , thus functioning as a major pathophysiologic extracellular protease.
A number of receptors for Plg/Plm are present on mammalian normal and tumor cells [10,11], as well as on bacterial cells [12,13]. Several candidate receptors have been identified to which Plg/Plm binds, utilizing the lysine-binding sites present in Plg/Plm kringles 1, 2, 4, and 5. Many Plg/Plm receptors, for example alpha-enolase  and annexin II , thus possess C-terminal lysines. Plg/Plm interactions with receptors have also been shown to occur via arrangements of internal side chains that are isosteric with lysine, an example of which is the binding of human Plg to the group A streptococcal virulence protein PAM [16,17]. Plg/Plm–cellular receptor interactions function to facilitate the assembly of the fibrinolytic machinery on cell surfaces. This allows for efficient generation of cell-associated Plm activity, which can regulate cell migration through degrading protein barriers, a function that is important for a number of pathophysiologic processes, such as angiogenesis associated with tumor growth and dissemination [18,19].
Mice with a targeted total deficiency of Plg have been generated in two laboratories [20,21]. These mice are viable and survive to adulthood, but some of the spontaneous phenotypes that arise include diminished growth rates, fibrin deposition in a number of organs, gastrointestinal ulcerations, and rectal prolapse . Since the generation of these mice, a number of challenge models have been developed to determine the importance of Plg in regulating normal and pathologic physiologies, and studies have been published that have investigated a Plg deficiency in models of vascular injury and repair, arthritis, glomerulonephritis, pulmonary fibrosis, inflammatory cell recruitment, cancer, wound healing, and neurologic-related processes (reviewed in ). Most in vivo investigations attempting to distinguish between the fibrinolytic activity of Plm and its other potential proteolytic and non-proteolytic functions have relied on double deletions of Plg and fibrinogen . Although this is a very valuable approach, it nonetheless complicates the pathophysiology of the animal because of the lack of two proteins with other functions. In an attempt to circumvent some of these issues, and to allow the definition of Plg-related functions attributable exclusively to Plm protease activity, we generated mice in which the murine Plm active site residue, Ser743 (identical to Ser741 in human Plg), has been mutated to Ala. In Plg/fibrinogen pleiotropy studies, this mouse line allows the Plg and fibrinogen genes to remain, while at the same time eliminating any possible proteolytic activity of Plm. Initial phenotypes of this new mouse line are reported in this communication.
For the generation of these mice, a plasmid (pPE7neoW-F2LF) that contains a single loxP site, two flippase recombination target (FRT) sites and a neomycin resistance (NEO) cassette (from K. Yusa, Osaka University, Osaka, Japan) was modified to provide a targeting vector (TV) backbone (pPE7neoW-F2L2) in which the NEO cassette was flanked by two loxP sites and two FRT sites.
A C57Bl/6 mouse BAC clone containing the Plg locus (RPRI 23-218G19; BACPAC Resources Center, Children’s Hospital Oakland Research Institute) was purified and electroporated into a modified Escherichia coli strain (SW106) that is capable of temperature-inducible homologous recombination and arabinose-inducible expression of Cre recombinase . The PlgS743A mutation was then introduced in Plg exon 19 of BAC Plg in SW106, using overlapping polymerase chain reaction (PCR) along with a selection–counterselection strategy with plasmid pRpsL-Neo (GeneBridges, Heidelberg, Germany) to select correctly targeted cells containing PlgS743A (218G19-PlgS743A). Following this, the loxP-FRT-NEO-FRT-loxP cassette from pPE7neoW-F2L2 was inserted between the 3′-end of Plg exon 19 and the 3′-end of Scl22a3 exon 11 in the BAC DNA from the cells containing 218G19-PlgS743A. Correctly targeted cells containing 218G19-PlgS743A-NEO (RPR-218G19-PlgS743A-NEO) were then selected. For the final TV, the modified Plg was retrieved from RPR-218G19-PlgS743A-NEO and used to construct the plasmid pMCS-DTA-Plg, containing Plg exon 17 and Scl22a3 exon 10 flanking an Spe1 site. This latter restriction site was used to insert the region from exon 18 of Plg to exon 10 of Scl22a3 of 218G19-PlgS743A-NEO into pMCS-DTA-Plg, yielding the final TV for PlgS743A, which contained the negative autotoxic diphtheria toxin A (DTA) gene , upstream of Plg exon 17 (Fig. 1A). This plasmid was then electroporated into C57Bl/6 embryonic stem (ES) cells. Those ES cells surviving the negative selection were positively selected by G418 resistance, and screened by PCR for proper recombination (Fig. 1B), and by Southern analysis using both external probes (data not shown). Final selection was made after sequencing of the lox and FRT 5′-flanks and 3′-flanks. Next, injections of the recombined ES cells into Balb/c blastocysts were performed. Chimeric males were identified and mated with female C57Bl/6 mice. The resulting F1 offspring were tested for proper germline transmission by PCR and sequence analyses.
F1 mice were then bred with mice expressing flippase, Tg-CAG_FLPe37, in order to remove the NEO cassette. Genomic DNA was obtained from tail snips of wild-type (WT) and PlgS743A mice, and exon 19 from both lines (N = 4 for each) was amplified with specific 5′-primers and 3′-primers and sequenced. The results confirmed that Ser743 (AGT) was mutated to Ala743 (GCC) in the genomic DNA. Mice with a double allele of this mutation expressed similar levels of Plg in plasma as WT mice (Fig. 1C). Plg was purified from the pooled plasmas of 12–15 WT and PlgS743A/S743A mice by affinity chromatography on Sepharose–lysine. Approximately 1 mg of Plg was obtained from approximately 8 mL of plasma from each line, further confirming the nearly equal expression levels of WT Plg and PlgS743A. The Plg samples were activated by u-PA. Whereas WT Plg provided two-chain Plm with amidolytic activity towards the Plm chromogenic substrate S2251, PlgS743A displayed no activity towards S2251, despite the formation of two-chain Plm (Fig. 1D). This latter result is consistent with previous in vitro studies, in which the active site serine of recombinant human Plg was mutated to alanine  or cysteine , yielding an inactive two-chain Plm with all Plg activators examined. These results demonstrate that the genomic DNA was successfully modified to express PlgS743A protein, that mice with the double allele for this mutation expressed PlgS743A to an equal extent as WT mice expressed Plg, that the PlgS743A was activated to its two-chain equivalent Plm, and that the PlmS743A was inactive.
The prothrombin times (PTs) (10.9 ± 0.3 s, n = 6, for Plg−/− mice; 10.6 ± 0.8 s, n = 3, for PlgS743A/S743A mice; and 11.6 ± 0.7 s, n = 4, for WT mice) and activated partial thromboplastin times (35 ± 4 s, n = 6, for Plg−/− mice; 42 ± 3 s, n = 5, for PlgS743A/S743A mice; and 34 ± 1 s, n = 4, for WT mice) were similar for the three mouse lines. However, resting plasma fibrinogen levels were higher in PlgS743A/S743A mice at all ages between 4 and 36 weeks, and the differences in plasma fibrinogen concentrations between the WT and PlgS743A/S743A mice became larger as the mice aged. This effect closely followed that in Pg−/− mice (Fig. 1E), and is probably an acute-phase response consistent with increased spontaneous disease progression in the absence of Plm and the inability of Plg−/− and PlgS743A/S743A mice to clear fibrin. In this regard, in both the PlgS743A/S743A and Plg−/− mice, histologically evident (N = 4) focal areas of fibrin deposition and microcalcification were observed in the liver (Fig. 1F). Also noted in both mutated lines were focal ulceration, inflammation, and fibrin deposition at the anorectal junction (not shown), as well as several enlarged lymph nodes proximal to the pancreas. The WT mice did not present these pathologies, and appeared to be normal microscopically.
Regarding additional spontaneous abnormalities, at 6 weeks PlgS743A/S743A mice were growth retarded (18.8 ± 0.5 g, n = 4) relative to WT mice (20.8 ± 0.5 g, n = 6, P = 0.03), and developed rectal prolapse during a time frame very similar to that seen with Plg−/− mice.
The results from this study demonstrate that an inactivating mutation of the latent active site serine of Plm in mice recapitulates many of the spontaneous phenotypes observed in Plg−/− mice, suggesting that plasminolytic activity is critical for normal maintenance of hemostasis in mice. This newly developed PlgS743A/S743A mouse line is important for studies aimed at selectively eliminating the protease activity of Plm, while maintaining the binding of effector molecules to Plg and Plm via their intact kringle domains.
The authors wish to thank M. Sandoval-Cooper for assistance with histology and D. L. Donahue for performing animal surgery. This study was supported by grants HL013423 and HL073750 from the NIH (to F. J. Castellino).
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.