Tissue Factor Prothrombotic Activity Is Regulated by Integrin-arf6 Trafficking

TY - JOUR

T1 - Tissue Factor Prothrombotic Activity Is Regulated by Integrin-arf6 Trafficking

AU - Rothmeier, Andrea S

AU - Marchese, Patrizia

AU - Langer, Florian

AU - Kamikubo, Yuichi

AU - Schaffner, Florence

AU - Cantor, Joseph

AU - Ginsberg, Mark H

AU - Ruggeri, Zaverio M

AU - Ruf, Wolfram

N1 - © 2017 American Heart Association, Inc.

PY - 2017/7

Y1 - 2017/7

N2 - OBJECTIVE: Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood.APPROACH AND RESULTS: Here, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation.CONCLUSIONS: These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.

AB - OBJECTIVE: Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood.APPROACH AND RESULTS: Here, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation.CONCLUSIONS: These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.

KW - ADP-Ribosylation Factors

KW - Adenosine Triphosphate

KW - Animals

KW - Blood Coagulation

KW - Cell Line, Tumor

KW - Cell-Derived Microparticles

KW - Factor VIIa

KW - Gene Knock-In Techniques

KW - Genotype

KW - Humans

KW - Integrin alpha4

KW - Integrin alpha4beta1

KW - Macrophages

KW - Mice, Inbred C57BL

KW - Mice, Transgenic

KW - Phenotype

KW - Phospholipids

KW - Protein Transport

KW - Receptors, Purinergic P2X7

KW - Signal Transduction

KW - Thromboplastin

KW - Thrombosis

KW - Transfection

KW - Journal Article

U2 - 10.1161/ATVBAHA.117.309315

DO - 10.1161/ATVBAHA.117.309315

M3 - SCORING: Journal article

C2 - 28495929

VL - 37

SP - 1323

EP - 1331

JO - ARTERIOSCL THROM VAS

JF - ARTERIOSCL THROM VAS

SN - 1079-5642

IS - 7

ER -