Activation of protein C and thrombin activable fibrinolysis inhibitor on cultured human endothelial cells

C Wu; P Y Kim; L L Swystun; P C Liaw; J I Weitz

doi:10.1111/jth.13222

Activation of protein C and thrombin activable fibrinolysis inhibitor on cultured human endothelial cells

J Thromb Haemost. 2016 Feb;14(2):366-74. doi: 10.1111/jth.13222. Epub 2016 Feb 1.

Authors

C Wu^{1

2}, P Y Kim^{1

2}, L L Swystun³, P C Liaw^{1

2}, J I Weitz^{1

2

4}

Affiliations

¹ Thrombosis and Atherosclerosis Research Institute, Hamilton, ON, Canada.
² Department of Medicine, McMaster University, Hamilton, ON, Canada.
³ Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada.
⁴ Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.

PMID: 26663133
DOI: 10.1111/jth.13222

Abstract

ESSENTIALS: It is unknown if thrombin activatable fibrinolysis inhibitor (TAFI) and protein C compete on cells. TAFI and protein C activation on endothelial cells was simultaneously quantified. TAFI and protein C do not compete for activation on endothelial cells. TAFI and protein C are independently recognized by the thrombin-thrombomodulin complex.

Background: When bound to thrombomodulin (TM), thrombin is a potent activator of protein C (PC) and thrombin activable fibrinolysis inhibitor (TAFI). By binding PC and presenting it to the thrombin-TM complex, endothelial cell PC receptor (EPCR) enhances PC activation. It is unknown whether PC and TAFI compete for the thrombin-TM complex on endothelial cells.

Objective: To compare PC and TAFI activation on the surface of cultured human endothelial cells in the absence or presence of JRK1535 and/or CTM1009, inhibitory antibodies directed against EPCR and TM, respectively, and to determine whether PC and TAFI compete with each other for activation.

Methods: PC and TAFI activation on endothelial cells were compared, and the effect of PC on TAFI activation and TAFI on PC activation was determined in the absence or presence of JRK1535 and/or CTM1009.

Results: In the absence of antibodies, activation of PC was four-fold faster than that of TAFI. Blocking EPCR with JRK1535 resulted in a 53-fold decrease in PC activation and no effect on TAFI activation. Blocking TM with CTM1009 inhibited both TAFI and PC activation. Neither TAFI nor PC competed with each other in the absence or presence of JRK1535.

Conclusions: PC and TAFI are concurrently activated in a TM-dependent manner and do not compete for the thrombin-TM complex, raising the possibility that they interact with distinct activation complexes. EPCR selectively enhances PC activation so that PC and TAFI activation kinetics become comparable on endothelial cells.

Keywords: carboxypeptidase B2; endothelial cells; enzyme activation; protein C; thrombin-activable fibrinolysis inhibitor; thrombomodulin.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Antigens, CD / metabolism*
Binding, Competitive
Carboxypeptidase B2 / metabolism*
Cells, Cultured
Endothelial Cells / enzymology*
Endothelial Protein C Receptor
Enzyme Activation
Human Umbilical Vein Endothelial Cells / enzymology
Humans
Kinetics
Protein Binding
Protein C / metabolism*
Receptors, Cell Surface / metabolism*
Thrombin / metabolism
Thrombomodulin / metabolism

Substances

Antigens, CD
Endothelial Protein C Receptor
PROCR protein, human
Protein C
Receptors, Cell Surface
THBD protein, human
Thrombomodulin
Carboxypeptidase B2
Thrombin

Grants and funding

MOP-97743/Canadian Institutes of Health Research/Canada