Participation of endothelial cells in the protein C-protein S anticoagulant pathway

The synthesis and release of protein S

David Stern, J. Brett, K. Harris, P. Nawroth

Research output: Contribution to journalArticle

99 Citations (Scopus)

Abstract

The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at M(r) ~ 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/105 cells per 24 h and contain 44 fmol/105 cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor V(a) inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by > 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 μM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.

Original languageEnglish (US)
Pages (from-to)1971-1978
Number of pages8
JournalJournal of Cell Biology
Volume102
Issue number5
DOIs
StatePublished - Jan 1 1986
Externally publishedYes

Fingerprint

Protein S
Protein C
Anticoagulants
Endothelial Cells
Endothelium
Calcium
Antigens
Blood Coagulation Factors
Factor V
Vitamin K
Calcium Ionophores
Rough Endoplasmic Reticulum
Serum-Free Culture Media
Calcimycin
Octoxynol
Golgi Apparatus
Warfarin
Cycloheximide
Lysosomes
Plasma Cells

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

Participation of endothelial cells in the protein C-protein S anticoagulant pathway : The synthesis and release of protein S. / Stern, David; Brett, J.; Harris, K.; Nawroth, P.

In: Journal of Cell Biology, Vol. 102, No. 5, 01.01.1986, p. 1971-1978.

Research output: Contribution to journalArticle

@article{349b1924bdf74983bac5fdba035b6273,
title = "Participation of endothelial cells in the protein C-protein S anticoagulant pathway: The synthesis and release of protein S",
abstract = "The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at M(r) ~ 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/105 cells per 24 h and contain 44 fmol/105 cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor V(a) inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by > 90{\%} and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 μM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.",
author = "David Stern and J. Brett and K. Harris and P. Nawroth",
year = "1986",
month = "1",
day = "1",
doi = "10.1083/jcb.102.5.1971",
language = "English (US)",
volume = "102",
pages = "1971--1978",
journal = "Journal of Cell Biology",
issn = "0021-9525",
publisher = "Rockefeller University Press",
number = "5",

}

TY - JOUR

T1 - Participation of endothelial cells in the protein C-protein S anticoagulant pathway

T2 - The synthesis and release of protein S

AU - Stern, David

AU - Brett, J.

AU - Harris, K.

AU - Nawroth, P.

PY - 1986/1/1

Y1 - 1986/1/1

N2 - The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at M(r) ~ 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/105 cells per 24 h and contain 44 fmol/105 cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor V(a) inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by > 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 μM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.

AB - The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at M(r) ~ 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/105 cells per 24 h and contain 44 fmol/105 cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor V(a) inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by > 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 μM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.

UR - http://www.scopus.com/inward/record.url?scp=0022444441&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022444441&partnerID=8YFLogxK

U2 - 10.1083/jcb.102.5.1971

DO - 10.1083/jcb.102.5.1971

M3 - Article

VL - 102

SP - 1971

EP - 1978

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 5

ER -