Purpose: To investigate the use of tissue plasminogen activator (tPA) and its effects on the ocular proteome as a therapeutic intervention for postoperative inflammation and fibrin formation following intraocular lens (IOL) insertion in a juvenile rabbit model. Methods: Twenty-six 6-7 week old rabbits underwent lensectomy with IOL insertion. Following examination on day 3, 100 microliters of either 25 micrograms of recombinant rabbit tPA or balanced salt solution (control) was injected into the anterior chamber. On postoperative day 4, rabbits underwent examination, and eyes were harvested and fixed for 9.4T magnetic resonance imaging (MRI). Three masked observers quantified fibrin scar volume using Horos Project Software. Aqueous humor (AH) was collected immediately prior to surgery and on postoperative days 3 and 4. Proteins related to coagulation and inflammation were assessed in AH samples using targeted mass spectrometry via parallel reaction monitoring. Results: tPA significantly reduced the volume of fibrin twenty-four hours following administration compared to control eyes (0.560mm3 vs. 3.29mm3; p < 0.0001). Despite the reduced fibrin scar, proteins related to the coagulation and complement cascade were not significantly different following tPA injection. Conclusions: tPA may be a safe candidate for reduction of postoperative fibrin scarring after intraocular surgery. MRI can provide a quantitative value for fibrin volume changes. Translational Relevance: tPA is a potential candidate to treat ocular fibrosis. MRI can quantify the efficacy of treatments in future dose-response studies. Targeted mass spectrometry can provide critical data necessary to help decipher the effect on the abundance of targeted proteins following pharmacological intervention.

10 microliters of each aqueous humor (AH) sample was aliquoted and diluted with 100mM ammonium bicarbonate, 40% Invitrosol, 20% acentonitrile (MeCN), and 5.52 mM tris(2-carboxyethyl)phosphine (TCEP) to bring the samples to the final concentration of TCEP to 5mM for reduction. Samples were then incubated for 30 minutes at 1400 rpm at 25 °C in a thermomixer (Eppendorf, Hamburg, Germany). 100 mM Iodoacetamide was added to bring samples to 10mM for alkylation and then mixed in the dark for 30 minutes at 1400rpm at 37 degrees Celsius in a thermomixer. Pierce Trypsin/Lys-C (Thermo Scientific, Waltham, MA) mix was added in a 1:20 trypsin to protein ratio for overnight incubation (~18 hours) at 37 degrees Celsius 1400 rpm in the thermomixer. Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (hydrophylic and hydrophobic; GE Life Sciences, Sheffield, UK) were prepared a 1:1 ratio to create a particle suspension, and peptides were prepared for proteomic analysis using the SP2 method as previously described (1). Briefly, 8 microliters of particle solution was added to the samples to create a 20:1 particle-to-peptide ratio, and 100% MeCN was added to bring the final concentration of MeCN to 95%. Samples were mixed by pipetting up and down 3-5x to make sure the particles were well dispersed, allowed to settle for two minutes, and placed on a magnetic rack. The supernatant was removed, and samples were washed with 100% MeCN to cover the particles completely. Samples were returned to the magnetic rack and let sit for 60 seconds, and another 100% MeCN wash was performed and the supernatant discarded. The particles were then reconstituted in a solution consisting of 2% MeCN and water, vortexed, and allowed to settle for one minute. The supernatant (now containing the peptides) was collected and transferred to a new microfuge tube. Samples were then acidified with 10% formic acid for a final concentration of 0.1% acid. The sample was then spun at 14000 rpm for 10 minutes and the supernatant was collected. The peptides were quantified using a Pierce Quantitative Fluorometric Peptide assay and diluted to 25ng/microliter in 2% MeCN with 0.1% formic acid. Peptide Retention Time Calibration (PRTC) Mixture (Thermo Fisher Scientific) was added to each sample at a final concentration of 4nM to enable retention time calibration and assessment of instrument performance throughout acquisition. Equal volumes of each sample were combined into a single “pooled quality-control (QC)” mixture. The final injection volume for all samples was 20 microliters. Based upon a previous study (2) a list of proteins related to coagulation, complement cascades, as well as unrelated proteins (as control proteins) were analyzed by mass spectrometry (MS). Targeted quantitation by parallel reaction monitoring (PRM) was performed using a Dionex UltiMate 3000 RSLCnano system in-line with an Orbitrap Fusion Lumos Tribrid mass spectrometer. Samples (as single replicate injections) were queued in a randomized order. Each peptide was scheduled with a set 10-minute scheduled retention time window, and resulting MS data were processed using Skyline. Retention times and ions were manually aligned and selected. (1) Waas M, Pereckas M, Jones Lipinski RA, Ashwood C, Gundry RL. SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses. J Proteome Res. 2019;18(4):1644-56. PMC6571012 (2) Young JB, Keppel TR, Waas M, Salmon AE, Buchberger AR, Skumatz CMB, Gundry RL, Kassem IS. Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and immunomodulatory proteins. Mol Omics. 2020;16(2):126-37. PMID:32031200

Aqueous humor is a clear fluid in the front part of the eye. We collected this fluid from juvenile rabbits before surgery, and three days following surgery, and four days following surgery. We sought to determine how administration of tissue plasminogen activator would alter the proteins in the aqueous humor twenty-four hours following drug administration.