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Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡
[Image: see text] Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646660/ https://www.ncbi.nlm.nih.gov/pubmed/18590276 http://dx.doi.org/10.1021/bi800593k |
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author | Hirbawi, Jamila Bukys, Michael A. Barhoover, Melissa A. Erdogan, Evrim Kalafatis, Michael |
author_facet | Hirbawi, Jamila Bukys, Michael A. Barhoover, Melissa A. Erdogan, Evrim Kalafatis, Michael |
author_sort | Hirbawi, Jamila |
collection | PubMed |
description | [Image: see text] Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680−709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor V(Δ680−709)) and a mutant molecule with the (695)DYDY(698) → AAAA substitutions (factor V(4A)). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor Va(Wt)). Using an assay employing purified reagents, we found that prothrombinase assembled with factor Va(Δ680−709) displayed an ∼39% increase in k(cat), while prothrombinase assembled with factor Va(4A) exhibited an ∼20% increase in k(cat) for the activation of prothrombin as compared to prothrombinase assembled with factor Va(Wt). Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg(271) and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase. |
format | Text |
id | pubmed-2646660 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-26466602009-03-20 Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡ Hirbawi, Jamila Bukys, Michael A. Barhoover, Melissa A. Erdogan, Evrim Kalafatis, Michael Biochemistry [Image: see text] Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680−709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor V(Δ680−709)) and a mutant molecule with the (695)DYDY(698) → AAAA substitutions (factor V(4A)). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor Va(Wt)). Using an assay employing purified reagents, we found that prothrombinase assembled with factor Va(Δ680−709) displayed an ∼39% increase in k(cat), while prothrombinase assembled with factor Va(4A) exhibited an ∼20% increase in k(cat) for the activation of prothrombin as compared to prothrombinase assembled with factor Va(Wt). Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg(271) and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase. American Chemical Society 2008-07-01 2008-07-29 /pmc/articles/PMC2646660/ /pubmed/18590276 http://dx.doi.org/10.1021/bi800593k Text en Copyright © 2008 American Chemical Society http://pubs.acs.org This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org. 40.75 |
spellingShingle | Hirbawi, Jamila Bukys, Michael A. Barhoover, Melissa A. Erdogan, Evrim Kalafatis, Michael Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡ |
title | Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡ |
title_full | Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡ |
title_fullStr | Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡ |
title_full_unstemmed | Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡ |
title_short | Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase‡ |
title_sort | role of the acidic hirudin-like cooh-terminal amino acid region of factor va heavy chain in the enhanced function of prothrombinase‡ |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646660/ https://www.ncbi.nlm.nih.gov/pubmed/18590276 http://dx.doi.org/10.1021/bi800593k |
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