Factor Xa-Antithrombin Complex Enhances Tissue Plasminogen Activator-Mediated Clot Lysis
Notice bibliographique
Résumé
Abstract Abstract 1180 Introduction: The current model of fibrinolysis accepts fibrin as the only cofactor involved in accelerating tissue plasminogen activator (tPA)-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease. Our previous work has demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin. In the presence of anionic phospholipid and calcium, intact FXa-alpha is converted to FXa-beta and then to non-covalently associated fragments of 33kDa and 13kDa (Xa33/13). Contrary to fibrinolysis dogma, Xa33/13 is a potent accelerator of tPA-dependent dissolution of purified fibrin because localized C-terminal lysine binding sites for plasminogen and tPA are exposed. Here we investigated whether Xa33/13 is produced in plasma during clot formation and dissolution. The normal fate of FXa produced in plasma is rapid irreversible association with its inhibitor, antithrombin (AT). Therefore, the effect of complex formation with AT on FXa fibrinolysis accelerator function was also evaluated. Methods: Purified FXa and Xa-AT plasmin cleavage profiles were monitored by Coomassie blue protein staining or by western blot using FX and AT-specific monoclonal antibodies. N-terminal sequencing was performed by automated Edman degradation to map FXa and AT cleavage sites.125I-plasminogen was used to probe plasminogen binding sites by ligand blotting. To follow purified plasminogen activation by tPA, plasmin generation was evaluated chromogenically (S2251). Fibrinolysis of plasma clots were measured by turbidity. Results: FX western blot analysis of plasma induced to clot showed the rapid formation of a band corresponding to a Xa-AT complex that correlated to the appearance of Xa33 antigen. An AT western blot detected the simultaneous appearance of a band of approximately 13 kDa higher apparent molecular weight than AT. Xa13 is known to contain the AT covalent linkage site, suggesting a Xa13-AT adduct. Purified Xa-AT was cleaved at least10-fold faster by plasmin compared to FXa and generated a Xa33/13 species. By electophoresis the resulting Xa33 derived from Xa-AT or FXa was not distinguishable, but sequencing revealed different cleavage sites at Lys-Met339 and Lys-Gly331, respectively. Cleavage of Xa-AT by FXa also liberated a Xa33 fragment with cleavage at Arg-Thr337. Sequencing confirmed the identity of the covalent, Xa13-AT, and non-covalent, Xa33/13-AT, complexes. Ligand blots revealed 125I-plasminogen binding to the Xa33 subunit of Xa33/13-AT generated by plasmin cleavage, which is consistent with the predicted exposure of a new C-terminal Lys338. Chromogenic assays demonstrated Xa-AT enhanced tPA-dependent plasmin generation by 7-fold. An excess of Xa-AT to overcome the endogenous FX and AT in plasma significantly enhanced the rate of fibrinolysis. Conclusions: For the first time we demonstrate the generation of Xa33/13 in plasma, supporting its physiological relevance as a tPA cofactor. Within this context, a novel function for AT is furthermore suggested by our results. In complex with FXa, AT accelerates the rate of conversion of FXa into the fibrinolytic form, Xa33/13. The subsequent exposure of new C-terminal lysine binding sites for plasminogen enhances fibrinolysis. These results are consistent with an auxiliary cofactor model of fibrinolysis in addition to the accelerating role of fibrin. Disclosures: No relevant conflicts of interest to declare.
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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 | 0,001 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,004 | 0,001 |
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 ».