Platelet-Mediated Mechanical Tensile Force Influences ADAMTS13 Localization and Regulation Of Thrombus Development At The Site Of Platelet Accumulation
Notice bibliographique
Résumé
Background The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion and aggregation at the site of vessel injury. The adhesive property of VWF is regulated by its multimer length, such that ultra large VWF (ULVWF) multimers, newly released from the endothelium, have greater hemostatic activity. multimer size is regulated by the metalloprotease ADAMTS13, which cleaves the A2 domain to reduce VWF multimer size and functional activity. static conditions, VWF maintains a globular conformation and the ADAMTS13 cleavage site is inaccessible. However, the exposure of endothelial-anchored VWF to tensile forces mediated by platelets and hydrodynamic shear enhance the cleavage of VWF by ADAMTS13. releases VWF of optimal hemostatic length from the endothelium into the plasma. We have previously reported using a flow chamber model which demonstrates that in addition to regulating VWF length and activity at the site of release, ADAMTS13 also associates with VWF at the site of thrombus formation. observed that under conditions of high and very high shear, ADAMTS13 reduced the size of thrombus volume., multi-coloured immunostaining revealed that ADAMTS13 co-localized with VWF and platelets at the top and middle layers of the thrombus, in the presence of very high shear. Aim To better understand the mechanism by which ADAMTS13 regulates thrombus size in our flow chamber model, we assessed the contribution of platelet tensile force to the localization of ADAMTS13 at the site of the thrombus. this model, the contributions of platelet GPIb, GPIIbIIIa, and P-selectin to ADAMTS13 localization were observed. Method Full length mouse VWF and ADAMTS13 cDNA were cloned into pCIneo and pcDNA3.1 plasmid, respectively. The gain of platelet GPIb binding mutation V1316M, and loss of GPIIbIIIa binding mutation (RGD to RGG) were introduced by site-directed-mutagenesis. mCherry was cloned at the C terminus of ADAMTS13 with a 12AA linker. Recombinant mVWF and mADAMTS13-mCherry proteins were produced via HEK293T cells by calcium phosphate transient transfection. mADAMTS13-mCherry (2 U/mL) and wild type or mutant mVWF (4 U/mL) was added to whole blood obtained from VWF-/-/ADAMTS13-/- double knockout mice. Whole blood containing DiOC6-labeled platelets was perfused over a collagen coated flow chamber at very high shear (7500s-1). The role of P-selectin was also analyzed by adding a P-selectin blocking antibody to blood obtained from ADAMTS13-/-knockout mice prior to the flow chamber experiment. After the perfusion, thrombi were fixed and immunostaining was performed to further analyze the distribution of platelets, VWF and ADAMTS13. Result As previously reported, ADAMTS13 localization was observed in the top and middle layers of the thrombus in the presence of wild type mVWF. The GPIb gain-of-function mutation V1316M increased both platelet (126%, p<0.0001) and VWF (190% and p<0.0001) accumulation at the thrombus site. ADAMTS13 localization was also increased (135%, p<0.001) relative to the binding to wild type VWF. Interestingly, with this gain-of-function VWF mutant, ADAMTS13 localization was found throughout the entire thrombus. In contrast, the GPIIbIIIa RGD binding mutant demonstrated decreased VWF (56%, p<0.01), and ADAMTS13 (82%, p<0.05) intensity, although platelet intensity was unaffected. to wild type, ADAMTS13 localized to the middle and top layers of the thrombus. Finally, inhibition of P-selectin significantly decreased VWF (46%, p<0.01) and ADAMTS13 (34%, p<0.01) localization to the thrombus, but again did not significantly alter platelet binding. Conclusion These studies demonstrate the central role of platelet-mediated mechanical tensile force on the regulation of thrombus growth at the site of platelet accumulation. Enhanced tensile force induced by increased GPIb binding resulted in increased ADAMTS13 localization, while reduced tensile force through loss of GPIIbIIIa or P-selectin binding decreased ADAMTS13 localization. This suggests that ADAMTS13 activity at the site of thrombus formation is maintained by the combination of hydrodynamic shear force and platelet tethering. aggregate, these studies suggest that under conditions of shear, ADAMTS13 regulates thrombus size by preserving the hemostatic function of the thrombus, and preventing dysregulated thrombus growth. 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 | 0,000 | 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) | 0,002 | 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 ».