Detection of Calpain Activity in Plasma via Fluorogenic Substrate as a Diagnostic for Traumatic Brain Injury

Introduction: : Traumatic brain injury (TBI) affects over 2.8 million Americans annually, causing 300,000 hospitalizations, yet current diagnostics remain inadequate for guiding therapeutic interventions. Clinicians use the Glasgow Coma Scale, CT/MRI scans, and blood biomarkers (GFAP/UCH-L1) to assess TBI severity and structural damage. However, no biomarkers interrogate biochemical pathways driving neurodegeneration—a critical limitation as novel neuroprotective therapies advance.

A key pathological mechanism in TBI is calpain activation, a calcium dependent protease, which can trigger the secondary injury cascade, including excitotoxicity, inflammation, and cytoskeletal degradation. Existing blood biomarkers detect proteolytic fragments of calpain cleavage, however their reliance on endogenous substrates introduces variability due to concentration, cleavage kinetics, and stability. To overcome this variability, we engineered a fluorogenic substrate that reports calpain activity through dequenching of a fluorophore-quencher pair upon calpain-specific cleavage. While we have evaluated this material as an injectable sensor, clinical translation requires non-invasive formats.
Here, we developed an assay to measure calpain activity in plasma, hypothesizing that calpain activity is elevated in blood after TBI and that our material can detect that increase. Our approach offers two key advantages over existing technologies: signal amplification as one calpain cleaves multiple substrates, and detection of a disease-driving mechanism, which could more accurately reflect injury progression. After optimizing assay conditions for human and mouse plasma, we positively identify TBI from blood samples collected in a mouse controlled cortical impact model. This work bridges a diagnostic gap, providing a tool to diagnose TBI by pathological activity rather than byproduct of disease.

Materials and

Methods: : Calpain Assay Optimization
We developed a fluorescence-based calpain activity assay that is compatible for measurements in blood plasma. The calpain-specific fluorogenic substrate was QSY21-QEVYGAMP-K(Cy5)-PEG2-GC-NH2 (CPC Scientific Inc.). At 2 mU of calpain-1 enzyme (Calbiochem 208713), we optimized components of assay: calpain-specific fluorogenic substrate (CS-1, 1-32 mM), pH buffer (HEPES or phosphate), calcium chloride (CaCl₂, 1.25–20 mM), sodium chloride (NaCl, 12.5–200 mM), EDTA (0.5–8 mM), and β-mercaptoethanol (bME, 5–20 mM). Substrate cleavage was measured through fluorescence measured via a plate reader (Tecan Spark). To measure the limit of detection, the calpain-1 enzyme was titrated between 2000–1 uU.
To establish relevance to human blood, we tested the assay in human plasma (Innovative Research), evaluating buffer compatibility and the stabilizing effects of bovine serum albumin (BSA). Each component was systematically titrated to determine optimal concentrations for maximal signal-to-noise ratio while minimizing interference.
In Vivo Application in a TBI Model
We applied the assay to blood samples from a controlled cortical impact (CCI) mouse model of TBI to assess calpain activity. Injuries were performed at -2.2, -2.2 mm from bregma at a depth of 2 mm and speed of 3 m/s. Blood samples were collected at 0.5, 1, 3, & 24 hours post-injury and separated into plasma for analysis.
Statistical Analysis
Fluorescence data were normalized to background and analyzed using Michaelis Menten enzyme kinetics. Error bars show SEM and statistical significance was determined via one-way ANOVA with post-hoc testing using GraphPad Prism V10.4.0.

Results, Conclusions, and Discussions:: Results &

Discussion:
We observed that our fluorogenic substrate had reduced performance in the presence of plasma (Fig. 1A). Initially, we evaluated buffer constituents and found phosphate buffer outperformed HEPES, with a ~1.4 fold increase and greater signal stability (Fig. 1B). Furthermore, we optimized the concentrations of substrate, chelator, salt, calcium, and reducing agent. The final optimized conditions for detecting 4 mU/uL (15.5 nM) calpain with 5 µL plasma included: 4 mM CS-1, 4 mM EDTA, 100 mM NaCl, 20 mM β-mercaptoethanol (bME), 10 mM CaCl₂, and 200 mM potassium phosphate. Our optimized assay conditions increased the signal by ~2.6 fold (Fig. 1B) and achieve a limit of detection of 0.2 mU/ul (1.56 nM) (Fig. 1C). Based on literature, we expect a concentration of ~20 nM of active calpain in the brain parenchyma, though blood levels are uncharacterized to our knowledge.
We next used our optimized assay to determine whether we could positively identify TBI from plasma collected from injured mice. We collected plasma before injury and at various times after injury (n=5) to determine the time frame in which calpain activity was elevated in the blood. We found calpain activity in plasma peaked at ~1 hour post-injury, with detectable activity at 0.5 and 3 hours, confirming TBI-dependent blood calpain activity (Fig. 1D). Receiver operating characteristic (ROC) analysis demonstrated strong diagnostic power (AUC > 0.9) at those three timepoints (Fig. 1E,F). This timeline is consistent to when TBI patients arrive in emergency rooms for diagnosis.

Conclusion
We developed a blood-based calpain activity measurement that could detect TBI in a mouse model with strong diagnostic power. Future studies will validate the presence of calpain activity in human plasma and whether our technology can identify TBI in human patients.

Acknowledgements and/or References (Optional)::