Limits...
Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

Choi MS, Parikh K, Saxena A, Chilukuri N - J Burns Wounds (2007)

Bottom Line: The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation.These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular Pharmacology, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.

ABSTRACT

Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity.

Methods: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.

Results: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree.

Conclusion: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

No MeSH data available.


Related in: MedlinePlus

Thermal stability of wt-KD1. Wt-KD1 (panels A and B) and mammalian full-length TFPI-2 (panels C and D) were incubated at different temperatures for varying periods of time and their ability to inhibit amidolytic activities of plasmin (panels A and C) and trypsin (panels B and D) were determined. All values were the mean ± SD of triplicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1937028&req=5

Figure 3: Thermal stability of wt-KD1. Wt-KD1 (panels A and B) and mammalian full-length TFPI-2 (panels C and D) were incubated at different temperatures for varying periods of time and their ability to inhibit amidolytic activities of plasmin (panels A and C) and trypsin (panels B and D) were determined. All values were the mean ± SD of triplicate samples.

Mentions: In these studies, thermal stability of wt-KD1 was assessed and compared to that of TFPI-2 over an 8-week period (Figure 3). Recombinant proteins were incubated at different degrees of temperature, and their ability to inhibit plasmin and trypsin amidolytic activities were analyzed. At 65°C, the inhibitory activity of wt-KD1 toward plasmin was completely lost in 4 days (Figure 3A) whereas trypsin inhibitory activity was lost in 1 day (Figure 3B). Wt-KD1 was thermally stable for 4 weeks at 42°C and for more than 8 weeks at room temperature (Figure 3A and 3B). Similar results were observed for TFPI-2 (Figure 3C and 3D). These results suggest that the refolded wt-KD1 is stable for extended periods of incubation.


Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

Choi MS, Parikh K, Saxena A, Chilukuri N - J Burns Wounds (2007)

Thermal stability of wt-KD1. Wt-KD1 (panels A and B) and mammalian full-length TFPI-2 (panels C and D) were incubated at different temperatures for varying periods of time and their ability to inhibit amidolytic activities of plasmin (panels A and C) and trypsin (panels B and D) were determined. All values were the mean ± SD of triplicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1937028&req=5

Figure 3: Thermal stability of wt-KD1. Wt-KD1 (panels A and B) and mammalian full-length TFPI-2 (panels C and D) were incubated at different temperatures for varying periods of time and their ability to inhibit amidolytic activities of plasmin (panels A and C) and trypsin (panels B and D) were determined. All values were the mean ± SD of triplicate samples.
Mentions: In these studies, thermal stability of wt-KD1 was assessed and compared to that of TFPI-2 over an 8-week period (Figure 3). Recombinant proteins were incubated at different degrees of temperature, and their ability to inhibit plasmin and trypsin amidolytic activities were analyzed. At 65°C, the inhibitory activity of wt-KD1 toward plasmin was completely lost in 4 days (Figure 3A) whereas trypsin inhibitory activity was lost in 1 day (Figure 3B). Wt-KD1 was thermally stable for 4 weeks at 42°C and for more than 8 weeks at room temperature (Figure 3A and 3B). Similar results were observed for TFPI-2 (Figure 3C and 3D). These results suggest that the refolded wt-KD1 is stable for extended periods of incubation.

Bottom Line: The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation.These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular Pharmacology, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.

ABSTRACT

Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity.

Methods: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.

Results: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree.

Conclusion: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

No MeSH data available.


Related in: MedlinePlus