Covalent Stabilization Of Coagulation Factor Xa Enhances Its Fibrinolytic Function In Vitro and In Vivo: A Novel Cofactor-Based Thrombolytic Agent
Bibliographic record
Abstract
Abstract The key “clot-busting” enzyme, tissue plasminogen activator (tPA), functions to activate plasminogen (Pg) to plasmin, which directly cleaves and solubilizes the clot. tPA requires a cofactor for physiologically significant plasmin generation. Fibrin is thought to be the only essential tPA cofactor and consequently localizes fibrinolysis. Limited proteolysis of fibrin enhances this tPA cofactor function by exposing C-terminal lysines that are integral to new Pg and tPA binding sites. Our recent work has suggested that the initiating steps of fibrinolysis are accelerated not just by fibrin, but also by localized auxiliary cofactors. These proteins can be cleaved by plasmin more efficiently than fibrin to reveal C-terminal lysine. Our focus has been clotting factor Xa (FXa), whose sequential plasmin-mediated derivatives FXaβ and Xa33/13 acquire C-terminal lysines that facilitate Pg binding and tPA-cofactor function. We now report that in plasma Xa33/13 is rapidly degraded, and hypothesize that preventing formation of Xa33/13 will prolong the fibrinolytic cofactor activity of FXaβ in plasma. To impair the production of Xa33/13 and stabilize the FXaβ activity, an ∼25 angstrom tetraethylene glycol (TEG) steric group was linked to the active site. A C-terminal lysine was tethered to the TEG to further enhance the activity (Xa-K). Treatment with purified plasmin showed that Xa-K was converted to the β-form without further proteolysis to the Xa33/13 analogue. Using conventional turbidity assays, the time for thrombin (3 nM)-induced plasma clots to reach 50% fibrinolysis (25 pM tPA) was shortened ∼8-fold by 1 nM Xa-K, whereas unmodified FXa at 200-fold higher concentration was insignificant. Identically modified trypsin-K had no effect on fibrinolysis. Using Doppler ultrasound to follow blood flow in surgically exposed mouse carotid arteries, the effect of Xa-K on shortening the time to reperfusion was evaluated after complete occlusion was induced by ferric chloride. The tPA variant Tenecteplase (TNKase) injected into the tail vein (17 µg/g) completely restored blood flow by ∼30 min, which was reduced to ∼14 min in the presence of Xa-K (0.5 µg/g, n=10). At a sub-therapeutic dose (9 µg/g, n=4) TNKase did not restore blood flow by the experimental end-point (60 minutes). However, complete reperfusion was observed by ∼20 min when combined with Xa-K (0.5 µg/g, n=8), showing that adjunctive Xa-K can reduce the required amount of TNKase. In the absence of TNKase, a dose-dependent effect on reperfusion was observed when Xa-K was injected alone (0.5 and 1.1 µg/g n=5), indicating an effect on endogenous tPA. Western blots of plasma from mice achieving complete reperfusion showed animals administered Xa-K alone, but not TNKase alone, had no systemic plasminogen or fibrinogen fragmentation. Furthermore, XaK appears to have significantly attenuated the systemic effect of TNKase during adjunctive treatment, possibly due to localization by Xa-K to the site of the clot. These results support a role for auxiliary cofactors in fibrinolysis and suggest that Xa-K may have novel thrombolytic applications. Disclosures: Pryzdial: Canadian Blood Services: Employment, Inventor, Inventor Patents & Royalties.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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) | 0.000 | 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 | 0.000 | 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".