Regular Article The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes Manjunath Goolyam Basavaraj a, ⁎, Franz X. Gruber b , Mikhail Sovershaev c , Hege I. Appelbom a , Bjarne Østerud d , Lars C. Petersen e , John-Bjarne Hansen a a Hematological Research Group (HERG), Department of Clinical Medicine, University of Tromsø, Tromsø, Norway b Department of Pharmacology, Institute of Pharmacy, University of Tromsø, Tromsø, Norway c Division of Internal Medicine, University Hospital of North Norway, Tromsø, Norway d Hematological Research Group (HERG), Department of Medical Biology, University of Tromsø, Tromsø, Norway e Biopharmaceuticals Research Unit, Novo Nordisk, Måløv, Denmark abstract article info Article history: Received 29 March 2010 Received in revised form 4 July 2010 Accepted 19 July 2010 Available online 17 August 2010 Keywords: Monocytes Tissue Factor Tissue factor pathway inhibitor Thrombin Introduction: 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. Materials and 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 6 hrs 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. © 2010 Elsevier Ltd. All rights reserved. Introduction Monocytes are important regulators of blood thrombogenicity through expression and presentation of tissue factor (TF) on their surface under various pathological conditions [1]. TF is an integral membrane protein with a molecular weight of 45 kDa, which initiates blood coagulation through binding to factor VII/VIIa on cell surfaces with subsequent limited proteolysis of factor IX and X ultimately leading to thrombin generation [2,3]. Although TF is normally absent from cells in contact with circulating blood, monocytes express cell- surface TF upon activation under certain pathological conditions, such as stable angina and unstable angina [4], acute myocardial infarction [4,5], and endotoxinemia [6]. Rupture of the culprit lesion followed by an immediate formation of intraluminal thrombus is the main cause of acute coronary events [7]. TF is also expressed in monocytes/ macrophages/foam cells resident in atherosclerotic plaques [8], and the thrombogenicity of the plaque is largely associated with intramural TF expression [9]. Tissue factor pathway inhibitor (TFPI) is the principal endogenous inhibitor of TF induced blood coagulation, which exerts its function by neutralizing FXa directly and by feedback inhibition of the FVIIa/TF catalytic complex in the presence of FXa [10,11]. To date, several Thrombosis Research 126 (2010) 418–425 Abbreviations: APC, allophycocyanin; PE, Phycoerythrin; TF, tissue factor; TFPI, tissue factor pathway inhibitor; PBMCs, peripheral blood mononuclear cells; CAT, calibrated automated thrombogram; LPS, lipopolysaccharide; FVIIa, factor VIIa; FXa, factor Xa; GPI, glycosylphosphatidylinositol; HEPES, 4-(2-hydroxyethyl)-1-piperazi- neethanesulfonic acid; PBS, phosphate buffered saline; w/v, weight per volume; BSA, bovine serum albumin; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative real-time polymerase chain reaction; MIQE, mini- mum information for publication of quantitative real-time PCR experiments; C q , quantification cycle; GUS, beta-glucuronidase; NTCN, normalized target copy number; MFI, median fluorescence intensity; AU, arbitrary unit; hr, hour; min, minute; sec, second. ⁎ Corresponding author. Hematological Research Group (HERG), Department of Clinical Medicine, University of Tromsø, N-9037 Tromsø, Norway. Tel.: +47 776 45479; fax: +47 77644650. E-mail address: manjunath.goolyam@uit.no (M.G. Basavaraj). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.07.014 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres