| Reconstitution of rabbit thrombomodulin into phospholipid vesicles. | |
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MedLine Citation:
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PMID: 3029069 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C. |
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Authors:
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J B Galvin; S Kurosawa; K Moore; C T Esmon; N L Esmon |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 262 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1987 Feb |
Date Detail:
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Created Date: 1987-03-30 Completed Date: 1987-03-30 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 2199-205 Citation Subset: IM |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Calcium / metabolism Calcium-Binding Proteins / metabolism Cattle Kinetics Liposomes / metabolism* Osteocalcin Peptide Fragments / pharmacology Phospholipids* Protein C / metabolism Protein Precursors / pharmacology Prothrombin / pharmacology Rabbits Receptors, Cell Surface / metabolism* Receptors, Thrombin |
| Grant Support | |
ID/Acronym/Agency:
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R01 HL29807/HL/NHLBI NIH HHS; R01 HL30340/HL/NHLBI NIH HHS; T32 HL 07202-10/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Calcium-Binding Proteins; 0/Liposomes; 0/Peptide Fragments; 0/Phospholipids; 0/Protein C; 0/Protein Precursors; 0/Receptors, Cell Surface; 0/Receptors, Thrombin; 104982-03-8/Osteocalcin; 72270-84-9/prothrombin fragment 1; 7440-70-2/Calcium; 9001-26-7/Prothrombin |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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