The Use of Prothrombin(S525C) Labeled with Fluorescein to Directly Study the Inhibition of Prothrombinase by Antithrombin during Prothrombin Activation
Bibliographic record
Abstract
Serine 525 of human prothrombin was mutated to cysteine and covalently labeled with fluorescein to make II(S525C)-fluorescein. Kinetics of cleavage of this derivative by prothrombinase are identical to those of wild-type prothrombin. Cleavage is coincident with a 50% increase in fluorescence intensity and the product is catalytically inactive. Thus, it allows convenient monitoring of prothrombin activation without generating active thrombin. The kinetics of inhibition of factor Xa (FXa) by antithrombin (AT) and AT-heparin were measured by monitoring activation of II(S525C)-fluorescein and the hydrolysis of the chromogenic substrate S2222 in the presence of AT. With S2222 as the substrate the rate constant for inhibition of FXa, Ca(2+), and unilamellar vesicles of phosphatidylcholine and phosphatidylserine (75:25) (PCPS) vesicles by AT was 3.51 x 10(3) m(-1) s(-1); when factor Va (FVa) was included the rate constant was 1.55 x 10(3) m(-1) s(-1). In the absence of FVa, II(S525C)-fluorescein had no effect on inhibition. When II(S525C)-fluorescein was the substrate, however, FVa at saturating concentrations profoundly protected FXa from inhibition by AT, increasing the half-life from 3 min with FXa, Ca(2+), PCPS, and II(S525C)-fluorescein, to greater than 69 min when FVa was included. Thus, both FVa and prothrombin are necessary for this level of protection. In the absence of prothrombin, FVa decreased the second order rate constant for inhibition by the AT-heparin complex from 1.58 x 10(7) m(-1) s(-1), for FXa, Ca(2+), and PCPS, to 7.72 x 10(6) m(-1) s(-1). II(S525C)-fluorescein and factor Va together reduced the rate constant to less than 1% of that for FXa, Ca(2+), and PCPS. At a heparin concentration of 0.2 unit/ml, this corresponds to a half-life increase from 1 s to 136 s.
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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 | 0.000 | 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 | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".