The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes.
BACKGROUND
Although the procoagulant reactivity of monocytes largely depends on expression and cell surface presentation of tissue factor (TF), little is known about the impact of tissue factor pathway inhibitor (TFPI) on regulation of TF function on the monocyte surface.


METHODS
Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy subjects and cryopreserved. We investigated TF and TFPI mRNA expression by reverse transcription-quantitative real-time PCR (RT-qPCR), surface presentation by flow cytometry and confocal microscopy, and TFPI-mediated regulation of TF functional activity on the surface of resting and LPS-stimulated PBMCs by TF activity assay and Calibrated Automated Thrombogram (CAT) assay.


RESULTS
Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPIα mRNA isoform expression was higher than TFPIβ. LPS stimulation caused a parallel and sustained upregulation of both TFPI isoforms, concomitant with increased surface presentation of TFPI antigen. Stronger, but transient upregulation of TF mRNA and surface antigen was observed at 6hrs of LPS stimulation. After LPS stimulation TF and TFPI were co-localized in the same areas of the monocyte membrane. Pre-incubation of PBMCs with anti-TFPI IgG significantly enhanced TF activity, shortened Lag-time, and increased thrombin generation. TFPI-dependent inhibition of TF was more prominent in resting than in LPS-stimulated cells.


CONCLUSIONS
Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes.
Basavaraj
Manjunath Goolyam
MG
Hematological Research Group (HERG), Department of Clinical Medicine, University of Tromsø, Tromsø, Norway. manjunath.goolyam@uit.no
Gruber
Franz X
FX
Sovershaev
Mikhail
M
Appelbom
Hege I
HI
Osterud
Bjarne
B
Petersen
Lars C
LC
Hansen
John-Bjarne
JB
D000328:Adult; D005434:Flow Cytometry; D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018613:Microscopy, Confocal; D008875:Middle Aged; D009000:Monocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013925:Thromboplastin / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013927:Thrombosis / Q000097:blood
D008074:Lipoproteins; D012333:RNA, Messenger; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin