Covalent Stabilization Of Coagulation Factor Xa Enhances Its Fibrinolytic Function In Vitro and In Vivo: A Novel Cofactor-Based Thrombolytic Agent
Notice bibliographique
Résumé
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.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».