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Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation

The prothrombinase complex processes prothrombin to thrombin through sequential cleavage at Arg320 followed by Arg271 when cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all bound to the same membrane surface. In the absence of the membrane or cofactor, cleavage occurs in th...

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Autores principales: Üstok, Fatma Işık, Huntington, James A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Hematology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9101250/
https://www.ncbi.nlm.nih.gov/pubmed/35148539
http://dx.doi.org/10.1182/blood.2021014878
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author Üstok, Fatma Işık
Huntington, James A.
author_facet Üstok, Fatma Işık
Huntington, James A.
author_sort Üstok, Fatma Işık
collection PubMed
description The prothrombinase complex processes prothrombin to thrombin through sequential cleavage at Arg320 followed by Arg271 when cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all bound to the same membrane surface. In the absence of the membrane or cofactor, cleavage occurs in the opposite order. For the less favorable cleavage site at Arg320 to be cleaved first, it is thought that prothrombin docks on fVa in a way that presents Arg320 and hides Arg271 from the active site of fXa. Based on the crystal structure of the prothrombinase complex from the venom of the Australian eastern brown snake, pseutarin C, we modeled an initial prothrombin docking mode, which involved an interaction with discrete portions of the A1 and A2 domains of fV and the loop connecting the 2 domains, known as the a1-loop. We interrogated the proposed interface by site-directed PEGylation and by swapping the a1-loop in pseutarin C with that of human fV and fVIII and measuring the effect on rate and pathway of thrombin generation. PEGylation of residues within our proposed binding site greatly reduced the rate of thrombin generation, without affecting the pathway, whereas those outside the proposed interface had no effect. PEGylation of residues within the a1-loop also reduced the rate of thrombin generation. The sequence of the a1-loop was found to play a critical role in prothrombin binding and in the presentation of Arg320 for initial cleavage.
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spelling pubmed-91012502022-06-04 Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation Üstok, Fatma Işık Huntington, James A. Blood Thrombosis and Hemostasis The prothrombinase complex processes prothrombin to thrombin through sequential cleavage at Arg320 followed by Arg271 when cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all bound to the same membrane surface. In the absence of the membrane or cofactor, cleavage occurs in the opposite order. For the less favorable cleavage site at Arg320 to be cleaved first, it is thought that prothrombin docks on fVa in a way that presents Arg320 and hides Arg271 from the active site of fXa. Based on the crystal structure of the prothrombinase complex from the venom of the Australian eastern brown snake, pseutarin C, we modeled an initial prothrombin docking mode, which involved an interaction with discrete portions of the A1 and A2 domains of fV and the loop connecting the 2 domains, known as the a1-loop. We interrogated the proposed interface by site-directed PEGylation and by swapping the a1-loop in pseutarin C with that of human fV and fVIII and measuring the effect on rate and pathway of thrombin generation. PEGylation of residues within our proposed binding site greatly reduced the rate of thrombin generation, without affecting the pathway, whereas those outside the proposed interface had no effect. PEGylation of residues within the a1-loop also reduced the rate of thrombin generation. The sequence of the a1-loop was found to play a critical role in prothrombin binding and in the presentation of Arg320 for initial cleavage. American Society of Hematology 2022-05-12 /pmc/articles/PMC9101250/ /pubmed/35148539 http://dx.doi.org/10.1182/blood.2021014878 Text en © 2022 by The American Society of Hematology. https://creativecommons.org/licenses/by-nc-nd/4.0/Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.
spellingShingle Thrombosis and Hemostasis
Üstok, Fatma Işık
Huntington, James A.
Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation
title Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation
title_full Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation
title_fullStr Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation
title_full_unstemmed Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation
title_short Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation
title_sort mapping the prothrombin-binding site of pseutarin c by site-directed pegylation
topic Thrombosis and Hemostasis
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9101250/
https://www.ncbi.nlm.nih.gov/pubmed/35148539
http://dx.doi.org/10.1182/blood.2021014878
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