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J Biol Chem. 2014 Nov 7;289(45):31647-61. doi: 10.1074/jbc.M114.569665. Epub 2014 Sep 28.

Platelets contain tissue factor pathway inhibitor-2 derived from megakaryocytes and inhibits fibrinolysis.

Author information

1
From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery.
2
the Department of Molecular and Medical Pharmacology.
3
the Department of Pediatrics, Division of Hematology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
4
the Department of Molecular and Medical Pharmacology, the Molecular Biology Institute, UCLA, Los Angeles, California 90095 and.
5
the Department of Medicine, Division of Pulmonology and Critical Care, and.
6
From the UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, the Molecular Biology Institute, UCLA, Los Angeles, California 90095 and pbajaj@mednet.ucla.edu.

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.

KEYWORDS:

Fibrinolysis; Hemostasis; Plasmin; Platelet; Protease Inhibitor

PMID:
25262870
PMCID:
PMC4223360
DOI:
10.1074/jbc.M114.569665
[Indexed for MEDLINE]
Free PMC Article

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