A rapid fluorometric assay for tear fluid plasmin activity

Area of Science:

• Ophthalmology
• Biochemistry
• Diagnostic Assay Development

Background:

• Plasmin plays a role in ocular tissue remodeling and inflammation.
• Measuring plasmin in tear fluid can offer insights into ocular disease pathology.
• Previous methods for plasmin detection in tears were limited.

Purpose of the Study:

• To develop a rapid, sensitive, and specific fluorometric assay for plasmin in tear fluid.
• To investigate plasmin activity and flux in patients with various ocular conditions.
• To establish plasmin flux as a potential diagnostic biomarker for eye diseases.

Main Methods:

• Development of a 5- to 10-minute fluorometric assay using H-D-Val-Leu-Lys.7-amido-4-methyl-coumarin (AMC) substrate.
• Optimization of assay conditions including pH 8.0, Michaelis-Menten constant (0.28 mM), and substrate concentration (1 mM).
• Inclusion of albumin to enhance sensitivity and correction for tear fluid flow to calculate plasmin flux.

Main Results:

• The assay demonstrated high sensitivity (0.6 IU/L detection limit) and precision (1.6% intra-assay, 4.4% inter-assay).
• Plasmin flux was significantly elevated in patients with microbial keratitis, corrosive trauma, ocular trauma, herpetic infection, and other diseases compared to healthy controls.
• Severely affected patients, particularly those with corneal ulcers, exhibited several hundredfold increases in plasmin flux.

Conclusions:

• The developed assay is a valuable tool for quantifying plasmin in small tear fluid volumes.
• Elevated plasmin flux is a significant indicator across a spectrum of ocular diseases.
• Plasmin flux measurement holds promise as a diagnostic biomarker for early detection and monitoring of eye conditions.