A Novel Thrombin Fluorogenic Substrate of High Affinity, Catalytic Efficiency and Selectivity.
Bibliographic record
Abstract
Abstract Thrombin is a serine protease with multiple functions, including the conversion of fibrinogen to fibrin, platelet activation, activation of Factor VIII and Factor V. Although many low molecular weight substrates have been developed for the study of thrombin catalytic activity, our interest in analyzing thrombin activity in the blood of a living mouse required development of a new class of thrombin substrates of high affinity and high selectivity whose product upon hydrolysis could be visualized by intravital fluorescence microscopy. We have developed a novel substrate for thrombin using the fluorochrome Alexa 488 and the quencher QSY35. Alexa 488 is conjugated to the N-terminus of a 12 amino acid peptide based upon the thrombin cleavage site in the α-chain of fibrinogen and the quencher is coupled to the C-terminus of the peptide, yielding Alexa 488-KGGVR-GPRVVEA-QSY35. We term this substrate FBG-12. Through fluorescence energy resonance transfer, the emission from Alexa 488 is absorbed by the quencher QSY 35, thus minimizing fluorescence, since the two moieties have overlapping spectral properties and are separated by a Förster radius less than 44 Å. Thrombin hydrolyzes the peptide yielding the N-terminal fragment, Alexa 488-KGGVR-COOH, which is highly fluorescent, and the C-terminal fragment NH2-GPRVVEA-QSY35 peptide which, being physically separated from the Alexa 488, no longer quenches the fluorochrome. The peptide was synthesized by solid phase peptide synthesis, its N-terminus and C-terminus were modified with Alexa 488 and QSY35 respectively, and its identity confirmed by mass spectroscopy and protein sequencing. The kinetic properties of FBG-12 were determined in vitro. Hydrolysis of the substrate by thrombin resulted in a linear increase in fluorescence at 525 nm over time and was dependent on enzyme concentration. The fluorescence of the product of thrombin hydrolysis of FBG-12 was 120-fold greater than that of the substrate. Michaelis-Menten kinetic analysis of thrombin hydrolysis of the fluorogenic substrate FBG-12 revealed a Km of 2 μM, a kcat of 759 s−1, and a kcat/Km of 3795 x 106 M−1 s−1. To determine the selectivity of this substrate, other plasma serine proteases were analyzed for their ability to hydrolyze FBG-12. Factor Xa, Factor VIIa, and activated protein C did not hydrolyze FBG-12. Factor XIa (Km 11 μM, kcat 20s−1, kcat/Km = 20 x 106 M−1 s−1) did hydrolyze FBG-12, although the reaction was inefficient compared to thrombin. Our characterization of FBG-12 suggests that this substrate is hydrolyzed efficiently and selectively by thrombin. Its spectral properties and solubility in a physiologic environment make FBG-12 a suitable substrate for the detection of thrombin activity via intravital imaging during thrombus formation in vivo.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".