title: Platelets contain tissue factor pathway inhibitor-2 derived from megakaryocytes and inhibits fibrinolysis.
abstract: Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pM) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with K(d) ~9 nM and binds FVa with K(d) ~100 nM. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with K(d) ~36-48 nM and bind FVa with K(d) ~252-456 nM. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (~7 nM) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.
authors.affiliation: From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery.
authors.forename: Kanagasabai
authors.initials: K
authors.lastname: Vadivel
authors.affiliation: From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery.
authors.forename: Sathya-Moorthy
authors.initials: SM
authors.lastname: Ponnuraj
authors.affiliation: From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Kumar
authors.affiliation: the Department of Molecular and Medical Pharmacology.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Zaiss
authors.affiliation: the Department of Pediatrics, Division of Hematology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Bunce
authors.affiliation: the Department of Pediatrics, Division of Hematology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Rodney M
authors.initials: RM
authors.lastname: Camire
authors.affiliation: From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery.
authors.forename: Ling
authors.initials: L
authors.lastname: Wu
authors.affiliation: From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery.
authors.forename: Denis
authors.initials: D
authors.lastname: Evseenko
authors.affiliation: the Department of Molecular and Medical Pharmacology, the Molecular Biology Institute, UCLA, Los Angeles, California 90095 and.
authors.forename: Harvey R
authors.initials: HR
authors.lastname: Herschman
authors.affiliation: the Department of Medicine, Division of Pulmonology and Critical Care, and.
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Bajaj
authors.affiliation: From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, the Molecular Biology Institute, UCLA, Los Angeles, California 90095 and pbajaj@mednet.ucla.edu.
authors.forename: S Paul
authors.initials: SP
authors.lastname: Bajaj
mesh_terms: D001777:Blood Coagulation; D001792:Blood Platelets / Q000166:cytology* / Q000201:enzymology; D001854:Bone Marrow Cells / Q000166:cytology; D005260:Female; D005312:Fetal Blood / Q000201:enzymology; D005342:Fibrinolysis / Q000502:physiology*; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006487:Hemostasis; D006801:Humans; D008024:Ligands; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008533:Megakaryocytes / Q000166:cytology*; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism*; D011247:Pregnancy; D011480:Protease Inhibitors / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
chemical_list: D006023:Glycoproteins; D008024:Ligands; D008074:Lipoproteins; D040201:Platelet Membrane Glycoprotein IIb; D011480:Protease Inhibitors; C051928:lipoprotein-associated coagulation inhibitor; C021240:tissue-factor-pathway inhibitor 2
keywords: Fibrinolysis; Hemostasis; Plasmin; Platelet; Protease Inhibitor