A rapid fluorometric assay for tear fluid plasmin activity.

A rapid (5- to 10-min), sensitive (detection limit 0.6 IU/L), and moderately specific fluorometric plasmin assay for small volume tear fluid samples was developed. Addition of albumin (up to 0.1% final concentration) to the assay buffer improved the sensitivity of the test so that plasmin activity in healthy controls could be detected. pH in the reaction buffer was 8.0, Michaelis-Menten constant for the substrate, H-D-Val-Leu-Lys.7-amido-4-methyl-coumarin (AMC), was 0.28 mM, and final substrate concentration in the reaction buffer was 1 mM. Intra- and interassay imprecisions were 1.6 and 4.4%, respectively at a plasmin level of 10 IU/L. Tear fluid flow was significantly higher in the patients than in the healthy controls, and this dilatory effect must be considered when using plasmin determination for diagnostic purposes. This effect was counteracted by correcting the plasmin activity values by tear fluid flow. Plasmin flux is plasmin activity (microIU) secreted in units of time (min). This parameter showed highly significant differences between the patients and controls. All patients with microbial keratitis, corrosive trauma, ocular trauma, herpetic infection, and other diseases showed highly significant elevation of plasmin flux compared with controls. The highest plasmin flux values (several hundredfold that of controls) were recorded in patients with severe corneal ulcers. Few patient samples showed some involvement of other proteases, which were not inhibited by aprotinin.