Exposure of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII.
Notice bibliographique
Résumé
Abstract The intrinsic tenase complex and the prothrombinase complex are composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. Incorporation of the protein cofactor in both complexes results in a substantial increase in the catalytic efficiency of both enzymes, factor IXa and factor Xa, for cleavage and activation of factor X and prothrombin respectively, resulting in normal hemostasis. The procofactors, factor V (FV) and factor VIII (fVIII), do not interact with the components of prothrombinase and intrinsic tenase respectively and must be activated. α-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate binding necessary for efficient catalysis. α-Thrombin catalyzes the activation of fV and fVIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by α-thrombin. We have used plasma-derived α-thrombin, β-thrombin (a thrombin molecule that has only ABE-II available) and a recombinant prothrombin molecule rMz-II (R155A/R284A/R271A) that can only be cleaved at Arg320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a sulfated pentapeptide (DY(SO3−)DY(SO3−)Q, named D5Q1,2) as exosite-directed inhibitor of thrombin. D5Q1,2 was found to increase thrombin time in a dose dependent manner yielding an eight-fold increase in thrombin time at 250 μM in the presence of 10 nM α-thrombin. This clotting time was equivalent to the thrombin time obtained with 10 nM β-thrombin alone. The clotting time of rMZ-IIa was increased four-fold compared to the clotting time of α-thrombin under similar experimental conditions. α-Thrombin readily activated fV following cleavages at Arg709, Arg1018, and Arg1545 and fVIII following proteolysis at Arg372, Arg740, and Arg1689. Cleavage of both procofactors by α-thrombin was significantly inhibited by D5Q1,2. In contrast, β-thrombin was unable to cleave fV at Arg1545 and fVIII at both Arg372 and Arg1689. The former is required for expression of factor Va (fVa) cofactor activity while the latter two cleavages are a prerequisite for expression of factor VIIIa (fVIIIa) cofactor activity. β-Thrombin was found to cleave fV at Arg709 and fVIII at Arg740, albeit less efficiently than α-thrombin. D5Q1,2 inhibited moderately both cleavages by β-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules with a rate similar to that observed for the activation of fV and fVIII by α-thrombin. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. These data demonstrate that ABE-I alone of α-thrombin can account for the interaction of both procofactors with α-thrombin resulting in their timely and efficient activation. Our data also show that a sulfated pentapeptide inhibits several procoagulant ABE-I-related functions of α-thrombin and provide a target as well as the scaffold for the synthesis of an exosite-directed anticoagulant molecule that could inhibit and/or attenuate therapeutically thrombin function in individuals with thrombotic tendencies.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
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 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 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 ».